%0 Journal Article %J bioRxiv %D 2024 %T Sensing of dietary amino acids and regulation of calcium dynamics in adipose tissues through Adipokinetic hormone in Drosophila %A M Ahmad %A Wu, S. %A Guo, X. %A Perrimon, N %A H. Li %X
Nutrient sensing and the subsequent metabolic responses are fundamental functions of animals, closely linked to diseases such as type 2 diabetes and various obesity-related diseases. Drosophila melanogaster has emerged as an excellent model for investigating metabolism and its associated disorders. In this study, we used live-cell imaging to demonstrate that the fly functional homolog of mammalian glucagon, Adipokinetic hormone (AKH), secreted from AKH hormone-producing cells (APCs) in the corpora cardiaca, stimulates intracellular Ca 2+ waves in the larval fat body/adipose tissue to promote lipid metabolism. Further, we show that specific dietary amino acids activate the APCs, leading to increased intracellular Ca 2+ and subsequent AKH secretion. Finally, a comparison of Ca 2+ dynamics in larval and adult fat bodies revealed different mechanisms of regulation, highlighting the interplay of pulses of AKH secretion, extracellular diffusion of the hormone, and intercellular communication through gap junctions. Our study underscores the suitability of Drosophila as a powerful model for exploring real-time nutrient sensing and inter-organ communication dynamics.
%B bioRxiv %V Mar :2024.03.04.583442 %P doi: 10.1101/2024.03.04.583442. %G eng %0 Journal Article %J Nature Communications %D 2024 %T An evolutionary mechanism to assimilate new nutrient sensors into the mTORC1 pathway %A Liu, G. %A Jouandin, P %A R Bahng %A Perrimon, N %A D Sabatini %X Animals sense and respond to nutrient availability in their environments, a task coordinated in part by the mTOR complex 1 (mTORC1) pathway. mTORC1 regulates growth in response to nutrients and, in mammals, senses specific amino acids through specialized sensors that bind the GATOR1/2 signaling hub. Given that animals can occupy diverse niches, we hypothesized that the pathway might evolve distinct sensors in different metazoan phyla. Whether such customization occurs, and how the mTORC1 pathway might capture new inputs, is unknown. Here, we identify the Drosophila melanogaster protein Unmet expectations (CG11596) as a species-restricted methionine sensor that directly binds the fly GATOR2 complex in a fashion antagonized by S-adenosylmethionine (SAM). We find that in Dipterans GATOR2 rapidly evolved the capacity to bind Unmet and to thereby repurpose a previously independent methyltransferase as a SAM sensor. Thus, the modular architecture of the mTORC1 pathway allows it to co-opt preexisting enzymes to expand its nutrient sensing capabilities, revealing a mechanism for conferring evolvability on an otherwise conserved system. %B Nature Communications %V March 21 %P 2517 %G eng %N 15(1) %0 Journal Article %J Nature Communications %D 2024 %T Tick hemocytes have a pleiotropic role in microbial infection and arthropod fitness %A A Rolandelli %A H Laukaitis-Yousey %A H Bogale %A N Singh %A S Samaddar %A A O'Neal %A C Ferraz %A M Butaru %A E Mameli %A B Xia %A M Tays Mendes %A L Rainer Bulter %A L Marnin %A F Cabrera-Paz %A L Valencia %A V Rana %A C Skerry %A P Utpal %A Mohr, SE %A Perrimon, N %A D Serre %A J H Pedra %X Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here we use and develop advanced techniques to describe immune cells (hemocytes) from the clinically relevant tick Ixodes scapularis at a single-cell resolution. We observe molecular alterations in hemocytes upon feeding and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We reveal hemocyte clusters exhibiting defined signatures related to immunity, metabolism, and proliferation. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, two I. scapularis hemocyte markers, impacting blood-feeding, molting behavior, and bacterial acquisition. Mechanistically, astakine alters hemocyte proliferation, whereas hemocytin affects the c-Jun N-terminal kinase (JNK) signaling pathway in I. scapularis. Altogether, we discover a role for tick hemocytes in immunophysiology and provide a valuable resource for comparative biology in arthropods. %B Nature Communications %V Mar 8 %P 2117 %G eng %N 15(1) %0 Journal Article %J Genetics %D 2024 %T FlyBase: updates to the Drosophila genes and genomes database %A A Öztürk-Çolak %A SJ Marygold %A G Antonazzo %A H Attrill %A Goutte-Gattat, D %A VK Jenkins %A BB Matthews %A G Millburn %A G Dos Santos %A CJ Tabone %A FlyBase Consortium, The %X FlyBase (flybase.org) is a model organism database and knowledge base about Drosophila melanogaster, commonly known as the fruit fly. Researchers from around the world rely on the genetic, genomic, and functional information available in FlyBase, as well as its tools to view and interrogate these data. In this article, we describe the latest developments and updates to FlyBase. These include the introduction of single-cell RNA sequencing data, improved content and display of functional information, updated orthology pipelines, new chemical reports, and enhancements to our outreach resources. %B Genetics %V Feb 1 %G eng %N iyad211 %0 Journal Article %J Nucleic Acids Research %D 2024 %T Expression Atlas update: Insights from sequencing data at both bulk and single cell level %A George, N %A Fexova, S %A Fuentes, A M %A Madrigal, P %A Bi, Y %A Iqbal, H %A Kumbham, U %A Nolte, N F %A Zhao, L. %A Thanki, A S %A Yu, I D %A Calles, J C %A Erdos, K %A Vilmovsky, L %A Kurri, S R %A Vathrakokoili-Pournara, A %A Osumi-Sutherland, D %A Prakash, A %A S. Wang %A Tello-Ruiz, M K %A Kumari, S %A Ware, D %A Goutte-Gattat, D %A Hu, Y. %A Brown, N %A Perrimon, N %A Vizcaíno, J A %A Burdett, T %A Teichmann, S %A Brazma, A %A Papatheodorou, I %X Expression Atlas (www.ebi.ac.uk/gxa) and its newest counterpart the Single Cell Expression Atlas (www.ebi.ac.uk/gxa/sc) are EMBL-EBI's knowledgebases for gene and protein expression and localisation in bulk and at single cell level. These resources aim to allow users to investigate their expression in normal tissue (baseline) or in response to perturbations such as disease or changes to genotype (differential) across multiple species. Users are invited to search for genes or metadata terms across species or biological conditions in a standardised consistent interface. Alongside these data, new features in Single Cell Expression Atlas allow users to query metadata through our new cell type wheel search. At the experiment level data can be explored through two types of dimensionality reduction plots, t-distributed Stochastic Neighbor Embedding (tSNE) and Uniform Manifold Approximation and Projection (UMAP), overlaid with either clustering or metadata information to assist users' understanding. Data are also visualised as marker gene heatmaps identifying genes that help confer cluster identity. For some data, additional visualisations are available as interactive cell level anatomograms and cell type gene expression heatmaps. %B Nucleic Acids Research %V Jan 5 %P D107-D114 %G eng %N 52(D1) %0 Journal Article %J mBio %D 2024 %T Genetic manipulation of an Ixodes scapularis cell line %A N Singh %A A Rolandelli %A A J O'Neal %A L R Butler %A S Samaddar %A H J Loukaitis-Yousey %A M Butnaru %A Mohr, SE %A Perrimon, N %A J H Pedra %X Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis. %B mBio %V Feb %P e0247923 %G eng %N 21 %0 Journal Article %J Nature Communications %D 2024 %T Mechanistic characterization of a Drosophila model of paraneoplastic nephrotic syndrome %A Xu, J %A Liu, Y. %A Yang, F %A Cao, Y %A W. Chen %A Li, J S S %A Zhang, S. %A Comjean, A %A Hu, Y. %A Perrimon, N %X Paraneoplastic syndromes occur in cancer patients and originate from dysfunction of organs at a distance from the tumor or its metastasis. A wide range of organs can be affected in paraneoplastic syndromes; however, the pathological mechanisms by which tumors influence host organs are poorly understood. Recent studies in the fly uncovered that tumor secreted factors target host organs, leading to pathological effects. In this study, using a Drosophila gut tumor model, we characterize a mechanism of tumor-induced kidney dysfunction. Specifically, we find that Pvf1, a PDGF/VEGF signaling ligand, secreted by gut tumors activates the PvR/JNK/Jra signaling pathway in the principal cells of the kidney, leading to mis-expression of renal genes and paraneoplastic renal syndrome-like phenotypes. Our study describes an important mechanism by which gut tumors perturb the function of the kidney, which might be of clinical relevance for the treatment of paraneoplastic syndromes. %B Nature Communications %V Feb 9 %P 1241 %G eng %N 15(1) %0 Journal Article %J Genetics %D 2024 %T Lipophorin receptors genetically modulate neurodegeneration caused by reduction of Psn expression in the aging Drosophila brain %A Kang, Jongkyun %A Zhang, Chen %A Wang, Yuhao %A Peng, Jian %A Berger, Bonnie %A Perrimon, Norbert %A Shen, Jie %X Mutations in the Presenilin (PSEN) genes are the most common cause of early-onset familial Alzheimer's disease (FAD). Studies in cell culture, in vitro biochemical systems, and knockin mice showed that PSEN mutations are loss-of-function mutations, impairing γ-secretase activity. Mouse genetic analysis highlighted the importance of Presenilin (PS) in learning and memory, synaptic plasticity and neurotransmitter release, and neuronal survival, and Drosophila studies further demonstrated an evolutionarily conserved role of PS in neuronal survival during aging. However, molecular pathways that interact with PS in neuronal survival remain unclear. To identify genetic modifiers that modulate PS-dependent neuronal survival, we developed a new DrosophilaPsn model that exhibits age-dependent neurodegeneration and increases of apoptosis. Following a bioinformatic analysis, we tested top ranked candidate genes by selective knockdown (KD) of each gene in neurons using two independent RNAi lines in Psn KD models. Interestingly, 4 of the 9 genes enhancing neurodegeneration in Psn KD flies are involved in lipid transport and metabolism. Specifically, neuron-specific KD of lipophorin receptors, lpr1 and lpr2, dramatically worsens neurodegeneration in Psn KD flies, and overexpression of lpr1 or lpr2 does not alleviate Psn KD-induced neurodegeneration. Furthermore, lpr1 or lpr2 KD alone also leads to neurodegeneration, increased apoptosis, climbing defects, and shortened lifespan. Lastly, heterozygotic deletions of lpr1 and lpr2 or homozygotic deletions of lpr1 or lpr2 similarly lead to age-dependent neurodegeneration and further exacerbate neurodegeneration in Psn KD flies. These findings show that LpRs modulate Psn-dependent neuronal survival and are critically important for neuronal integrity in the aging brain. %B Genetics %V 226 %P iyad202 %G eng %N 1 %0 Journal Article %J bioRxiv %D 2023 %T Updates to the Alliance of Genome Resources Central Infrastructure Alliance of Genome Resources Consortium %X The Alliance of Genome Resources (Alliance) is an extensible coalition of knowledgebases focused on the genetics and genomics of intensively-studied model organisms. The Alliance is organized as individual knowledge centers with strong connections to their research communities and a centralized software infrastructure, discussed here. Model organisms currently represented in the Alliance are budding yeast, C. elegans, Drosophila, zebrafish, frog, laboratory mouse, laboratory rat, and the Gene Ontology Consortium. The project is in a rapid development phase to harmonize knowledge, store it, analyze it, and present it to the community through a web portal, direct downloads, and APIs. Here we focus on developments over the last two years. Specifically, we added and enhanced tools for browsing the genome (JBrowse), downloading sequences, mining complex data (AllianceMine), visualizing pathways, full-text searching of the literature (Textpresso), and sequence similarity searching (SequenceServer). We enhanced existing interactive data tables and added an interactive table of paralogs to complement our representation of orthology. To support individual model organism communities, we implemented species-specific “landing pages” and will add disease-specific portals soon; in addition, we support a common community forum implemented in Discourse. We describe our progress towards a central persistent database to support curation, the data modeling that underpins harmonization, and progress towards a state-of-the art literature curation system with integrated Artificial Intelligence and Machine Learning (AI/ML). %B bioRxiv %V Nov 22 %P doi: 10.1101/2023.11.20.567935 %G eng %N 2023.11.20.567935 %0 Journal Article %J EMBO Reports %D 2023 %T Protein phosphatase 1 regulates core PCP signaling %A Song, S %A Cho, B %A Weiner, A %A Nissen, SB %A Naharros, IO %A Bosch, PS %A Suyama, K %A Hu, Y. %A He, L %A Svinkina, T %A Udeshi, ND %A Carr, SA %A Perrimon, N %A Axelrod, J D %X Planar cell polarity (PCP) signaling polarizes epithelial cells within the plane of an epithelium. Core PCP signaling components adopt asymmetric subcellular localizations within cells to both polarize and coordinate polarity between cells. Achieving subcellular asymmetry requires additional effectors, including some mediating post-translational modifications of core components. Identification of such proteins is challenging due to pleiotropy. We used mass spectrometry-based proximity labeling proteomics to identify such regulators in the Drosophila wing. We identified the catalytic subunit of protein phosphatase1, Pp1-87B, and show that it regulates core protein polarization. Pp1-87B interacts with the core protein Van Gogh and at least one serine/threonine kinase, Dco/CKIε, that is known to regulate PCP. Pp1-87B modulates Van Gogh subcellular localization and directs its dephosphorylation in vivo. PNUTS, a Pp1 regulatory subunit, also modulates PCP. While the direct substrate(s) of Pp1-87B in control of PCP is not known, our data support the model that cycling between phosphorylated and unphosphorylated forms of one or more core PCP components may regulate acquisition of asymmetry. Finally, our screen serves as a resource for identifying additional regulators of PCP signaling. %B EMBO Reports %V Nov %P e56997 %G eng %N 17 %0 Journal Article %J Genetics %D 2023 %T Finding information about uncharacterized Drosophila melanogaster genes %A Mohr, S %A Kim, A-R %A Hu, Y. %A Perrimon, N %B Genetics %V 10.1093/genetics/iyad187 %G eng %0 Journal Article %J Cell Reports %D 2023 %T Molecular and functional characterization of the Drosophila melanogaster conserved smORFeome. %A Bosch, J %A Keith, N %A Escobedo, F %A Fisher, W. F. %A LaGraff, J. T %A Rabasco, J %A Wan, K %A Weissman, R %A Hu, Y. %A Kondo, S %A Brown, J. B. %A Perrimon, N %A Celniker, S %X Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 298 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, and ∼182 are conserved in plants. We observe remarkably heterogeneous spatial and temporal expression patterns of smORF transcripts-indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains reveals a predicted enrichment of smORF polypeptides localizing to mitochondria. We conduct an embryonic ribosome profiling experiment and find support for translation of 137 of these smORFs during embryogenesis. We further embark on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity. %B Cell Reports %V 42 %P doi: 10.1016/j.celrep.2023.113311. PMID: 37889754 %G eng %N 113311 %0 Journal Article %J Nature Communications %D 2023 %T The endoribonuclease Arlr is required to maintain lipid homeostasis by downregulating lipolytic genes during aging %A Sun, Xiaowei %A Shen, Jie %A Perrimon, Norbert %A Kong, Xue %A Wang, Dan %B Nature Communications %V 14 %G eng %N 6254 %0 Journal Article %J Nature %D 2023 %T Cholinergic neurons trigger epithelial Ca2+ currents to heal the gut %A Petsakou, Afroditi %A Liu, Yifang %A Liu, Ying %A Comjean, Aram %A Hu, Yanhui %A Perrimon, Norbert %X A fundamental and unresolved question in regenerative biology is how tissues return to homeostasis after injury. Answering this question is essential for understanding the etiology of chronic disorders such as inflammatory bowel diseases and cancer1. We used the Drosophila midgut2 to investigate this and discovered that during regeneration a subpopulation of cholinergic3 neurons triggers Ca2+ currents among intestinal epithelial cells, the enterocytes, to promote return to homeostasis. We found that down-regulation of the conserved cholinergic enzyme Acetylcholinesterase4 in the gut epithelium enables acetylcholine from specific TNF/Egr5-sensing cholinergic neurons to activate nicotinic receptors in innervated enterocytes. This activation triggers high Ca2+ that spreads in the epithelium through Inx2/Inx7 gap junctions6, promoting enterocyte maturation followed by reduction of proliferation and inflammation. Disrupting this process causes chronic injury consisting of ion imbalance, Yki/Yap activation7, cell death and increase of inflammatory cytokines reminiscent of inflammatory bowel diseases8. Altogether, the conserved cholinergic pathway facilitates epithelial Ca2+ currents that heal the intestinal epithelium. Our findings demonstrate nerve- and bioelectric9-dependent intestinal regeneration and advance our current understanding of how a tissue returns to homeostasis after injury. %B Nature %V Sep 18 %P doi: 10.1038/s41586-023-06627-y. %G eng %0 Journal Article %J Nature Communications %D 2023 %T REPTOR and CREBRF encode key regulators of muscle energy metabolism %A Saavedra, P %A Dumesic, P A %A Hu, Y. %A Filine, E %A Jouandin, P %A Binari, R %A Wilensky, S E %A Rodiger, J %A Wang, H. %A Chen, W. %A Liu, Y. %A Spiegelman, B M %A Perrimon, N %X Metabolic flexibility of muscle tissue describes the adaptive capacity to use different energy substrates according to their availability. The disruption of this ability associates with metabolic disease. Here, using a Drosophila model of systemic metabolic dysfunction triggered by yorkie-induced gut tumors, we show that the transcription factor REPTOR is an important regulator of energy metabolism in muscles. We present evidence that REPTOR is activated in muscles of adult flies with gut yorkie-tumors, where it modulates glucose metabolism. Further, in vivo studies indicate that sustained activity of REPTOR is sufficient in wildtype muscles to repress glycolysis and increase tricarboxylic acid (TCA) cycle metabolites. Consistent with the fly studies, higher levels of CREBRF, the mammalian ortholog of REPTOR, reduce glycolysis in mouse myotubes while promoting oxidative metabolism. Altogether, our results define a conserved function for REPTOR and CREBRF as key regulators of muscle energy metabolism. %B Nature Communications %V Aug 15 %P 4943 %G eng %N 14(1) %0 Journal Article %J Elife %D 2023 %T Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for Drosophila research %A Coleman-Gosser, N %A Raghuvansh, S %A Stitzinger, S %A Hu, T %A W. Chen %A Luhur, A %A Mariyappa, D %A Josifov, M %A Zelhof, A %A Mohr, SE %A Perrimon, N %A Simcox, A %X Expression of activated Ras, RasV12, provides Drosophila cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted RasV12 expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad RasV12 expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Drosophila Genomic Resource Center. %B Elife %V Jul 20 %P e85814 %G eng %N 12 %0 Journal Article %J Cell Discovery %D 2023 %T Gut AstA mediates sleep deprivation-induced energy wasting in Drosophila %A Li, Y. %A X Zhou %A Ding, G %A Zhao, P %A Tan, K. %A Cheng, L %A Perrimon, N %A Veenstra, J A %A Zhang, L %A Song, W %X Severe sleep deprivation (SD) has been highly associated with systemic energy wasting, such as lipid loss and glycogen depletion. Despite immune dysregulation and neurotoxicity observed in SD animals, whether and how the gut-secreted hormones participate in SD-induced disruption of energy homeostasis remains largely unknown. Using Drosophila as a conserved model organism, we characterize that production of intestinal Allatostatin A (AstA), a major gut-peptide hormone, is robustly increased in adult flies bearing severe SD. Interestingly, the removal of AstA production in the gut using specific drivers significantly improves lipid loss and glycogen depletion in SD flies without affecting sleep homeostasis. We reveal the molecular mechanisms whereby gut AstA promotes the release of an adipokinetic hormone (Akh), an insulin counter-regulatory hormone functionally equivalent to mammalian glucagon, to mobilize systemic energy reserves by remotely targeting its receptor AstA-R2 in Akh-producing cells. Similar regulation of glucagon secretion and energy wasting by AstA/galanin is also observed in SD mice. Further, integrating single-cell RNA sequencing and genetic validation, we uncover that severe SD results in ROS accumulation in the gut to augment AstA production via TrpA1. Altogether, our results demonstrate the essential roles of the gut-peptide hormone AstA in mediating SD-associated energy wasting. %B Cell Discovery %V 9 %P 49 %G eng %N 1 %0 Journal Article %J PNAS %D 2023 %T split-intein Gal4 provides intersectional genetic labeling that is repressible by Gal80 %A Ewen-Campen, Ben %A Luan, Haojiang %A Xu, Jun %A Singh, Rohit %A Joshi, Neha %A Thakkar, Tanuj %A Berger, Bonnie %A White, Benjamin H %A Perrimon, Norbert %X The split-Gal4 system allows for intersectional genetic labeling of highly specific cell types and tissues in Drosophila. However, the existing split-Gal4 system, unlike the standard Gal4 system, cannot be repressed by Gal80, and therefore cannot be controlled temporally. This lack of temporal control precludes split-Gal4 experiments in which a genetic manipulation must be restricted to specific timepoints. Here, we describe a split-Gal4 system based on a self-excising split-intein, which drives transgene expression as strongly as the current split-Gal4 system and Gal4 reagents, yet which is repressible by Gal80. We demonstrate the potent inducibility of “split-intein Gal4” in vivo using both fluorescent reporters and via reversible tumor induction in the gut. Further, we show that our split-intein Gal4 can be extended to the drug-inducible GeneSwitch system, providing an independent method for intersectional labeling with inducible control. We also show that the split-intein Gal4 system can be used to generate highly cell type–specific genetic drivers based on in silico predictions generated by single-cell RNAseq (scRNAseq) datasets, and we describe an algorithm (“Two Against Background” or TAB) to predict cluster-specific gene pairs across multiple tissue-specific scRNA datasets. We provide a plasmid toolkit to efficiently create split-intein Gal4 drivers based on either CRISPR knock-ins to target genes or using enhancer fragments. Altogether, the split-intein Gal4 system allows for the creation of highly specific intersectional genetic drivers that are inducible/repressible. %B PNAS %V 120 %P e2304730120 %G eng %N 24 %0 Journal Article %J Nature %D 2023 %T A phosphate-sensing organelle regulates phosphate and tissue homeostasis %A Xu, Chiwei %A Xu, Jun %A Tang, Hong-Wen %A Ericsson, Maria %A Weng, Jui-Hsia %A DiRusso, Jonathan %A Hu, Yanhui %A Ma, Wenzhe %A Asara, John M %A Perrimon, Norbert %X Inorganic phosphate (Pi) is one of the essential molecules for life. However, little is known about intracellular Pi metabolism and signalling in animal tissues1. Following the observation that chronic Pi starvation causes hyperproliferation in the digestive epithelium of Drosophila melanogaster, we determined that Pi starvation triggers the downregulation of the Pi transporter PXo. In line with Pi starvation, PXo deficiency caused midgut hyperproliferation. Interestingly, immunostaining and ultrastructural analyses showed that PXo specifically marks non-canonical multilamellar organelles (PXo bodies). Further, by Pi imaging with a Förster resonance energy transfer (FRET)-based Pi sensor2, we found that PXo restricts cytosolic Pi levels. PXo bodies require PXo for biogenesis and undergo degradation following Pi starvation. Proteomic and lipidomic characterization of PXo bodies unveiled their distinct feature as an intracellular Pi reserve. Therefore, Pi starvation triggers PXo downregulation and PXo body degradation as a compensatory mechanism to increase cytosolic Pi. Finally, we identified connector of kinase to AP-1 (Cka), a component of the STRIPAK complex and JNK signalling3, as the mediator of PXo knockdown- or Pi starvation-induced hyperproliferation. Altogether, our study uncovers PXo bodies as a critical regulator of cytosolic Pi levels and identifies a Pi-dependent PXo-Cka-JNK signalling cascade controlling tissue homeostasis. %B Nature %V May 3 %P doi: 10.1038/s41586-023-06039-y %G eng %0 Journal Article %J Cell %D 2023 %T Cachexia: A systemic consequence of progressive, unresolved disease %A Ferrer, Miriam %A et al. %X
Cachexia, a systemic wasting condition, is considered a late consequence of diseases, including cancer, organ failure, or infections, and contributes to significant morbidity and mortality. The induction process and mechanistic progression of cachexia are incompletely understood. Refocusing academic efforts away from advanced cachexia to the etiology of cachexia may enable discoveries of new therapeutic approaches. Here, we review drivers, mechanisms, organismal predispositions, evidence for multi-organ interaction, model systems, clinical research, trials, and care provision from early onset to late cachexia. Evidence is emerging that distinct inflammatory, metabolic, and neuro-modulatory drivers can initiate processes that ultimately converge on advanced cachexia.
%B Cell %V Apr 27;186 %P 1824-1845. doi: 10.1016/j.cell.2023.03.028 %G eng %N 9 %0 Journal Article %J Cell Metabolism %D 2023 %T Very-long-chain fatty acids induce glial-derived sphingosine-1-phosphate synthesis, secretion, and neuroinflammation %A Chung, Hyung-Lok %A Ye, Qi %A Park, ye-Jin %A Zuo, Zhongyuan %A Mok, Jung-Wan %A Kanca, Oguz %A Tattikota, Sudhir Gopal %A Lu, Shenzhao %A Perrimon, Norbert %A Lee, Hyun Kyoung %A Bellen, Hugo J %X VLCFAs (very-long-chain fatty acids) are the most abundant fatty acids in myelin. Hence, during demyelination or aging, glia are exposed to higher levels of VLCFA than normal. We report that glia convert these VLCFA into sphingosine-1-phosphate (S1P) via a glial-specific S1P pathway. Excess S1P causes neuroinflammation, NF-κB activation, and macrophage infiltration into the CNS. Suppressing the function of S1P in fly glia or neurons, or administration of Fingolimod, an S1P receptor antagonist, strongly attenuates the phenotypes caused by excess VLCFAs. In contrast, elevating the VLCFA levels in glia and immune cells exacerbates these phenotypes. Elevated VLCFA and S1P are also toxic in vertebrates based on a mouse model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Indeed, reducing VLCFA with bezafibrate ameliorates the phenotypes. Moreover, simultaneous use of bezafibrate and fingolimod synergizes to improve EAE, suggesting that lowering VLCFA and S1P is a treatment avenue for MS. %B Cell Metabolism %V May 2;35 %P 855-874.e5. doi: 10.1016/j.cmet.2023.03.022. Epub 2023 Apr 20. %G eng %N 5 %0 Journal Article %J Elife %D 2023 %T Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in Drosophila cells %A Xia, Baolong %A Viswanatha, Raghuvir %A Hu, Yanhui %A Mohr, Stephanie %A Perrimon, Norbert %X Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In Drosophila cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in Drosophila cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (CG8468), a member of the lipocalin protein family (CG5399), and a zinc finger C2H2 transcription factor (CG9932). Mechanistically, we demonstrate that CG5399 overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by CG5399 requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in Drosophila cells. %B Elife %V Apr 20 %P e85542. doi: 10.7554/eLife.85542 %G eng %N 12 %0 Journal Article %J Nature Communications %D 2023 %T Next-generation large-scale binary protein interaction network for Drosophila melanogaster %A Tang, Hong-Wen %A et al. %X Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the 'FlyBi' dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function. %B Nature Communications %V Apr 15;14 %P 2162. doi: 10.1038/s41467-023-37876-0 %G eng %N 1 %0 Journal Article %J Nature Medicine %D 2023 %T Body composition and lung cancer-associated cachexia in TRACERx %A Al-Sawaf, Othman %A et al %X Cancer-associated cachexia (CAC) is a major determinant of morbidity and mortality in patients with non-small cell lung cancer (NSCLC). Key features of CAC include alterations in body composition and body weight. Here, we explore the association between body composition and body weight with survival and delineate possible biological processes and mediators that contribute to the development of CAC. Computed tomography-based (CT) body composition analysis of 651 patients in TRACERx suggested that patients with low skeletal muscle or adipose tissue area at the time of lung cancer diagnosis, represented by the bottom 20th percentile, had significantly shorter lung cancer-specific survival (LCSS) and overall survival (OS). This finding was validated in 420 patients in the independent Boston Lung Cancer Study. In a longitudinal subset of 272 patients in TRACERx who experienced disease relapse, loss of adipose tissue, skeletal muscle, or body weight in the interval between diagnosis and relapse, was significantly associated with shorter LCSS and OS. Patients with one or more features encompassing loss of adipose or muscle tissue, or BMI-adjusted weight loss according to specific thresholds were classified as having developed CAC and were found to have distinct tumour genomic and transcriptomic profiles compared with patients who did not develop such features at relapse. Primary NSCLCs from patients in the CAC group were characterised by enrichment of inflammatory signalling and epithelial-mesenchymal transitional pathways, and differentially expressed genes upregulated in these tumours included cancer-testis antigen MAGEA6 and matrix metalloproteinases, such as ADAMTS3. In an exploratory analysis of putative circulating cachexia mediators performed in a subset of 256 baseline and relapse plasma samples from TRACERx, proteomic analysis revealed a significant association between circulating GDF15 and loss of body weight, skeletal muscle, and adipose tissue at relapse, supporting the potential therapeutic relevance of targeting GDF15 in the management of CAC. %B Nature Medicine %V Apr;29 %P 846-858 doi: 10.1038/s41591-023-02232-8 %G eng %N 4 %0 Journal Article %J Nucleic Acids Research %D 2023 %T PANGEA: A New Gene Set Enrichment Tool for Drosophila and Common Research Organisms %A Hu, Yanhui %A Comjean, Aram %A Attrill, Helen %A Antonazzo, Giulia %A Thurmond, Jim %A Fangge, Li %A Tiffany Chao %A Mohr, Stephanie E %A Brown, Nicholas H %A Perrimon, Norbert %X Gene set enrichment analysis (GSEA) plays an important role in large-scale data analysis, helping scientists discover the underlying biological patterns over-represented in a gene list resulting from, for example, an 'omics' study. Gene Ontology (GO) annotation is the most frequently used classification mechanism for gene set definition. Here we present a new GSEA tool, PANGEA (PAthway, Network and Gene-set Enrichment Analysis; https://www.flyrnai.org/tools/pangea/ ), developed to allow a more flexible and configurable approach to data analysis using a variety of classification sets. PANGEA allows GO analysis to be performed on different sets of GO annotations, for example excluding high-throughput studies. Beyond GO, gene sets for pathway annotation and protein complex data from various resources as well as expression and disease annotation from the Alliance of Genome Resources (Alliance). In addition, visualisations of results are enhanced by providing an option to view network of gene set to gene relationships. The tool also allows comparison of multiple input gene lists and accompanying visualisation tools for quick and easy comparison. This new tool will facilitate GSEA for Drosophila and other major model organisms based on high-quality annotated information available for these species. %B Nucleic Acids Research %V May 1 %P doi: 10.1093/nar/gkad331 %G eng %N gkad331 %0 Journal Article %J Developmental Cell %D 2023 %T Synthetic Notch receptors and their applications to study cell-cell contacts in vivo %A He, Li %A Perrimon Norbert %X Physical cell-cell interaction is a fundamental mechanism that controls the development and physiology of multicellular organisms. However, methods to study cell contact within complex tissues are limited. In a recent issue of Science, Zhang et al. developed synthetic Notch (synNotch)-based tools that can monitor and trace cell-cell contacts in mammals. %B Developmental Cell %V 58 %P 171-173 %G eng %N 3 %0 Journal Article %J bioRxiv %D 2022 %T Molecular and functional characterization of the Drosophila melanogaster conserved smORFome %A Bosch, J %A Keith, N %A Escobedo, F %A Fisher, W %A Thai La Graff, J %A Rabasco, J %A Wan, Kenneth H %A Weiszmann, R %A Hu, Y. %A Kondo, S %A Brown, J B %A Perrimon, N %X Short polypeptides encoded by small open reading frames (smORFs) are ubiquitously found in eukaryotic genomes and are important regulators of physiology, development, and mitochondrial processes. Here, we focus on a subset of 194 smORFs that are evolutionarily conserved between Drosophila melanogaster and humans. Many of these smORFs are conserved broadly in the bilaterian lineage, with ∼60 conserved in plants. Within these conserved smORFs, we observed remarkably heterogenous spatial and temporal expression patterns – indicating wide-spread tissue-specific and stage-specific mitochondrial architectures. In addition, an analysis of annotated functional domains revealed a predicted enrichment of smORF polypeptides localizing to mitochondria. We conducted an embryonic ribosome profiling experiment and applied a machine learning approach, finding support for translation of 82 of these smORFs during embryogenesis. We further embarked on functional characterization using CRISPR knockout/activation, RNAi knockdown, and cDNA overexpression, revealing diverse phenotypes. This study underscores the importance of identifying smORF function in disease and phenotypic diversity. %B bioRxiv %V doi: https://doi.org/10.1101/2022.04.24.489283 %G eng %0 Journal Article %J bioRxiv %D 2022 %T Antagonistic regulation of Drosophila mitochondrial uncoupling protein UCP4b by cold and BMP signaling %A Chatterjee, N %A Song, W %A Dumesic, P A %A Spiegelman, B %A Perrimon, N %X
Regulation of energy metabolism and response to cold are intimately linked in mammals. Central to these two processes are the mitochondrial uncoupling proteins (UCPs), which by promoting proton leakage across the inner mitochondrial membrane lead to the generation of heat instead of ATP synthesis. In addition to heat generation, UCPs also influence energy storage and can protect against obesity and diabetes. Cold-blooded animals like flies also contain UCPs that protect from cold, however their regulations are poorly understood. We find that Drosophila UCP4b is induced by cold in a cell-intrinsic manner and protects against cold and obesity in fly models. Mechanistically, cold regulates UCP4b expression through calcium signaling and Spargel (Srl), the Drosophila ortholog of mammalian PGC1α. To the opposite, MAD, acting downstream of the BMP branch of the TGFβ signaling pathway, represses UCP4b expression independently of cold. Interestingly, the two mechanisms of UCP4b regulation are integrated as MAD binding to the UCP4b promoter is displaced by cold in a Srl-dependent manner. We discuss the similarities between the regulation of mammalian and Drosophila UCPs.
Significance Mitochondrial uncoupling proteins (UCPs) that uncouple the mitochondrial respiration from ATP synthesis regulate energy metabolism, non-shivering thermogenesis, and redox balance in vertebrates and invertebrates. However, their regulation in Drosophila is poorly understood. We found that Drosophila uncoupling protein UCP4b is induced by cold in a cell-autonomous fashion. Conversely, MAD, acting downstream of BMP signaling, inhibits UCP4b expression. MAD is displaced from the upstream regions of the UCP4b gene by cold. UCP4b protects Drosophila against cold and diet-induced obesity. The regulation of UCP4b by cold and BMP signaling is reminiscent of the regulation of mammalian uncoupling protein UCP1. Altogether, we discovered an important regulator of Drosophila energy metabolism which is controlled by regulatory processes that are similar between Drosophila and mammals.
%B bioRxiv %V doi: https://doi.org/10.1101/2022.01.27.477603 %G eng %0 Journal Article %J Current Protocols %D 2022 %T NanoTag Nanobody Tools for Drosophila in vitro and in vivo Studies. %A Kim, AR %A Xu, J %A Mohr, SE %A Zirin, J %A Ploegh, H.L. %A Perrimon, N %X Nanobodies have emerged as powerful protein-binding tools to uncover protein functions. Using functionalized protein binders, proteins of interest can be visualized, degraded, delocalized, or post-translationally modified in vivo. We recently reported the use of two short peptide tags, 10-aa 127D01 and 14-aa VHH05, and their corresponding nanobodies, Nb127D01 and NbVHH05, for both in vitro and in vivo studies in Drosophila. Here, we provide detailed protocols for nanobody production and for visualization of proteins of interest in either fixed or live samples. In addition, we include protocols for endogenous protein tagging using CRISPR-mediated genome engineering. © 2022 Wiley Periodicals LLC. %B Current Protocols %V December %G eng %U https://doi.org/10.1002/cpz1.628 %N 26 %0 Journal Article %J Computational and Structural Biotechnology Journal %D 2022 %T Paralog Explorer: A resource for mining information about paralogs in common research organisms %A Hu, Yanhui %A Ewen-Campen, Ben %A Comjean, Aram %A Rodiger, Jonathan %A Mohr, Stephanie E %A Perrimon, Norbert %X Paralogs are genes which arose via gene duplication, and when such paralogs retain overlapping or redundant function, this poses a challenge to functional genetics research. Recent technological advancements have made it possible to systematically probe gene function for redundant genes using dual or multiplex gene perturbation, and there is a need for a simple bioinformatic tool to identify putative paralogs of a gene(s) of interest. We have developed Paralog Explorer (https://www.flyrnai.org/tools/paralogs/), an online resource that allows researchers to quickly and accurately identify candidate paralogous genes in the genomes of the model organisms D. melanogaster, C. elegans, D. rerio, M. musculus, and H. sapiens. Paralog Explorer deploys an effective between-species ortholog prediction software, DIOPT, to analyze within-species paralogs. Paralog Explorer allows users to identify candidate paralogs, and to navigate relevant databases regarding gene co-expression, protein–protein and genetic interaction, as well as gene ontology and phenotype annotations. Altogether, this tool extends the value of current ortholog prediction resources by providing sophisticated features useful for identification and study of paralogous genes. 2022 The Authors. Published by Elsevier B.V. on behalf of Research Network of Computational and Structural Biotechnology. This is an open access article under the CC BY license (http://creativecommons.org/liscenses/by/4.0/). %B Computational and Structural Biotechnology Journal %P 6570-6577 %G eng %N 20 %0 Journal Article %J PLoS Genetics %D 2022 %T A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress %A Dalton, Hans %A Viswanatha, Roderick %A Zuno, Jae Sophia %A Berman, Alexys R. %A Rushforth, Rebekah %A Mohr, Stephanie E %A Perrimon, Norbert %A Chow, Clement Y %X Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1- CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually causes CDGs. While both in vivo models ostensibly cause cellular stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress. %B PLoS Genetics %V 18 %P e1010430 %G eng %N 9 %0 Journal Article %J Trends in Cell Biology %D 2022 %T Bioelectric regulation of intestinal stem cells %A Petsakou, Afroditi %A Perrimon, Norbert %X
Proper regulation of ion balance across the intestinal epithelium is essential for physiological functions, while ion imbalance causes intestinal disorders with dire health consequences. Ion channels, pumps, and exchangers are vital for regulating ion movements (i.e., bioelectric currents) that control epithelial absorption and secretion. Recent in vivo studies used the Drosophila gut to identify conserved pathways that link regulators of Ca2+, Na+ and Cl- with intestinal stem cell (ISC) proliferation. These studies laid a foundation for using the Drosophila gut to identify conserved proliferative responses triggered by bioelectric regulators. Here, we review these studies, discuss their significance, as well as the advantages of using Drosophila to unravel conserved bioelectrically induced molecular pathways in the intestinal epithelium under physiological, pathophysiological, and regenerative conditions.
Keywords: Drosophila; bioelectric signaling; gut; intestinal stem cells; ion channels.
%B Trends in Cell Biology %V Nov 14;S0962-8924 %P 234-3 %G eng %N 22 %0 Journal Article %J Elife %D 2022 %T Two neuronal peptides encoded from a single transcript regulate mitochondrial complex III in %A Bosch, Justin A %A Ugur, Berrak %A Pichardo-Casas, Israel %A Rabasco, Jordan %A Escobedo, Felipe %A Zuo, Zhongyuan %A Brown, Ben %A Celniker, Susan %A Sinclair, David A %A Bellen, Hugo J %A Perrimon, Norbert %X Naturally produced peptides (<100 amino acids) are important regulators of physiology, development, and metabolism. Recent studies have predicted that thousands of peptides may be translated from transcripts containing small open reading frames (smORFs). Here, we describe two peptides in Drosophila encoded by conserved smORFs, Sloth1 and Sloth2. These peptides are translated from the same bicistronic transcript and share sequence similarities, suggesting that they encode paralogs. Yet, Sloth1 and Sloth2 are not functionally redundant, and loss of either peptide causes animal lethality, reduced neuronal function, impaired mitochondrial function, and neurodegeneration. We provide evidence that Sloth1/2 are highly expressed in neurons, imported to mitochondria, and regulate mitochondrial complex III assembly. These results suggest that phenotypic analysis of smORF genes in Drosophila can provide a wealth of information on the biological functions of this poorly characterized class of genes. %B Elife %V 11 %8 2022 Nov 08 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/36346220?dopt=Abstract %R 10.7554/eLife.82709 %0 Journal Article %J Nat Metab %D 2022 %T No sugar, just protein please - says the fly %A Petsakou, Afroditi %A Perrimon, Norbert %B Nat Metab %8 2022 Nov 07 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/36344763?dopt=Abstract %R 10.1038/s42255-022-00665-y %0 Journal Article %J Nature %D 2022 %T CRISPR Screens in Drosophila Cells Identify Vsg as a Tc Toxin Receptor %A Xu, Ying %A Viswanatha, Raghuvir %A Sitsel, Oleg %A Roderer, Daniel %A Zhao, Haifang %A Ashwood, Christopher %A Voelcker, Cecilia %A Tian, Songhai %A Raunser, Stefan %A Perrimon, Norbert %A Dong, Min %K Animals %K Bacterial Toxins %K Biological Control Agents %K Drosophila %K Drosophila melanogaster %K Humans %K Mucins %K Photorhabdus %K Proline %K Serine %K Threonine %K Virulence Factors %X Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens. %B Nature %V 610 %P 349-355 %8 2022 10 %G eng %N 7931 %1 http://www.ncbi.nlm.nih.gov/pubmed/36171290?dopt=Abstract %R 10.1038/s41586-022-05250-7 %0 Journal Article %J EMBO Molecular Medicine %D 2022 %T FIBCD1 is an Endocytic GAG Receptor Associated With a Novel Neurodevelopmental Disorder %A Fell, Christopher W %A Hagelkruys, Astrid %A Cicvaric, Ana %A Horrer, Marion %A Liu, Lucy %A Li, Joshua Shing Shun %A Stadlmann, Johannes %A Polyansky, Anton A %A Mereiter, Stefan %A Tejada, Miguel Angel %A Kokotović, Tomislav %A Achuta, Venkat Swaroop %A Scaramuzza, Angelica %A Twyman, Kimberly A %A Morrow, Michelle M %A Juusola, Jane %A Yan, Huifang %A Wang, Jingmin %A Burmeister, Margit %A Choudhury, Biswa %A Andersen, Thomas Levin %A Wirnsberger, Gerald %A Holmskov, Uffe %A Perrimon, Norbert %A Žagrović, Bojan %A Monje, Francisco J %A Moeller, Jesper Bonnet %A Penninger, Josef M %A Nagy, Vanja %K Animals %K Endocytosis %K Extracellular Matrix %K Humans %K Mice %K Neurodevelopmental Disorders %K Receptors, Cell Surface %X Whole-exome sequencing of two patients with idiopathic complex neurodevelopmental disorder (NDD) identified biallelic variants of unknown significance within FIBCD1, encoding an endocytic acetyl group-binding transmembrane receptor with no known function in the central nervous system. We found that FIBCD1 preferentially binds and endocytoses glycosaminoglycan (GAG) chondroitin sulphate-4S (CS-4S) and regulates GAG content of the brain extracellular matrix (ECM). In silico molecular simulation studies and GAG binding analyses of patient variants determined that such variants are loss-of-function by disrupting FIBCD1-CS-4S association. Gene knockdown in flies resulted in morphological disruption of the neuromuscular junction and motor-related behavioural deficits. In humans and mice, FIBCD1 is expressed in discrete brain regions, including the hippocampus. Fibcd1 KO mice exhibited normal hippocampal neuronal morphology but impaired hippocampal-dependent learning. Further, hippocampal synaptic remodelling in acute slices from Fibcd1 KO mice was deficient but restored upon enzymatically modulating the ECM. Together, we identified FIBCD1 as an endocytic receptor for GAGs in the brain ECM and a novel gene associated with an NDD, revealing a critical role in nervous system structure, function and plasticity. %B EMBO Molecular Medicine %V 14 %P e15829 %8 2022 09 07 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/35916241?dopt=Abstract %R 10.15252/emmm.202215829 %0 Journal Article %J Open Biology %D 2022 %T A genetic model for in vivo proximity labelling of the mammalian secretome %A Yang, Rui %A Meyer, Amanda S %A Droujinine, Ilia A %A Udeshi, Namrata D %A Hu, Yanhui %A Guo, JinJin %A McMahon, Jill A %A Carey, Dominique K %A Xu, Charles %A Fang, Qiao %A Sha, Jihui %A Qin, Shishang %A Rocco, David %A Wohlschlegel, James %A Ting, Alice Y %A Carr, Steven A %A Perrimon, Norbert %A McMahon, Andrew P %K Animals %K Biotinylation %K Mammals %K Mass Spectrometry %K Mice %K Models, Genetic %K Proteomics %K Secretome %X Organ functions are highly specialized and interdependent. Secreted factors regulate organ development and mediate homeostasis through serum trafficking and inter-organ communication. Enzyme-catalysed proximity labelling enables the identification of proteins within a specific cellular compartment. Here, we report a BirA*G3 mouse strain that enables CRE-dependent promiscuous biotinylation of proteins trafficking through the endoplasmic reticulum. When broadly activated throughout the mouse, widespread labelling of proteins was observed within the secretory pathway. Streptavidin affinity purification and peptide mapping by quantitative mass spectrometry (MS) proteomics revealed organ-specific secretory profiles and serum trafficking. As expected, secretory proteomes were highly enriched for signal peptide-containing proteins, highlighting both conventional and non-conventional secretory processes, and ectodomain shedding. Lower-abundance proteins with hormone-like properties were recovered and validated using orthogonal approaches. Hepatocyte-specific activation of BirA*G3 highlighted liver-specific biotinylated secretome profiles. The BirA*G3 mouse model demonstrates enhanced labelling efficiency and tissue specificity over viral transduction approaches and will facilitate a deeper understanding of secretory protein interplay in development, and in healthy and diseased adult states. %B Open Biology %V 12 %P 220149 %8 2022 08 %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/35946312?dopt=Abstract %R 10.1098/rsob.220149 %0 Journal Article %J Nucleic Acids Research %D 2022 %T Local assembly of long reads enables phylogenomics of transposable elements in a polyploid cell line %A Han, Shunhua %A Dias, Guilherme B %A Basting, Preston J %A Viswanatha, Raghuvir %A Perrimon, Norbert %A Bergman, Casey M %X Animal cell lines often undergo extreme genome restructuring events, including polyploidy and segmental aneuploidy that can impede de novo whole-genome assembly (WGA). In some species like Drosophila, cell lines also exhibit massive proliferation of transposable elements (TEs). To better understand the role of transposition during animal cell culture, we sequenced the genome of the tetraploid Drosophila S2R+ cell line using long-read and linked-read technologies. WGAs for S2R+ were highly fragmented and generated variable estimates of TE content across sequencing and assembly technologies. We therefore developed a novel WGA-independent bioinformatics method called TELR that identifies, locally assembles, and estimates allele frequency of TEs from long-read sequence data (https://github.com/bergmanlab/telr). Application of TELR to a ∼130x PacBio dataset for S2R+ revealed many haplotype-specific TE insertions that arose by transposition after initial cell line establishment and subsequent tetraploidization. Local assemblies from TELR also allowed phylogenetic analysis of paralogous TEs, which revealed that proliferation of TE families in vitro can be driven by single or multiple source lineages. Our work provides a model for the analysis of TEs in complex heterozygous or polyploid genomes that are recalcitrant to WGA and yields new insights into the mechanisms of genome evolution in animal cell culture. %B Nucleic Acids Research %8 2022 Sep 26 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/36156149?dopt=Abstract %R 10.1093/nar/gkac794 %0 Journal Article %J BMC Genomics %D 2022 %T Modeling exercise using optogenetically contractible Drosophila larvae %A Ghosh, Arpan C %A Hu, Yanhui %A Tattikota, Sudhir Gopal %A Liu, Yifang %A Comjean, Aram %A Perrimon, Norbert %K Animals %K Drosophila %K Humans %K Larva %K Muscle Contraction %K Muscle, Skeletal %K Physical Conditioning, Animal %X The pathophysiological effects of a number of metabolic and age-related disorders can be prevented to some extent by exercise and increased physical activity. However, the molecular mechanisms that contribute to the beneficial effects of muscle activity remain poorly explored. Availability of a fast, inexpensive, and genetically tractable model system for muscle activity and exercise will allow the rapid identification and characterization of molecular mechanisms that mediate the beneficial effects of exercise. Here, we report the development and characterization of an optogenetically-inducible muscle contraction (OMC) model in Drosophila larvae that we used to study acute exercise-like physiological responses. To characterize muscle-specific transcriptional responses to acute exercise, we performed bulk mRNA-sequencing, revealing striking similarities between acute exercise-induced genes in flies and those previously identified in humans. Our larval muscle contraction model opens a path for rapid identification and characterization of exercise-induced factors. %B BMC Genomics %V 23 %P 623 %8 2022 Aug 30 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/36042416?dopt=Abstract %R 10.1186/s12864-022-08845-6 %0 Journal Article %J Methods in Molecular Biology %D 2022 %T Prime Editing for Precise Genome Engineering in Drosophila %A Bosch, Justin A %A Perrimon, Norbert %K Animals %K Animals, Genetically Modified %K CRISPR-Cas Systems %K Drosophila %K Gene Editing %K Genome, Insect %K Mammals %X Editing the Drosophila genome is incredibly useful for gene functional analysis. However, compared to gene knockouts, precise gene editing is difficult to achieve. Prime editing, a recently described CRISPR/Cas9-based technique, has the potential to make precise editing simpler and faster, and produce less errors than traditional methods. Initially described in mammalian cells, prime editing is functional in Drosophila somatic and germ cells. Here, we outline steps to design, generate, and express prime editing components in transgenic flies. Furthermore, we highlight a crossing scheme to produce edited fly stocks in less than 3 months. %B Methods in Molecular Biology %V 2540 %P 113-134 %8 2022 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/35980575?dopt=Abstract %R 10.1007/978-1-0716-2541-5_5 %0 Journal Article %J Nature %D 2022 %T Sestrin Mediates Detection Of and Adaptation To Low-Leucine Diets in Drosophila %A Gu, Xin %A Jouandin, Patrick %A Lalgudi, Pranav V %A Binari, Rich %A Valenstein, Max L %A Reid, Michael A %A Allen, Annamarie E %A Nolan Kamitaki %A Locasale, Jason W %A Perrimon, Norbert %A Sabatini, David M %K Adaptation, Physiological %K Animal Feed %K Animals %K Autophagy %K Diet %K Drosophila melanogaster %K Drosophila Proteins %K Food Preferences %K Leucine %K Mechanistic Target of Rapamycin Complex 1 %K Mutant Proteins %K Neuroglia %K Sestrins %K Signal Transduction %X Mechanistic target of rapamycin complex 1 (mTORC1) regulates cell growth and metabolism in response to multiple nutrients, including the essential amino acid leucine1. Recent work in cultured mammalian cells established the Sestrins as leucine-binding proteins that inhibit mTORC1 signalling during leucine deprivation2,3, but their role in the organismal response to dietary leucine remains elusive. Here we find that Sestrin-null flies (Sesn-/-) fail to inhibit mTORC1 or activate autophagy after acute leucine starvation and have impaired development and a shortened lifespan on a low-leucine diet. Knock-in flies expressing a leucine-binding-deficient Sestrin mutant (SesnL431E) have reduced, leucine-insensitive mTORC1 activity. Notably, we find that flies can discriminate between food with or without leucine, and preferentially feed and lay progeny on leucine-containing food. This preference depends on Sestrin and its capacity to bind leucine. Leucine regulates mTORC1 activity in glial cells, and knockdown of Sesn in these cells reduces the ability of flies to detect leucine-free food. Thus, nutrient sensing by mTORC1 is necessary for flies not only to adapt to, but also to detect, a diet deficient in an essential nutrient. %B Nature %V 608 %P 209-216 %8 2022 08 %G eng %N 7921 %1 http://www.ncbi.nlm.nih.gov/pubmed/35859173?dopt=Abstract %R 10.1038/s41586-022-04960-2 %0 Journal Article %J Elife %D 2022 %T An expanded toolkit for gene tagging using synthesized homology donor constructs for CRISPR-mediated homologous recombination %A Kanca, Oguz %A Zirin, Jonathan %A Hu, Yanhui %A Tepe, Burak %A Dutta, Debdeep %A Lin, Wen-Wen %A Ma, Liwen %A Ge, Ming %A Zuo, Zhongyuan %A Liu, Lu-Ping %A Levis, Robert W %A Perrimon, Norbert %A Bellen, Hugo J %K Animals %K Clustered Regularly Interspaced Short Palindromic Repeats %K CRISPR-Cas Systems %K Drosophila %K Exons %K Homologous Recombination %K Plasmids %X Previously, we described a large collection of Drosophila strains that each carry an artificial exon containing a T2AGAL4 cassette inserted in an intron of a target gene based on CRISPR-mediated homologous recombination. These alleles permit numerous applications and have proven to be very useful. Initially, the homologous recombination-based donor constructs had long homology arms (>500 bps) to promote precise integration of large constructs (>5 kb). Recently, we showed that in vivo linearization of the donor constructs enables insertion of large artificial exons in introns using short homology arms (100-200 bps). Shorter homology arms make it feasible to commercially synthesize homology donors and minimize the cloning steps for donor construct generation. Unfortunately, about 58% of Drosophila genes lack a suitable coding intron for integration of artificial exons in all of the annotated isoforms. Here, we report the development of new set of constructs that allow the replacement of the coding region of genes that lack suitable introns with a KozakGAL4 cassette, generating a knock-out/knock-in allele that expresses GAL4 similarly as the targeted gene. We also developed custom vector backbones to further facilitate and improve transgenesis. Synthesis of homology donor constructs in custom plasmid backbones that contain the target gene sgRNA obviates the need to inject a separate sgRNA plasmid and significantly increases the transgenesis efficiency. These upgrades will enable the targeting of nearly every fly gene, regardless of exon-intron structure, with a 70-80% success rate. %B Elife %V 11 %8 2022 06 20 %G eng %U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9239680/ %1 http://www.ncbi.nlm.nih.gov/pubmed/35723254?dopt=Abstract %R 10.7554/eLife.76077 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2022 %T Transcriptional and functional motifs defining renal function revealed by single-nucleus RNA sequencing %A Xu, Jun %A Liu, Yifang %A Li, Hongjie %A Tarashansky, Alexander J %A Kalicki, Colin H %A Hung, Ruei-Jiun %A Hu, Yanhui %A Comjean, Aram %A Kolluru, Sai Saroja %A Wang, Bo %A Quake, Stephen R %A Liqun Luo %A McMahon, Andrew P %A Dow, Julian A T %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Hepatocyte Nuclear Factor 4 %K Kidney %K Malpighian Tubules %K Mice %K Nerve Tissue Proteins %K Regeneration %K Sequence Analysis, RNA %K Single-Cell Analysis %K Stem Cells %K Transcription Factors %X Recent advances in single-cell sequencing provide a unique opportunity to gain novel insights into the diversity, lineage, and functions of cell types constituting a tissue/organ. Here, we performed a single-nucleus study of the adult Drosophila renal system, consisting of Malpighian tubules and nephrocytes, which shares similarities with the mammalian kidney. We identified 11 distinct clusters representing renal stem cells, stellate cells, regionally specific principal cells, garland nephrocyte cells, and pericardial nephrocytes. Characterization of the transcription factors specific to each cluster identified fruitless (fru) as playing a role in stem cell regeneration and Hepatocyte nuclear factor 4 (Hnf4) in regulating glycogen and triglyceride metabolism. In addition, we identified a number of genes, including Rho guanine nucleotide exchange factor at 64C (RhoGEF64c), Frequenin 2 (Frq2), Prip, and CG1093 that are involved in regulating the unusual star shape of stellate cells. Importantly, the single-nucleus dataset allows visualization of the expression at the organ level of genes involved in ion transport and junctional permeability, providing a systems-level view of the organization and physiological roles of the tubules. Finally, a cross-species analysis allowed us to match the fly kidney cell types to mouse kidney cell types and planarian protonephridia, knowledge that will help the generation of kidney disease models. Altogether, our study provides a comprehensive resource for studying the fly kidney. %B Proc Natl Acad Sci U S A %V 119 %P e2203179119 %8 2022 Jun 21 %G eng %U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9231607/ %N 25 %1 http://www.ncbi.nlm.nih.gov/pubmed/35696569?dopt=Abstract %R 10.1073/pnas.2203179119 %0 Journal Article %J iScience %D 2022 %T Trans-omics analysis of insulin action reveals a cell growth subnetwork which co-regulates anabolic processes %A Terakawa, Akira %A Hu, Yanhui %A Kokaji, Toshiya %A Yugi, Katsuyuki %A Morita, Keigo %A Ohno, Satoshi %A Pan, Yifei %A Bai, Yunfan %A Parkhitko, Andrey A %A Ni, Xiaochun %A Asara, John M %A Bulyk, Martha L %A Perrimon, Norbert %A Kuroda, Shinya %X Insulin signaling promotes anabolic metabolism to regulate cell growth through multi-omic interactions. To obtain a comprehensive view of the cellular responses to insulin, we constructed a trans-omic network of insulin action in Drosophila cells that involves the integration of multi-omic data sets. In this network, 14 transcription factors, including Myc, coordinately upregulate the gene expression of anabolic processes such as nucleotide synthesis, transcription, and translation, consistent with decreases in metabolites such as nucleotide triphosphates and proteinogenic amino acids required for transcription and translation. Next, as cell growth is required for cell proliferation and insulin can stimulate proliferation in a context-dependent manner, we integrated the trans-omic network with results from a CRISPR functional screen for cell proliferation. This analysis validates the role of a Myc-mediated subnetwork that coordinates the activation of genes involved in anabolic processes required for cell growth. %B iScience %V 25 %P 104231 %8 2022 May 20 %G eng %U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9044165/ %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/35494245?dopt=Abstract %R 10.1016/j.isci.2022.104231 %0 Journal Article %J Front. Insect Sci. %D 2022 %T Roles of Insect Oenocytes in Physiology and Their Relevance to Human Metabolic Diseases %A Huang, Kerui %A Liu, Ying %A Perrimon, Norbert %X
Oenocytes are large secretory cells present in the abdomen of insects known to synthesize very-long-chain fatty acids to produce hydrocarbons and pheromones that mediate courtship behavior in adult flies. In recent years, oenocytes have been implicated in the regulation of energy metabolism. These hepatocyte-like cells accumulate lipid droplets under starvation and can non-autonomously regulate tracheal waterproofing and adipocyte lipid composition. Here, we summarize evidence, mostly from Drosophila, establishing that oenocytes perform liver-like functions. We also compare the functional differences in oenocytes and the fat body, another lipid storage tissue which also performs liver-like functions. Lastly, we examine signaling pathways that regulate oenocyte metabolism derived from other metabolic tissues, as well as oenocyte-derived signals that regulate energy homeostasis.
Regulating energy utilization and storage is central to animal physiology and adaptation to environmental challenges. Under conditions of nutrition surplus, glucose is converted to fatty acids, which are then synthesized into triglycerides (TGs) and stored as lipid droplets. Excessive lipid stores can be detrimental and have been associated with various metabolic diseases, such as cardiovascular diseases (CVDs), non-alcoholic fatty liver disease (NAFLD), obesity and insulin resistance, making understanding lipid metabolism of great importance to human health.
The liver is the major detoxifying organ of the body and plays a central role in regulating the metabolism of carbohydrates, proteins and lipids. Moreover, the liver is the major site for glycogen storage and very low-density lipoprotein (VLDL) secretion (1, 2). During starvation, adipocytes undergo lipolysis to produce free fatty acids (FFAs). FFAs are processed by hepatic oxidation to generate ketone bodies in the liver which are then used as fuels for other tissues. If mobilization of FFAs exceeds the rate of lipid oxidation, re-esterification of surplus FFAs to TGs occurs in the liver, leading to an increase in intrahepatic TG content, i.e., steatosis. NAFLD, a common manifestation of the metabolic syndrome, is characterized by steatosis in the absence of starvation. Nonalcoholic hepatic steatosis is present in approximately 25% of the adult population worldwide, and NAFLD is the most common liver disease in Western societies. Thus, understanding how hepatic diseases regulate cellular processes in peripheral organs and how other organs contribute to steatosis is of interest to human metabolic diseases.
Major metabolic and endocrine pathways are conserved in Drosophila, making this model organism well suited to dissect the cellular and molecular mechanisms underlying physiology (3–5). The fly fat body is equivalent to the vertebrate white adipose tissue (WAT), which stores excess fat as TGs. In addition, fly oenocytes, which are similar to hepatocyte cells, are important for mobilizing stored lipids from the fly fat body (6). Like mammals, flies convert excess carbohydrates into TGs through de novo lipogenesis (7, 8). In addition, excess carbohydrates and amino acids can also be processed into UDP-glucose, which fuels glycogen synthesis (9). Regulation of energy storage in flies involves several signaling pathways, including insulin/insulin-like growth factor (IGF) signaling, which is similar to the insulin signaling in mammals (10). However, unlike mammals, there are eight different Drosophila insulin-like peptides (dILPs). Most of these modulate the IGF pathway through a single insulin receptor, InR (10, 11). Under nutrient-deprivation or energy demanding conditions, lipids are released from the fat body through increased lipolysis (12), and are further processed in oenocytes (6, 13). Signaling that regulates catabolism of lipids and carbohydrates include adipokinetic hormone (Akh), which is similar to glucagon in mammals and ecdysone, which antagonizes insulin signaling (14, 15).
In this review, we explore the potential of Drosophila oenocytes as a model for hepatic diseases. We summarize the different roles of oenocytes and the fat body in regulating carbohydrate and lipid metabolism under normal or starved conditions. We also discuss the intricate interplay of oenocytes with other tissues, including the fat body and muscles, in shaping organismal lipid storage.
%B Front. Insect Sci. %G eng %U https://www.frontiersin.org/articles/10.3389/finsc.2022.859847/full %0 Journal Article %J Dis Model Mech %D 2022 %T Cancer cachexia: lessons from Drosophila %A Liu, Ying %A Saavedra, Pedro %A Perrimon, Norbert %X Cachexia, a wasting syndrome that is often associated with cancer, is one of the primary causes of death in cancer patients. Cancer cachexia occurs largely due to systemic metabolic alterations stimulated by tumors. Despite the prevalence of cachexia, our understanding of how tumors interact with host tissues and how they affect metabolism is limited. Among the challenges of studying tumor-host tissue crosstalk are the complexity of cancer itself and our insufficient knowledge of the factors that tumors release into the blood. Drosophila is emerging as a powerful model in which to identify tumor-derived factors that influence systemic metabolism and tissue wasting. Strikingly, studies that are characterizing factors derived from different fly tumor cachexia models are identifying both common and distinct cachectic molecules, suggesting that cachexia is more than one disease and that fly models can help identify these differences. Here, we review what has been learned from studies of tumor-induced organ wasting in Drosophila and discuss the open questions. %B Dis Model Mech %V 15 %8 2022 Mar 01 %G eng %U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8961677/ %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/35319749?dopt=Abstract %R 10.1242/dmm.049298 %0 Journal Article %J Genetics %D 2022 %T Harmonizing model organism data in the Alliance of Genome Resources %A AGRC %K Alleles %K Animals %K Databases, Genetic %K Gene Ontology %K Humans %K Internet %K Mice %K Molecular Sequence Annotation %K Rats %K Zebrafish %X The Alliance of Genome Resources (the Alliance) is a combined effort of 7 knowledgebase projects: Saccharomyces Genome Database, WormBase, FlyBase, Mouse Genome Database, the Zebrafish Information Network, Rat Genome Database, and the Gene Ontology Resource. The Alliance seeks to provide several benefits: better service to the various communities served by these projects; a harmonized view of data for all biomedical researchers, bioinformaticians, clinicians, and students; and a more sustainable infrastructure. The Alliance has harmonized cross-organism data to provide useful comparative views of gene function, gene expression, and human disease relevance. The basis of the comparative views is shared calls of orthology relationships and the use of common ontologies. The key types of data are alleles and variants, gene function based on gene ontology annotations, phenotypes, association to human disease, gene expression, protein-protein and genetic interactions, and participation in pathways. The information is presented on uniform gene pages that allow facile summarization of information about each gene in each of the 7 organisms covered (budding yeast, roundworm Caenorhabditis elegans, fruit fly, house mouse, zebrafish, brown rat, and human). The harmonized knowledge is freely available on the alliancegenome.org portal, as downloadable files, and by APIs. We expect other existing and emerging knowledge bases to join in the effort to provide the union of useful data and features that each knowledge base currently provides. %B Genetics %V 220 %8 2022 Apr 04 %G eng %U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8982023/ %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/35380658?dopt=Abstract %R 10.1093/genetics/iyac022 %0 Journal Article %J Science %D 2022 %T Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly %A Li, Hongjie %A Janssens, Jasper %A De Waegeneer, Maxime %A Kolluru, Sai Saroja %A Davie, Kristofer %A Gardeux, Vincent %A Saelens, Wouter %A David, Fabrice P A %A Brbić, Maria %A Spanier, Katina %A Leskovec, Jure %A McLaughlin, Colleen N %A Xie, Qijing %A Jones, Robert C %A Brueckner, Katja %A Shim, Jiwon %A Tattikota, Sudhir Gopal %A Schnorrer, Frank %A Rust, Katja %A Nystul, Todd G %A Carvalho-Santos, Zita %A Ribeiro, Carlos %A Pal, Soumitra %A Mahadevaraju, Sharvani %A Przytycka, Teresa M %A Allen, Aaron M %A Goodwin, Stephen F %A Berry, Cameron W %A Fuller, Margaret T %A White-Cooper, Helen %A Matunis, Erika L %A DiNardo, Stephen %A Galenza, Anthony %A O'Brien, Lucy Erin %A Dow, Julian A T %A FCA Consortium§ %A Jasper, Heinrich %A Oliver, Brian %A Perrimon, Norbert %A Deplancke, Bart %A Quake, Stephen R %A Liqun Luo %A Aerts, Stein %A Agarwal, Devika %A Ahmed-Braimah, Yasir %A Arbeitman, Michelle %A Ariss, Majd M %A Augsburger, Jordan %A Ayush, Kumar %A Baker, Catherine C %A Banisch, Torsten %A Birker, Katja %A Bodmer, Rolf %A Bolival, Benjamin %A Brantley, Susanna E %A Brill, Julie A %A Brown, Nora C %A Buehner, Norene A %A Cai, Xiaoyu Tracy %A Cardoso-Figueiredo, Rita %A Casares, Fernando %A Chang, Amy %A Clandinin, Thomas R %A Crasta, Sheela %A Desplan, Claude %A Detweiler, Angela M %A Dhakan, Darshan B %A Donà, Erika %A Engert, Stefanie %A Floc'hlay, Swann %A George, Nancy %A González-Segarra, Amanda J %A Groves, Andrew K %A Gumbin, Samantha %A Guo, Yanmeng %A Harris, Devon E %A Heifetz, Yael %A Holtz, Stephen L %A Horns, Felix %A Hudry, Bruno %A Hung, Ruei-Jiun %A Jan, Yuh Nung %A Jaszczak, Jacob S %A Jefferis, Gregory S X E %A Karkanias, Jim %A Karr, Timothy L %A Katheder, Nadja Sandra %A Kezos, James %A Kim, Anna A %A Kim, Seung K %A Kockel, Lutz %A Konstantinides, Nikolaos %A Kornberg, Thomas B %A Krause, Henry M %A Labott, Andrew Thomas %A Laturney, Meghan %A Lehmann, Ruth %A Leinwand, Sarah %A Li, Jiefu %A Li, Joshua Shing Shun %A Li, Kai %A Ke Li %A Li, Liying %A Li, Tun %A Litovchenko, Maria %A Liu, Han-Hsuan %A Liu, Yifang %A Lu, Tzu-Chiao %A Manning, Jonathan %A Mase, Anjeli %A Matera-Vatnick, Mikaela %A Matias, Neuza Reis %A McDonough-Goldstein, Caitlin E %A McGeever, Aaron %A McLachlan, Alex D %A Moreno-Roman, Paola %A Neff, Norma %A Neville, Megan %A Ngo, Sang %A Nielsen, Tanja %A O'Brien, Caitlin E %A Osumi-Sutherland, David %A Özel, Mehmet Neset %A Papatheodorou, Irene %A Petkovic, Maja %A Pilgrim, Clare %A Pisco, Angela Oliveira %A Reisenman, Carolina %A Sanders, Erin Nicole %A Dos Santos, Gilberto %A Scott, Kristin %A Sherlekar, Aparna %A Shiu, Philip %A Sims, David %A Sit, Rene V %A Slaidina, Maija %A Smith, Harold E %A Sterne, Gabriella %A Su, Yu-Han %A Sutton, Daniel %A Tamayo, Marco %A Tan, Michelle %A Tastekin, Ibrahim %A Treiber, Christoph %A Vacek, David %A Vogler, Georg %A Waddell, Scott %A Wang, Wanpeng %A Wilson, Rachel I. %A Wolfner, Mariana F %A Wong, Yiu-Cheung E %A Xie, Anthony %A Xu, Jun %A Yamamoto, Shinya %A Yan, Jia %A Yao, Zepeng %A Yoda, Kazuki %A Zhu, Ruijun %A Zinzen, Robert P %X For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution. %B Science %V 375 %P eabk2432 %8 2022 Mar 04 %G eng %N 6584 %1 http://www.ncbi.nlm.nih.gov/pubmed/35239393?dopt=Abstract %R 10.1126/science.abk2432 %0 Journal Article %J Cell Rep %D 2022 %T A salivary gland-secreted peptide regulates insect systemic growth %A Li, Zheng %A Qian, Wenliang %A Song, Wei %A Zhao, Tujing %A Yang, Yan %A Wang, Weina %A Wei, Ling %A Zhao, Dongchao %A Li, Yaoyao %A Perrimon, Norbert %A Xia, Qingyou %A Cheng, Daojun %X Insect salivary glands have been previously shown to function in pupal attachment and food lubrication by secreting factors into the lumen via an exocrine way. Here, we find in Drosophila that a salivary gland-derived secreted factor (Sgsf) peptide regulates systemic growth via an endocrine way. Sgsf is specifically expressed in salivary glands and secreted into the hemolymph. Sgsf knockout or salivary gland-specific Sgsf knockdown decrease the size of both the body and organs, phenocopying the effects of genetic ablation of salivary glands, while salivary gland-specific Sgsf overexpression increases their size. Sgsf promotes systemic growth by modulating the secretion of the insulin-like peptide Dilp2 from the brain insulin-producing cells (IPCs) and affecting mechanistic target of rapamycin (mTOR) signaling in the fat body. Altogether, our study demonstrates that Sgsf mediates the roles of salivary glands in Drosophila systemic growth, establishing an endocrine function of salivary glands. %B Cell Rep %V 38 %P 110397 %8 2022 Feb 22 %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/35196492?dopt=Abstract %R 10.1016/j.celrep.2022.110397 %0 Journal Article %J Science %D 2022 %T Lysosomal cystine mobilization shapes the response of TORC1 and tissue growth to fasting %A Jouandin, Patrick %A Marelja, Zvonimir %A Shih, Yung-Hsin %A Parkhitko, Andrey A %A Dambowsky, Miriam %A Asara, John M %A Nemazanyy, Ivan %A Dibble, Christian C %A Simons, Matias %A Perrimon, Norbert %X Adaptation to nutrient scarcity involves an orchestrated response of metabolic and signaling pathways to maintain homeostasis. We find that in the fat body of fasting Drosophila, lysosomal export of cystine coordinates remobilization of internal nutrient stores with reactivation of the growth regulator target of rapamycin complex 1 (TORC1). Mechanistically, cystine was reduced to cysteine and metabolized to acetyl-coenzyme A (acetyl-CoA) by promoting CoA metabolism. In turn, acetyl-CoA retained carbons from alternative amino acids in the form of tricarboxylic acid cycle intermediates and restricted the availability of building blocks required for growth. This process limited TORC1 reactivation to maintain autophagy and allowed animals to cope with starvation periods. We propose that cysteine metabolism mediates a communication between lysosomes and mitochondria, highlighting how changes in diet divert the fate of an amino acid into a growth suppressive program. %B Science %V 375 %P eabc4203 %8 2022 Feb 18 %G eng %N 6582 %1 http://www.ncbi.nlm.nih.gov/pubmed/35175796?dopt=Abstract %R 10.1126/science.abc4203 %0 Journal Article %J Elife %D 2022 %T Protein visualization and manipulation in through the use of epitope tags recognized by nanobodies %A Xu, Jun %A Kim, Ah-Ram %A Cheloha, Ross W %A Fischer, Fabian A %A Li, Joshua Shing Shun %A Feng, Yuan %A Stoneburner, Emily %A Binari, Richard %A Mohr, Stephanie E %A Zirin, Jonathan %A Ploegh, Hidde L %A Perrimon, Norbert %X Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags linked to proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we characterize two short (<15aa) NanoTag epitopes, 127D01 and VHH05, and their corresponding high-affinity nanobodies. We demonstrate their use in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond. %B Elife %V 11 %8 2022 01 25 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/35076390?dopt=Abstract %R 10.7554/eLife.74326 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2022 %T The Yun/Prohibitin complex regulates adult intestinal stem cell proliferation through the transcription factor E2F1 %A Zhao, Hang %A Shi, Lin %A Li, Zhengran %A Kong, Ruiyan %A Ren, Xuejing %A Ma, Rui %A Jia, Lemei %A Ma, Meifang %A Lu, Shan %A Xu, Ran %A Binari, Richard %A Wang, Jian-Hua %A Dong, Meng-Qiu %A Perrimon, Norbert %A Li, Zhouhua %X Stem cells constantly divide and differentiate to maintain adult tissue homeostasis, and uncontrolled stem cell proliferation leads to severe diseases such as cancer. How stem cell proliferation is precisely controlled remains poorly understood. Here, from an RNA interference (RNAi) screen in adult Drosophila intestinal stem cells (ISCs), we identify a factor, Yun, required for proliferation of normal and transformed ISCs. Yun is mainly expressed in progenitors; our genetic and biochemical evidence suggest that it acts as a scaffold to stabilize the Prohibitin (PHB) complex previously implicated in various cellular and developmental processes and diseases. We demonstrate that the Yun/PHB complex is regulated by and acts downstream of EGFR/MAPK signaling. Importantly, the Yun/PHB complex interacts with and positively affects the levels of the transcription factor E2F1 to regulate ISC proliferation. In addition, we find that the role of the PHB complex in cell proliferation is evolutionarily conserved. Thus, our study uncovers a Yun/PHB-E2F1 regulatory axis in stem cell proliferation. %B Proc Natl Acad Sci U S A %V 119 %8 2022 02 08 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/35115400?dopt=Abstract %R 10.1073/pnas.2111711119 %0 Journal Article %J Genetics %D 2021 %T FlyPhoneDB: an integrated web-based resource for cell-cell communication prediction in Drosophila %A Liu, Yifang %A Li, Joshua Shing Shun %A Rodiger, Jonathan %A Comjean, Aram %A Attrill, Helen %A Antonazzo, Giulia %A Brown, Nicholas H %A Hu, Yanhui %A Perrimon, Norbert %X Multicellular organisms rely on cell-cell communication to exchange information necessary for developmental processes and metabolic homeostasis. Cell-cell communication pathways can be inferred from transcriptomic datasets based on ligand-receptor expression. Recently, data generated from single-cell RNA sequencing have enabled ligand-receptor interaction predictions at an unprecedented resolution. While computational methods are available to infer cell-cell communication in vertebrates such a tool does not yet exist for Drosophila. Here, we generated a high-confidence list of ligand-receptor pairs for the major fly signaling pathways and developed FlyPhoneDB, a quantification algorithm that calculates interaction scores to predict ligand-receptor interactions between cells. At the FlyPhoneDB user interface, results are presented in a variety of tabular and graphical formats to facilitate biological interpretation. To illustrate that FlyPhoneDB can effectively identify active ligands and receptors to uncover cell-cell communication events, we applied FlyPhoneDB to Drosophila single-cell RNA sequencing data sets from adult midgut, abdomen, and blood, and demonstrate that FlyPhoneDB can readily identify previously characterized cell-cell communication pathways. Altogether, FlyPhoneDB is an easy-to-use framework that can be used to predict cell-cell communication between cell types from single-cell RNA sequencing data in Drosophila. %B Genetics %8 2021 Dec 31 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/35100387?dopt=Abstract %R 10.1093/genetics/iyab235 %0 Journal Article %J Nat Commun %D 2021 %T Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos %A Viswanatha, Raghuvir %A Mameli, Enzo %A Rodiger, Jonathan %A Merckaert, Pierre %A Feitosa-Suntheimer, Fabiana %A Colpitts, Tonya M %A Mohr, Stephanie E %A Hu, Yanhui %A Perrimon, Norbert %K Animals %K Anopheles %K Cell Line %K Computational Biology %K CRISPR-Cas Systems %K Gene Knockout Techniques %K Gene Library %K Genes, Essential %K Humans %K Mosquito Control %K Mosquito Vectors %K Pest Control, Biological %K RNA, Guide %K Vector Borne Diseases %X Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species. %B Nat Commun %V 12 %P 6825 %8 2021 11 24 %G eng %U https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8613219/ %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/34819517?dopt=Abstract %R 10.1038/s41467-021-27129-3 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2021 %T A genetic model of methionine restriction extends health- and lifespan %A Parkhitko, Andrey A %A Wang, Lin %A Filine, Elizabeth %A Jouandin, Patrick %A Leshchiner, Dmitry %A Binari, Richard %A Asara, John M %A Rabinowitz, Joshua D %A Perrimon, Norbert %K Aging %K Alzheimer Disease %K Amino Acids %K Animals %K Animals, Genetically Modified %K Carbon-Sulfur Lyases %K Drosophila %K Food %K Humans %K Longevity %K Methionine %K Models, Genetic %X Loss of metabolic homeostasis is a hallmark of aging and is characterized by dramatic metabolic reprogramming. To analyze how the fate of labeled methionine is altered during aging, we applied 13C5-Methionine labeling to Drosophila and demonstrated significant changes in the activity of different branches of the methionine metabolism as flies age. We further tested whether targeted degradation of methionine metabolism components would "reset" methionine metabolism flux and extend the fly lifespan. Specifically, we created transgenic flies with inducible expression of Methioninase, a bacterial enzyme capable of degrading methionine and revealed methionine requirements for normal maintenance of lifespan. We also demonstrated that microbiota-derived methionine is an alternative and important source in addition to food-derived methionine. In this genetic model of methionine restriction (MetR), we also demonstrate that either whole-body or tissue-specific Methioninase expression can dramatically extend Drosophila health- and lifespan and exerts physiological effects associated with MetR. Interestingly, while previous dietary MetR extended lifespan in flies only in low amino acid conditions, MetR from Methioninase expression extends lifespan independently of amino acid levels in the food. Finally, because impairment of the methionine metabolism has been previously associated with the development of Alzheimer's disease, we compared methionine metabolism reprogramming between aging flies and a Drosophila model relevant to Alzheimer's disease, and found that overexpression of human Tau caused methionine metabolism flux reprogramming similar to the changes found in aged flies. Altogether, our study highlights Methioninase as a potential agent for health- and lifespan extension. %B Proc Natl Acad Sci U S A %V 118 %8 2021 10 05 %G eng %N 40 %1 http://www.ncbi.nlm.nih.gov/pubmed/34588310?dopt=Abstract %R 10.1073/pnas.2110387118 %0 Journal Article %J Trends Genet %D 2021 %T State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila %A Zirin, Jonathan %A Bosch, Justin %A Viswanatha, Raghuvir %A Mohr, Stephanie E %A Perrimon, Norbert %X For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered regularly interspaced short palindromic repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens. %B Trends Genet %8 2021 Dec 18 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/34933779?dopt=Abstract %R 10.1016/j.tig.2021.11.006 %0 Journal Article %J Bio-Protocol %D 2021 %T Preparation of Drosophila Larval Blood Cells for Single-cell RNA Sequencing %A Tattikota, SG %A Perrimon, N %B Bio-Protocol %V 11 %G eng %N 16 %0 Journal Article %J Cell Rep %D 2021 %T Coordination of tumor growth and host wasting by tumor-derived Upd3 %A Ding, Guangming %A Xiang, Xiaoxiang %A Hu, Yanhui %A Xiao, Gen %A Chen, Yuchen %A Binari, Richard %A Comjean, Aram %A Li, Jiaying %A Rushworth, Elisabeth %A Fu, Zhenming %A Mohr, Stephanie E %A Perrimon, Norbert %A Song, Wei %X yki-induced gut tumors in Drosophila are associated with host wasting, including muscle dysfunction, lipid loss, and hyperglycemia, a condition reminiscent of human cancer cachexia. We previously used this model to identify tumor-derived ligands that contribute to host wasting. To identify additional molecular networks involved in host-tumor interactions, we develop PathON, a web-based tool analyzing the major signaling pathways in Drosophila, and uncover the Upd3/Jak/Stat axis as an important modulator. We find that yki-gut tumors secrete Upd3 to promote self-overproliferation and enhance Jak/Stat signaling in host organs to cause wasting, including muscle dysfunction, lipid loss, and hyperglycemia. We further reveal that Upd3/Jak/Stat signaling in the host organs directly triggers the expression of ImpL2, an antagonistic binding protein for insulin-like peptides, to impair insulin signaling and energy balance. Altogether, our results demonstrate that yki-gut tumors produce a Jak/Stat pathway ligand, Upd3, that regulates both self-growth and host wasting. %B Cell Rep %V 36 %P 109553 %8 2021 Aug 17 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/34407411?dopt=Abstract %R 10.1016/j.celrep.2021.109553 %0 Journal Article %J Ann N Y Acad Sci %D 2021 %T Metabolic decisions in development and disease-a Keystone Symposia report %A Cable, Jennifer %A Pourquié, Olivier %A Wellen, Kathryn E %A Finley, Lydia W S %A Aulehla, Alexander %A Gould, Alex P. %A Teleman, Aurelio %A Tu, William B %A Garrett, Wendy Sarah %A Miguel-Aliaga, Irene %A Perrimon, Norbert %A Hooper, Lora V %A Walhout, A J Marian %A Wei, Wei %A Alexandrov, Theodore %A Erez, Ayelet %A Ralser, Markus %A Rabinowitz, Joshua D %A Hemalatha, Anupama %A Gutiérrez-Pérez, Paula %A Chandel, Navdeep S %A Rutter, Jared %A Locasale, Jason W %A Landoni, Juan C %A Christofk, Heather %X There is an increasing appreciation for the role of metabolism in cell signaling and cell decision making. Precise metabolic control is essential in development, as evident by the disorders caused by mutations in metabolic enzymes. The metabolic profile of cells is often cell-type specific, changing as cells differentiate or during tumorigenesis. Recent evidence has shown that changes in metabolism are not merely a consequence of changes in cell state but that metabolites can serve to promote and/or inhibit these changes. Metabolites can link metabolic pathways with cell signaling pathways via several mechanisms, for example, by serving as substrates for protein post-translational modifications, by affecting enzyme activity via allosteric mechanisms, or by altering epigenetic markers. Unraveling the complex interactions governing metabolism, gene expression, and protein activity that ultimately govern a cell's fate will require new tools and interactions across disciplines. On March 24 and 25, 2021, experts in cell metabolism, developmental biology, and human disease met virtually for the Keystone eSymposium, "Metabolic Decisions in Development and Disease." The discussions explored how metabolites impact cellular and developmental decisions in a diverse range of model systems used to investigate normal development, developmental disorders, dietary effects, and cancer-mediated changes in metabolism. %B Ann N Y Acad Sci %8 2021 Aug 19 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/34414571?dopt=Abstract %R 10.1111/nyas.14678 %0 Journal Article %J Nucleic Acids Res %D 2021 %T TIMEOR: a web-based tool to uncover temporal regulatory mechanisms from multi-omics data %A Conard, Ashley Mae %A Goodman, Nathaniel %A Hu, Yanhui %A Perrimon, Norbert %A Singh, Ritambhara %A Lawrence, Charles %A Larschan, Erica %X Uncovering how transcription factors regulate their targets at DNA, RNA and protein levels over time is critical to define gene regulatory networks (GRNs) and assign mechanisms in normal and diseased states. RNA-seq is a standard method measuring gene regulation using an established set of analysis stages. However, none of the currently available pipeline methods for interpreting ordered genomic data (in time or space) use time-series models to assign cause and effect relationships within GRNs, are adaptive to diverse experimental designs, or enable user interpretation through a web-based platform. Furthermore, methods integrating ordered RNA-seq data with protein-DNA binding data to distinguish direct from indirect interactions are urgently needed. We present TIMEOR (Trajectory Inference and Mechanism Exploration with Omics data in R), the first web-based and adaptive time-series multi-omics pipeline method which infers the relationship between gene regulatory events across time. TIMEOR addresses the critical need for methods to determine causal regulatory mechanism networks by leveraging time-series RNA-seq, motif analysis, protein-DNA binding data, and protein-protein interaction networks. TIMEOR's user-catered approach helps non-coders generate new hypotheses and validate known mechanisms. We used TIMEOR to identify a novel link between insulin stimulation and the circadian rhythm cycle. TIMEOR is available at https://github.com/ashleymaeconard/TIMEOR.git and http://timeor.brown.edu. %B Nucleic Acids Res %8 2021 Jun 14 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/34125906?dopt=Abstract %R 10.1093/nar/gkab384 %0 Journal Article %J Sci Adv %D 2021 %T What fuels the fly: Energy metabolism in and its application to the study of obesity and diabetes %A Chatterjee, Nirmalya %A Perrimon, Norbert %X The organs and metabolic pathways involved in energy metabolism, and the process of ATP production from nutrients, are comparable between humans and Drosophila melanogaster This level of conservation, together with the power of Drosophila genetics, makes the fly a very useful model system to study energy homeostasis. Here, we discuss the major organs involved in energy metabolism in Drosophila and how they metabolize different dietary nutrients to generate adenosine triphosphate. Energy metabolism in these organs is controlled by cell-intrinsic, paracrine, and endocrine signals that are similar between Drosophila and mammals. We describe how these signaling pathways are regulated by several physiological and environmental cues to accommodate tissue-, age-, and environment-specific differences in energy demand. Last, we discuss several genetic and diet-induced fly models of obesity and diabetes that can be leveraged to better understand the molecular basis of these metabolic diseases and thereby promote the development of novel therapies. %B Sci Adv %V 7 %8 2021 Jun %G eng %N 24 %1 http://www.ncbi.nlm.nih.gov/pubmed/34108216?dopt=Abstract %R 10.1126/sciadv.abg4336 %0 Journal Article %J Comput Struct Biotechnol J %D 2021 %T DRscDB: A single-cell RNA-seq resource for data mining and data comparison across species %A Hu, Yanhui %A Tattikota, Sudhir Gopal %A Liu, Yifang %A Comjean, Aram %A Gao, Yue %A Forman, Corey %A Kim, Grace %A Rodiger, Jonathan %A Papatheodorou, Irene %A Dos Santos, Gilberto %A Mohr, Stephanie E %A Perrimon, Norbert %X With the advent of single-cell RNA sequencing (scRNA-seq) technologies, there has been a spike in studies involving scRNA-seq of several tissues across diverse species including Drosophila. Although a few databases exist for users to query genes of interest within the scRNA-seq studies, search tools that enable users to find orthologous genes and their cell type-specific expression patterns across species are limited. Here, we built a new search database, DRscDB (https://www.flyrnai.org/tools/single_cell/web/), to address this need. DRscDB serves as a comprehensive repository for published scRNA-seq datasets for Drosophila and relevant datasets from human and other model organisms. DRscDB is based on manual curation of Drosophila scRNA-seq studies of various tissue types and their corresponding analogous tissues in vertebrates including zebrafish, mouse, and human. Of note, our search database provides most of the literature-derived marker genes, thus preserving the original analysis of the published scRNA-seq datasets. Finally, DRscDB serves as a web-based user interface that allows users to mine gene expression data from scRNA-seq studies and perform cell cluster enrichment analyses pertaining to various scRNA-seq studies, both within and across species. %B Comput Struct Biotechnol J %V 19 %P 2018-2026 %8 2021 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33995899?dopt=Abstract %R 10.1016/j.csbj.2021.04.021 %0 Journal Article %J Nat Commun %D 2021 %T Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes %A Feng, Xuechun %A López Del Amo, Víctor %A Mameli, Enzo %A Lee, Megan %A Bishop, Alena L %A Perrimon, Norbert %A Gantz, Valentino M %X Culex mosquitoes are a global vector for multiple human and animal diseases, including West Nile virus, lymphatic filariasis, and avian malaria, posing a constant threat to public health, livestock, companion animals, and endangered birds. While rising insecticide resistance has threatened the control of Culex mosquitoes, advances in CRISPR genome-editing tools have fostered the development of alternative genetic strategies such as gene drive systems to fight disease vectors. However, though gene-drive technology has quickly progressed in other mosquitoes, advances have been lacking in Culex. Here, we develop a Culex-specific Cas9/gRNA expression toolkit and use site-directed homology-based transgenesis to generate and validate a Culex quinquefasciatus Cas9-expressing line. We show that gRNA scaffold variants improve transgenesis efficiency in both Culex quinquefasciatus and Drosophila melanogaster and boost gene-drive performance in the fruit fly. These findings support future technology development to control Culex mosquitoes and provide valuable insight for improving these tools in other species. %B Nat Commun %V 12 %P 2960 %8 2021 05 20 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/34017003?dopt=Abstract %R 10.1038/s41467-021-23239-0 %0 Journal Article %J Nat Commun %D 2021 %T Proteomics of protein trafficking by in vivo tissue-specific labeling %A Droujinine, Ilia A %A Meyer, Amanda S %A Wang, Dan %A Udeshi, Namrata D %A Hu, Yanhui %A Rocco, David %A McMahon, Jill A %A Yang, Rui %A Guo, JinJin %A Mu, Luye %A Carey, Dominique K %A Svinkina, Tanya %A Zeng, Rebecca %A Branon, Tess %A Tabatabai, Areya %A Bosch, Justin A %A Asara, John M %A Ting, Alice Y %A Carr, Steven A %A McMahon, Andrew P %A Perrimon, Norbert %K Animals %K Animals, Genetically Modified %K Biotin %K Biotinylation %K Carbon-Nitrogen Ligases %K Cell Line %K Disease Models, Animal %K Drosophila %K Embryonic Stem Cells %K Escherichia coli Proteins %K Female %K Humans %K Male %K Mice %K Protein Engineering %K Protein Transport %K Proteomics %K Repressor Proteins %K Staining and Labeling %K Tandem Mass Spectrometry %K Teratoma %X Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease. %B Nat Commun %V 12 %P 2382 %8 2021 04 22 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/33888706?dopt=Abstract %R 10.1038/s41467-021-22599-x %0 Journal Article %J Genetics %D 2021 %T Methods and tools for spatial mapping of single-cell RNAseq clusters in Drosophila %A Mohr, Stephanie E %A Tattikota, Sudhir Gopal %A Xu, Jun %A Zirin, Jonathan %A Hu, Yanhui %A Perrimon, Norbert %X Single-cell RNA sequencing (scRNAseq) experiments provide a powerful means to identify clusters of cells that share common gene expression signatures. A major challenge in scRNAseq studies is to map the clusters to specific anatomical regions along the body and within tissues. Existing data, such as information obtained from large-scale in situ RNA hybridization studies, cell type specific transcriptomics, gene expression reporters, antibody stainings, and fluorescent tagged proteins, can help to map clusters to anatomy. However, in many cases, additional validation is needed to precisely map the spatial location of cells in clusters. Several approaches are available for spatial resolution in Drosophila, including mining of existing datasets, and use of existing or new tools for direct or indirect detection of RNA, or direct detection of proteins. Here, we review available resources and emerging technologies that will facilitate spatial mapping of scRNAseq clusters at high resolution in Drosophila. Importantly, we discuss the need, available approaches, and reagents for multiplexing gene expression detection in situ, as in most cases scRNAseq clusters are defined by the unique coexpression of sets of genes. %B Genetics %8 2021 Mar 13 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33713129?dopt=Abstract %R 10.1093/genetics/iyab019 %0 Journal Article %J Mol Cell %D 2021 %T mTORC1 promotes cell growth via mA-dependent mRNA degradation %A Cho, Sungyun %A Lee, Gina %A Pickering, Brian F %A Jang, Cholsoon %A Park, Jin H %A He, Long %A Mathur, Lavina %A Kim, Seung-Soo %A Jung, Sunhee %A Tang, Hong-Wen %A Monette, Sebastien %A Rabinowitz, Joshua D %A Perrimon, Norbert %A Jaffrey, Samie R %A Blenis, John %X Dysregulated mTORC1 signaling alters a wide range of cellular processes, contributing to metabolic disorders and cancer. Defining the molecular details of downstream effectors is thus critical for uncovering selective therapeutic targets. We report that mTORC1 and its downstream kinase S6K enhance eIF4A/4B-mediated translation of Wilms' tumor 1-associated protein (WTAP), an adaptor for the N-methyladenosine (mA) RNA methyltransferase complex. This regulation is mediated by 5' UTR of WTAP mRNA that is targeted by eIF4A/4B. Single-nucleotide-resolution mA mapping revealed that MAX dimerization protein 2 (MXD2) mRNA contains mA, and increased mA modification enhances its degradation. WTAP induces cMyc-MAX association by suppressing MXD2 expression, which promotes cMyc transcriptional activity and proliferation of mTORC1-activated cancer cells. These results elucidate a mechanism whereby mTORC1 stimulates oncogenic signaling via mA RNA modification and illuminates the WTAP-MXD2-cMyc axis as a potential therapeutic target for mTORC1-driven cancers. %B Mol Cell %8 2021 Mar 19 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33756105?dopt=Abstract %R 10.1016/j.molcel.2021.03.010 %0 Journal Article %J PLoS Genet %D 2021 %T Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells %A Parkhitko, Andrey A %A Singh, Arashdeep %A Hsieh, Sharon %A Hu, Yanhui %A Binari, Richard %A Lord, Christopher J %A Hannenhalli, Sridhar %A Ryan, Colm J %A Perrimon, Norbert %X The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer. %B PLoS Genet %V 17 %P e1009354 %8 2021 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/33591981?dopt=Abstract %R 10.1371/journal.pgen.1009354 %0 Journal Article %J Curr Opin Insect Sci %D 2021 %T Defining cell types and lineage in the Drosophila midgut using single cell transcriptomics %A Hung, Ruei-Jiun %A Li, Joshua Shing Shun %A Liu, Yifang %A Perrimon, Norbert %X The Drosophila midgut has emerged in recent years as a model system to study stem cell renewal and differentiation and tissue homeostasis. Histological, genetic and gene expression studies have provided a wealth of information on gut cell types, regionalization, genes and pathways involved in cell proliferation and differentiation, stem cell renewal, and responses to changes in environmental factors such as the microbiota and nutrients. Here, we review the contribution of single cell transcriptomic methods to our understanding of gut cell type diversity, lineage and behavior. %B Curr Opin Insect Sci %V 47 %P 12-17 %8 2021 Feb 17 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33609768?dopt=Abstract %R 10.1016/j.cois.2021.02.008 %0 Journal Article %J Nat Commun %D 2021 %T Endonuclease G promotes autophagy by suppressing mTOR signaling and activating the DNA damage response %A Wang, Wenjun %A Li, Jianshuang %A Tan, Junyang %A Wang, Miaomiao %A Yang, Jing %A Zhang, Zhi-Min %A Li, Chuanzhou %A Basnakian, Alexei G %A Tang, Hong-Wen %A Perrimon, Norbert %A Zhou, Qinghua %K 14-3-3 Proteins %K Animals %K Apoptosis %K Autophagy %K Caenorhabditis elegans %K Cell Line %K Class III Phosphatidylinositol 3-Kinases %K DNA Damage %K Drosophila %K Endodeoxyribonucleases %K Gene Knockout Techniques %K Hep G2 Cells %K Humans %K Liver %K Mice %K Mice, Knockout %K Mitochondria %K Phosphorylation %K Signal Transduction %K TOR Serine-Threonine Kinases %K Transcriptome %K Tuberous Sclerosis Complex 2 Protein %X Endonuclease G (ENDOG), a mitochondrial nuclease, is known to participate in many cellular processes, including apoptosis and paternal mitochondrial elimination, while its role in autophagy remains unclear. Here, we report that ENDOG released from mitochondria promotes autophagy during starvation, which we find to be evolutionally conserved across species by performing experiments in human cell lines, mice, Drosophila and C. elegans. Under starvation, Glycogen synthase kinase 3 beta-mediated phosphorylation of ENDOG at Thr-128 and Ser-288 enhances its interaction with 14-3-3γ, which leads to the release of Tuberin (TSC2) and Phosphatidylinositol 3-kinase catalytic subunit type 3 (Vps34) from 14-3-3γ, followed by mTOR pathway suppression and autophagy initiation. Alternatively, ENDOG activates DNA damage response and triggers autophagy through its endonuclease activity. Our results demonstrate that ENDOG is a crucial regulator of autophagy, manifested by phosphorylation-mediated interaction with 14-3-3γ, and its endonuclease activity-mediated DNA damage response. %B Nat Commun %V 12 %P 476 %8 2021 01 20 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/33473107?dopt=Abstract %R 10.1038/s41467-020-20780-2 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2021 %T mTORC1-chaperonin CCT signaling regulates mA RNA methylation to suppress autophagy %A Tang, Hong-Wen %A Weng, Jui-Hsia %A Lee, Wen Xing %A Hu, Yanhui %A Gu, Lei %A Cho, Sungyun %A Lee, Gina %A Binari, Richard %A Li, Cathleen %A Cheng, Min En %A Kim, Ah-Ram %A Xu, Jun %A Shen, Zhangfei %A Xu, Chiwei %A Asara, John M %A Blenis, John %A Perrimon, Norbert %X Mechanistic Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism that senses and integrates nutritional and environmental cues with cellular responses. Recent studies have revealed critical roles of mTORC1 in RNA biogenesis and processing. Here, we find that the mA methyltransferase complex (MTC) is a downstream effector of mTORC1 during autophagy in and human cells. Furthermore, we show that the Chaperonin Containing Tailless complex polypeptide 1 (CCT) complex, which facilitates protein folding, acts as a link between mTORC1 and MTC. The mTORC1 activates the chaperonin CCT complex to stabilize MTC, thereby increasing mA levels on the messenger RNAs encoding autophagy-related genes, leading to their degradation and suppression of autophagy. Altogether, our study reveals an evolutionarily conserved mechanism linking mTORC1 signaling with mA RNA methylation and demonstrates their roles in suppressing autophagy. %B Proc Natl Acad Sci U S A %V 118 %8 2021 Mar 09 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/33649236?dopt=Abstract %R 10.1073/pnas.2021945118 %0 Journal Article %J Nucleic Acids Res %D 2021 %T FlyBase: updates to the Drosophila melanogaster knowledge base %A Larkin, Aoife %A Marygold, Steven J %A Antonazzo, Giulia %A Attrill, Helen %A Dos Santos, Gilberto %A Garapati, Phani V %A Goodman, Joshua L %A Gramates, L Sian %A Millburn, Gillian %A Strelets, Victor B %A Tabone, Christopher J %A Thurmond, Jim %K Animals %K Computational Biology %K Databases, Genetic %K Drosophila melanogaster %K Genes, Insect %K Genome, Insect %K Genomics %K Knowledge Bases %K Molecular Sequence Annotation %K Search Engine %K Web Browser %X FlyBase (flybase.org) is an essential online database for researchers using Drosophila melanogaster as a model organism, facilitating access to a diverse array of information that includes genetic, molecular, genomic and reagent resources. Here, we describe the introduction of several new features at FlyBase, including Pathway Reports, paralog information, disease models based on orthology, customizable tables within reports and overview displays ('ribbons') of expression and disease data. We also describe a variety of recent important updates, including incorporation of a developmental proteome, upgrades to the GAL4 search tab, additional Experimental Tool Reports, migration to JBrowse for genome browsing and improvements to batch queries/downloads and the Fast-Track Your Paper tool. %B Nucleic Acids Res %V 49 %P D899-D907 %8 2021 01 08 %G eng %N D1 %1 http://www.ncbi.nlm.nih.gov/pubmed/33219682?dopt=Abstract %R 10.1093/nar/gkaa1026 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2021 %T Precise genome engineering in using prime editing %A Bosch, Justin A %A Birchak, Gabriel %A Perrimon, Norbert %X Precise genome editing is a valuable tool to study gene function in model organisms. Prime editing, a precise editing system developed in mammalian cells, does not require double-strand breaks or donor DNA and has low off-target effects. Here, we applied prime editing for the model organism and developed conditions for optimal editing. By expressing prime editing components in cultured cells or somatic cells of transgenic flies, we precisely introduce premature stop codons in three classical visible marker genes, , , and Furthermore, by restricting editing to germ cells, we demonstrate efficient germ-line transmission of a precise edit in to 36% of progeny. Our results suggest that prime editing is a useful system in to study gene function, such as engineering precise point mutations, deletions, or epitope tags. %B Proc Natl Acad Sci U S A %V 118 %8 2021 Jan 05 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/33443210?dopt=Abstract %R 10.1073/pnas.2021996118 %0 Journal Article %J Nucleic Acids Res %D 2021 %T FlyRNAi.org-the database of the Drosophila RNAi screening center and transgenic RNAi project: 2021 update %A Hu, Yanhui %A Comjean, Aram %A Rodiger, Jonathan %A Liu, Yifang %A Gao, Yue %A Chung, Verena %A Zirin, Jonathan %A Perrimon, Norbert %A Mohr, Stephanie E %K Animals %K Animals, Genetically Modified %K Computational Biology %K Databases, Genetic %K Drosophila melanogaster %K Drosophila Proteins %K Gene Expression Profiling %K Genome, Insect %K Genomics %K Information Storage and Retrieval %K Internet %K RNA Interference %X The FlyRNAi database at the Drosophila RNAi Screening Center and Transgenic RNAi Project (DRSC/TRiP) provides a suite of online resources that facilitate functional genomics studies with a special emphasis on Drosophila melanogaster. Currently, the database provides: gene-centric resources that facilitate ortholog mapping and mining of information about orthologs in common genetic model species; reagent-centric resources that help researchers identify RNAi and CRISPR sgRNA reagents or designs; and data-centric resources that facilitate visualization and mining of transcriptomics data, protein modification data, protein interactions, and more. Here, we discuss updated and new features that help biological and biomedical researchers efficiently identify, visualize, analyze, and integrate information and data for Drosophila and other species. Together, these resources facilitate multiple steps in functional genomics workflows, from building gene and reagent lists to management, analysis, and integration of data. %B Nucleic Acids Res %V 49 %P D908-D915 %8 2021 01 08 %G eng %N D1 %1 http://www.ncbi.nlm.nih.gov/pubmed/33104800?dopt=Abstract %R 10.1093/nar/gkaa936 %0 Journal Article %J Wiley Interdiscip Rev Dev Biol %D 2021 %T Proximity-dependent labeling methods for proteomic profiling in living cells: An update %A Bosch, Justin A %A Chen, Chiao-Lin %A Perrimon, Norbert %X Characterizing the proteome composition of organelles and subcellular regions of living cells can facilitate the understanding of cellular organization as well as protein interactome networks. Proximity labeling-based methods coupled with mass spectrometry (MS) offer a high-throughput approach for systematic analysis of spatially restricted proteomes. Proximity labeling utilizes enzymes that generate reactive radicals to covalently tag neighboring proteins. The tagged endogenous proteins can then be isolated for further analysis by MS. To analyze protein-protein interactions or identify components that localize to discrete subcellular compartments, spatial expression is achieved by fusing the enzyme to specific proteins or signal peptides that target to particular subcellular regions. Although these technologies have only been introduced recently, they have already provided deep insights into a wide range of biological processes. Here, we provide an updated description and comparison of proximity labeling methods, as well as their applications and improvements. As each method has its own unique features, the goal of this review is to describe how different proximity labeling methods can be used to answer different biological questions. This article is categorized under: Technologies > Analysis of Proteins. %B Wiley Interdiscip Rev Dev Biol %V 10 %P e392 %8 2021 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/32909689?dopt=Abstract %R 10.1002/wdev.392 %0 Journal Article %J bioRxiv %D 2020 %T CRISPR-Cas13 mediated Knock Down in Drosophila cultured cells %A Viswanatha, R %A Zaffagni, M %A Zirin, J %A Perrimon, N %A Kadener, S %X Manipulation of gene expression is one of the best approaches for studying gene function in vivo. CRISPR-Cas13 has the potential to be a powerful technique for manipulating RNA expression in diverse animal systems in vivo, including Drosophila melanogaster. Studies using Cas13 in mammalian cell lines for gene knockdown showed increased on-target efficiency and decreased off-targeting relative to RNAi. Moreover, catalytically inactive Cas13 fusions can be used to image RNA molecules, install precise changes to the epitranscriptome, or alter splicing. However, recent studies have suggested that there may be limitations to the deployment of these tools in Drosophila, so further optimization of the system is required. Here, we report a new set of PspCas13b and RfxCas13d expression constructs and use these reagents to successfully knockdown both reporter and endogenous transcripts in Drosophila cells. As toxicity issues have been reported with high level of Cas13, we effectively decreased PspCas13b expression without impairing its function by tuning down translation. Furthermore, we altered the spatial activity of both PspCas13b and RfxCas13d by introducing Nuclear Exportation Sequences (NES) and Nuclear Localization Sequences (NLS) while maintaining activity. Finally, we generated a stable cell line expressing RfxCas13d under the inducible metallothionein promoter, establishing a useful tool for high-throughput genetic screening. Thus, we report new reagents for performing RNA CRISPR-Cas13 experiments in Drosophila, providing additional Cas13 expression constructs that retain activity. %B bioRxiv %V https://doi.org/10.1101/2020.11.01.364166 %G eng %0 Journal Article %J Bio Protoc %D 2020 %T Probe-Seq: Method for RNA Sequencing of Specific Cell Types from Animal Tissue %A Ryoji Amamoto %A Garcia, Mauricio D %A West, Emma R %A Choi, Jiho %A Lapan, Sylvain W %A Lane, Elizabeth A %A Perrimon, Norbert %A Cepko, Constance L %X Most organs and tissues are composed of many types of cells. To characterize cellular state, various transcription profiling approaches are currently available, including whole-tissue bulk RNA sequencing, single cell RNA sequencing (scRNA-Seq), and cell type-specific RNA sequencing. What is missing in this repertoire is a simple, versatile method for bulk transcriptional profiling of cell types for which cell type-specific genetic markers or antibodies are not readily available. We therefore developed Probe-Seq, which uses hybridization of gene-specific probes to RNA markers for isolation of specific types of cells, to enable downstream FACS isolation and bulk RNA sequencing. We show that this method can enable isolation and profiling of specific cell types from mouse retina, frozen human retina, midgut, and developing chick retina, suggesting that it is likely useful for most organisms. %B Bio Protoc %V 10 %P e3749 %8 2020 Sep 20 %G eng %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/33659409?dopt=Abstract %R 10.21769/BioProtoc.3749 %0 Journal Article %J G3 (Bethesda) %D 2020 %T SNP-CRISPR: A Web Tool for SNP-Specific Genome Editing %A Chen, Chiao-Lin %A Rodiger, Jonathan %A Chung, Verena %A Viswanatha, Raghuvir %A Mohr, Stephanie E %A Hu, Yanhui %A Perrimon, Norbert %K Animals %K Clustered Regularly Interspaced Short Palindromic Repeats %K Diptera %K Gene Editing %K Humans %K Internet %K Mice %K Polymorphism, Single Nucleotide %K Rats %K RNA, Guide %K Software %K Zebrafish %X CRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction. %B G3 (Bethesda) %V 10 %P 489-494 %8 2020 02 06 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/31822517?dopt=Abstract %R 10.1534/g3.119.400904 %0 Journal Article %J G3 (Bethesda) %D 2020 %T BioLitMine: Advanced Mining of Biomedical and Biological Literature About Human Genes and Genes from Major Model Organisms %A Hu, Yanhui %A Chung, Verena %A Comjean, Aram %A Rodiger, Jonathan %A Nipun, Fnu %A Perrimon, Norbert %A Mohr, Stephanie E %X The accumulation of biological and biomedical literature outpaces the ability of most researchers and clinicians to stay abreast of their own immediate fields, let alone a broader range of topics. Although available search tools support identification of relevant literature, finding relevant and key publications is not always straightforward. For example, important publications might be missed in searches with an official gene name due to gene synonyms. Moreover, ambiguity of gene names can result in retrieval of a large number of irrelevant publications. To address these issues and help researchers and physicians quickly identify relevant publications, we developed BioLitMine, an advanced literature mining tool that takes advantage of the medical subject heading (MeSH) index and gene-to-publication annotations already available for PubMed literature. Using BioLitMine, a user can identify what MeSH terms are represented in the set of publications associated with a given gene of the interest, or start with a term and identify relevant publications. Users can also use the tool to find co-cited genes and a build a literature co-citation network. In addition, BioLitMine can help users build a gene list relevant to a MeSH term, such as a list of genes relevant to "stem cells" or "breast neoplasms." Users can also start with a gene or pathway of interest and identify authors associated with that gene or pathway, a feature that makes it easier to identify experts who might serve as collaborators or reviewers. Altogether, BioLitMine extends the value of PubMed-indexed literature and its existing expert curation by providing a robust and gene-centric approach to retrieval of relevant information. %B G3 (Bethesda) %V 10 %P 4531-4539 %8 2020 Dec 03 %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/33028629?dopt=Abstract %R 10.1534/g3.120.401775 %0 Journal Article %J Elife %D 2020 %T Downregulation of the tyrosine degradation pathway extends lifespan %A Parkhitko, Andrey A %A Ramesh, Divya %A Wang, Lin %A Leshchiner, Dmitry %A Filine, Elizabeth %A Binari, Richard %A Olsen, Abby L %A Asara, John M %A Cracan, Valentin %A Rabinowitz, Joshua D %A Brockmann, Axel %A Perrimon, Norbert %X Aging is characterized by extensive metabolic reprogramming. To identify metabolic pathways associated with aging, we analyzed age-dependent changes in the metabolomes of long-lived . Among the metabolites that changed, levels of tyrosine were increased with age in long-lived flies. We demonstrate that the levels of enzymes in the tyrosine degradation pathway increase with age in wild-type flies. Whole-body and neuronal-specific downregulation of enzymes in the tyrosine degradation pathway significantly extends lifespan, causes alterations of metabolites associated with increased lifespan, and upregulates the levels of tyrosine-derived neuromediators. Moreover, feeding wild-type flies with tyrosine increased their lifespan. Mechanistically, we show that suppression of ETC complex I drives the upregulation of enzymes in the tyrosine degradation pathway, an effect that can be rescued by tigecycline, an FDA-approved drug that specifically suppresses mitochondrial translation. In addition, tyrosine supplementation partially rescued lifespan of flies with ETC complex I suppression. Altogether, our study highlights the tyrosine degradation pathway as a regulator of longevity. %B Elife %V 9 %8 2020 12 15 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33319750?dopt=Abstract %R 10.7554/eLife.58053 %0 Journal Article %J Cell Rep %D 2020 %T Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression %A Ingaramo, María Clara %A Sánchez, Juan Andrés %A Perrimon, Norbert %A Dekanty, Andrés %X The tumor suppressor p53 regulates multiple metabolic pathways at the cellular level. However, its role in the context of a whole animal response to metabolic stress is poorly understood. Using Drosophila, we show that AMP-activated protein kinase (AMPK)-dependent Dmp53 activation is critical for sensing nutrient stress, maintaining metabolic homeostasis, and extending organismal survival. Under both nutrient deprivation and high-sugar diet, Dmp53 activation in the fat body represses expression of the Drosophila Leptin analog, Unpaired-2 (Upd2), which remotely controls Dilp2 secretion in insulin-producing cells. In starved Dmp53-depleted animals, elevated Upd2 expression in adipose cells and activation of Upd2 receptor Domeless in the brain result in sustained Dilp2 circulating levels and impaired autophagy induction at a systemic level, thereby reducing nutrient stress survival. These findings demonstrate an essential role for the AMPK-Dmp53 axis in nutrient stress responses and expand the concept that adipose tissue acts as a sensing organ that orchestrates systemic adaptation to nutrient status. %B Cell Rep %V 33 %P 108321 %8 2020 Oct 27 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/33113367?dopt=Abstract %R 10.1016/j.celrep.2020.108321 %0 Journal Article %J Elife %D 2020 %T PDGF/VEGF signaling from muscles to hepatocyte-like cells protects against obesity %A Ghosh, Arpan C %A Tattikota, Sudhir Gopal %A Liu, Yifang %A Comjean, Aram %A Hu, Yanhui %A Barrera, Victor %A Ho Sui, Shannan J %A Perrimon, Norbert %X PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine, and endocrine mechanisms. We investigated organ-specific metabolic roles of PDGF/VEGF-like factors (Pvfs). We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf1 signals to the hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. We find that adult muscle-specific expression of increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. Our findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells. %B Elife %V 9 %8 2020 10 27 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33107824?dopt=Abstract %R 10.7554/eLife.56969 %0 Journal Article %J Ageing Res Rev %D 2020 %T Targeting metabolic pathways for extension of lifespan and healthspan across multiple species %A Parkhitko, Andrey A %A Filine, Elizabeth %A Mohr, Stephanie E %A Moskalev, Alexey %A Perrimon, Norbert %K Aging %K Animals %K Longevity %K Metabolic Networks and Pathways %K Mice %K Models, Biological %K Mutation %X Metabolism plays a significant role in the regulation of aging at different levels, and metabolic reprogramming represents a major driving force in aging. Metabolic reprogramming leads to impaired organismal fitness, an age-dependent increase in susceptibility to diseases, decreased ability to mount a stress response, and increased frailty. The complexity of age-dependent metabolic reprogramming comes from the multitude of levels on which metabolic changes can be connected to aging and regulation of lifespan. This is further complicated by the different metabolic requirements of various tissues, cross-organ communication via metabolite secretion, and direct effects of metabolites on epigenetic state and redox regulation; however, not all of these changes are causative to aging. Studies in yeast, flies, worms, and mice have played a crucial role in identifying mechanistic links between observed changes in various metabolic traits and their effects on lifespan. Here, we review how changes in the organismal and organ-specific metabolome are associated with aging and how targeting of any one of over a hundred different targets in specific metabolic pathways can extend lifespan. An important corollary is that restriction or supplementation of different metabolites can change activity of these metabolic pathways in ways that improve healthspan and extend lifespan in different organisms. Due to the high levels of conservation of metabolism in general, translating findings from model systems to human beings will allow for the development of effective strategies for human health- and lifespan extension. %B Ageing Res Rev %V 64 %P 101188 %8 2020 12 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/33031925?dopt=Abstract %R 10.1016/j.arr.2020.101188 %0 Journal Article %J Nat Protoc %D 2020 %T CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells %A Xia, Baolong %A Amador, Gabriel %A Viswanatha, Raghuvir %A Zirin, Jonathan %A Mohr, Stephanie E %A Perrimon, Norbert %K Alleles %K Animals %K Base Sequence %K Clustered Regularly Interspaced Short Palindromic Repeats %K CRISPR-Cas Systems %K Drosophila %K Endonucleases %K Gene Editing %K Gene Knockout Techniques %K Homozygote %K Polyploidy %K RNA, Guide %X Precise and efficient genome modifications provide powerful tools for biological studies. Previous CRISPR gene knockout methods in cell lines have relied on frameshifts caused by stochastic insertion/deletion in all alleles. However, this method is inefficient for genes with high copy number due to polyploidy or gene amplification because frameshifts in all alleles can be difficult to generate and detect. Here we describe a homology-directed insertion method to knockout genes in the polyploid Drosophila S2R+ cell line. This protocol allows generation of homozygous mutant cell lines using an insertion cassette which autocatalytically generates insertion mutations in all alleles. Knockout cells generated using this method can be directly identified by PCR without a need for DNA sequencing. This protocol takes 2-3 months and can be applied to other polyploid cell lines or high-copy-number genes. %B Nat Protoc %V 15 %P 3478-3498 %8 2020 10 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/32958931?dopt=Abstract %R 10.1038/s41596-020-0383-8 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2020 %T Expanding the horizons of genome editing in the fruit fly with Cas12a %A Ewen-Campen, Ben %A Perrimon, Norbert %K Animals %K CRISPR-Associated Proteins %K CRISPR-Cas Systems %K Drosophila %K Gene Editing %K RNA, Guide %B Proc Natl Acad Sci U S A %V 117 %P 24019-24021 %8 2020 09 29 %G eng %N 39 %1 http://www.ncbi.nlm.nih.gov/pubmed/32883881?dopt=Abstract %R 10.1073/pnas.2016446117 %0 Journal Article %J Cell Rep %D 2020 %T No Evidence that Wnt Ligands Are Required for Planar Cell Polarity in Drosophila %A Ewen-Campen, Ben %A Comyn, Typhaine %A Vogt, Eric %A Perrimon, Norbert %X The frizzled (fz) and dishevelled (dsh) genes are highly conserved members of both the planar cell polarity (PCP) pathway and the Wnt signaling pathway. Given these dual functions, several studies have examined whether Wnt ligands provide a tissue-scale orientation cue for PCP establishment during development, and these studies have reached differing conclusions. Here, we re-examine this issue in the Drosophila melanogaster wing and notum using split-Gal4 co-expression analysis, multiplex somatic CRISPR, and double RNAi experiments. Pairwise loss-of-function experiments targeting wg together with other Wnt genes, via somatic CRISPR or RNAi, do not produce PCP defects in the wing or notum. In addition, somatic CRISPR against evi (aka wntless), which is required for the secretion of Wnt ligands, did not produce detectable PCP phenotypes. Altogether, our results do not support the hypothesis that Wnt ligands contribute to PCP signaling in the Drosophila wing or notum. %B Cell Rep %V 32 %P 108121 %8 2020 09 08 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/32905771?dopt=Abstract %R 10.1016/j.celrep.2020.108121 %0 Journal Article %J Nat Commun %D 2020 %T Single-cell transcriptome maps of myeloid blood cell lineages in Drosophila %A Cho, Bumsik %A Yoon, Sang-Ho %A Lee, Daewon %A Koranteng, Ferdinand %A Tattikota, Sudhir Gopal %A Cha, Nuri %A Shin, Mingyu %A Do, Hobin %A Hu, Yanhui %A Oh, Sue Young %A Lee, Daehan %A Vipin Menon, A %A Moon, Seok Jun %A Perrimon, Norbert %A Nam, Jin-Wu %A Shim, Jiwon %K Animals %K Animals, Genetically Modified %K Cell Differentiation %K Cell Lineage %K Drosophila melanogaster %K Ectoparasitic Infestations %K Gene Expression Profiling %K Hematopoiesis %K Hemocytes %K Host-Parasite Interactions %K Lymphoid Tissue %K RNA-Seq %K Single-Cell Analysis %K Transcriptome %K Wasps %X The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution. %B Nat Commun %V 11 %P 4483 %8 2020 09 08 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/32900993?dopt=Abstract %R 10.1038/s41467-020-18135-y %0 Journal Article %J Cell Discov %D 2020 %T CG14906 (mettl4) mediates mA methylation of U2 snRNA in %A Gu, Lei %A Wang, Longfei %A Chen, Hao %A Hong, Jiaxu %A Shen, Zhangfei %A Dhall, Abhinav %A Lao, Taotao %A Liu, Chaozhong %A Wang, Zheng %A Xu, Yifan %A Tang, Hong-Wen %A Chakraborty, Damayanti %A Chen, Jiekai %A Liu, Zhihua %A Rogulja, Dragana %A Perrimon, Norbert %A Wu, Hao %A Shi, Yang %B Cell Discov %V 6 %P 44 %8 2020 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/32637152?dopt=Abstract %R 10.1038/s41421-020-0178-7 %0 Journal Article %J Dis Model Mech %D 2020 %T A model of oral peptide therapeutics for adult intestinal stem cell tumors %A Bajpai, Anjali %A Quazi, Taushif Ahmad %A Tang, Hong-Wen %A Manzar, Nishat %A Singh, Virender %A Thakur, Ashwani %A Ateeq, Bushra %A Perrimon, Norbert %A Sinha, Pradip %X Peptide therapeutics, unlike small-molecule drugs, display crucial advantages of target specificity and the ability to block large interacting interfaces, such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yorkie (Yki), has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian vestigial-like (VGLL) proteins, specifically their TONDU domain, competitively inhibit YAP-TEAD interaction, resulting in arrest of tumor growth. Here, we show that overexpression of the TONDU peptide or its oral uptake leads to suppression of Yki-driven intestinal stem cell tumors in the adult midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with chromatin immunoprecipitation analysis, reveal that integrin pathway members are part of the Yki-oncogenic network. Collectively, our findings establish as a reliable platform to screen for cancer oral therapeutic peptides and reveal a tumor suppressive role for integrins in Yki-driven tumors.This article has an associated First Person interview with the first author of the paper. %B Dis Model Mech %V 13 %8 2020 07 23 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/32540914?dopt=Abstract %R 10.1242/dmm.044420 %0 Journal Article %J Elife %D 2020 %T A single-cell survey of blood %A Tattikota, Sudhir Gopal %A Cho, Bumsik %A Liu, Yifang %A Hu, Yanhui %A Barrera, Victor %A Steinbaugh, Michael J %A Yoon, Sang-Ho %A Comjean, Aram %A Li, Fangge %A Dervis, Franz %A Hung, Ruei-Jiun %A Nam, Jin-Wu %A Ho Sui, Shannan %A Shim, Jiwon %A Perrimon, Norbert %X blood cells, called hemocytes, are classified into plasmatocytes, crystal cells, and lamellocytes based on the expression of a few marker genes and cell morphologies, which are inadequate to classify the complete hemocyte repertoire. Here, we used single-cell RNA sequencing (scRNA-seq) to map hemocytes across different inflammatory conditions in larvae. We resolved plasmatocytes into different states based on the expression of genes involved in cell cycle, antimicrobial response, and metabolism together with the identification of intermediate states. Further, we discovered rare subsets within crystal cells and lamellocytes that express fibroblast growth factor (FGF) ligand and receptor , respectively. We demonstrate that these FGF components are required for mediating effective immune responses against parasitoid wasp eggs, highlighting a novel role for FGF signaling in inter-hemocyte crosstalk. Our scRNA-seq analysis reveals the diversity of hemocytes and provides a rich resource of gene expression profiles for a systems-level understanding of their functions. %B Elife %V 9 %8 2020 05 12 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/32396065?dopt=Abstract %R 10.7554/eLife.54818 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2020 %T as a model for studying cystic fibrosis pathophysiology of the gastrointestinal system %A Kim, Kevin %A Lane, Elizabeth A %A Saftien, Aurelia %A Wang, Haiyun %A Xu, Yue %A Wirtz-Peitz, Frederik %A Perrimon, Norbert %K Animals %K Cystic Fibrosis %K Cystic Fibrosis Transmembrane Conductance Regulator %K Disease Models, Animal %K Drosophila melanogaster %K Drosophila Proteins %K High-Throughput Nucleotide Sequencing %K Homeostasis %K Humans %K Intestines %K Mucins %K Mutation %K Phenotype %K Stem Cells %X Cystic fibrosis (CF) is a recessive disease caused by mutations in the () gene. The most common symptoms include progressive lung disease and chronic digestive conditions. CF is the first human genetic disease to benefit from having five different species of animal models. Despite the phenotypic differences among the animal models and human CF, these models have provided invaluable insight into understanding disease mechanisms at the organ-system level. Here, we identify a member of the ABCC4 family, CG5789, that has the structural and functional properties expected for encoding the equivalent of human CFTR, and thus refer to it as (). We show that knockdown of in the adult intestine disrupts osmotic homeostasis and displays CF-like phenotypes that lead to intestinal stem cell hyperplasia. We also show that expression of wild-type human , but not mutant variants of CFTR that prevent plasma membrane expression, rescues the mutant phenotypes of Furthermore, we performed RNA sequencing (RNA-Seq)-based transcriptomic analysis using fly intestine and identified a mucin gene, , which is required for proper intestinal barrier protection. Altogether, our findings suggest that can be a powerful model organism for studying CF pathophysiology. %B Proc Natl Acad Sci U S A %V 117 %P 10357-10367 %8 2020 05 12 %G eng %N 19 %1 http://www.ncbi.nlm.nih.gov/pubmed/32345720?dopt=Abstract %R 10.1073/pnas.1913127117 %0 Journal Article %J Metallomics %D 2020 %T Intestinal response to dietary manganese depletion in Drosophila %A Vásquez-Procopio, Johana %A Osorio, Beatriz %A Cortés-Martínez, Leticia %A Hernández-Hernández, Fidel %A Medina-Contreras, Oscar %A Ríos-Castro, Emmanuel %A Comjean, Aram %A Li, Fangge %A Hu, Yanhui %A Mohr, Stephanie %A Perrimon, Norbert %A Missirlis, Fanis %X Manganese is considered essential for animal growth. Manganese ions serve as cofactors to three mitochondrial enzymes: superoxide dismutase (Sod2), arginase and glutamine synthase, and to glycosyltransferases residing in the Golgi. In Drosophila melanogaster, manganese has also been implicated in the formation of ceramide phosphoethanolamine, the insect's sphingomyelin analogue, a structural component of cellular membranes. Manganese overload leads to neurodegeneration and toxicity in both humans and Drosophila. Here, we report specific absorption and accumulation of manganese during the first week of adulthood in flies, which correlates with an increase in Sod2 activity during the same period. To test the requirement of dietary manganese for this accumulation, we generated a Drosophila model of manganese deficiency. Due to the lack of manganese-specific chelators, we used chemically defined media to grow the flies and deplete them of the metal. Dietary manganese depletion reduced Sod2 activity. We then examined gene and protein expression changes in the intestines of manganese depleted flies. We found adaptive responses to the presumed loss of known manganese-dependent enzymatic activities: less glutamine synthase activity (amination of glutamate to glutamine) was compensated by 50% reduction in glutaminase (deamination of glutamine to glutamate); less glycosyltransferase activity, predicted to reduce protein glycosylation, was compensated by 30% reduction in lysosomal mannosidases (protein deglycosylating enzymes); less ceramide phosphoethanolamine synthase activity was compensated by 30% reduction in the Drosophila sphingomyeline phospodiesterase, which could catabolize ceramide phosphoethanolamine in flies. Reduced Sod2 activity, predicted to cause superoxide-dependent iron-sulphur cluster damage, resulted in cellular iron misregulation. %B Metallomics %V 12 %P 218-240 %8 2020 Feb 01 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/31799578?dopt=Abstract %R 10.1039/c9mt00218a %0 Journal Article %J Proc Natl Acad Sci U S A %D 2020 %T A cell atlas of the adult midgut %A Hung, Ruei-Jiun %A Hu, Yanhui %A Rory Kirchner %A Liu, Yifang %A Xu, Chiwei %A Comjean, Aram %A Tattikota, Sudhir Gopal %A Li, Fangge %A Song, Wei %A Ho Sui, Shannan %A Perrimon, Norbert %K Animals %K Digestive System %K Drosophila %K Drosophila Proteins %K Enterocytes %K Enteroendocrine Cells %K Epithelial Cells %K Epithelium %K Gene Expression Regulation %K Hormones %K Intestines %K Stem Cells %K Transcription Factors %K Transcriptome %X Studies of the adult midgut have led to many insights in our understanding of cell-type diversity, stem cell regeneration, tissue homeostasis, and cell fate decision. Advances in single-cell RNA sequencing provide opportunities to identify new cell types and molecular features. We used single-cell RNA sequencing to characterize the transcriptome of midgut epithelial cells and identified 22 distinct clusters representing intestinal stem cells, enteroblasts, enteroendocrine cells (EEs), and enterocytes. This unbiased approach recovered most of the known intestinal stem cells/enteroblast and EE markers, highlighting the high quality of the dataset, and led to insights on intestinal stem cell biology, cell type-specific organelle features, the roles of new transcription factors in progenitors and regional variation along the gut, 5 additional EE gut hormones, EE hormonal expression diversity, and paracrine function of EEs. To facilitate mining of this rich dataset, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing functions of genes in the midgut epithelium. %B Proc Natl Acad Sci U S A %V 117 %P 1514-1523 %8 2020 01 21 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/31915294?dopt=Abstract %R 10.1073/pnas.1916820117 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2020 %T An in vivo RNAi screen uncovers the role of AdoR signaling and adenosine deaminase in controlling intestinal stem cell activity %A Xu, Chiwei %A Franklin, Brian %A Tang, Hong-Wen %A Regimbald-Dumas, Yannik %A Hu, Yanhui %A Ramos, Justine %A Bosch, Justin A %A Villalta, Christians %A He, Xi %A Perrimon, Norbert %X Metabolites are increasingly appreciated for their roles as signaling molecules. To dissect the roles of metabolites, it is essential to understand their signaling pathways and their enzymatic regulations. From an RNA interference (RNAi) screen for regulators of intestinal stem cell (ISC) activity in the midgut, we identified () as a top candidate gene required for ISC proliferation. We demonstrate that Ras/MAPK and Protein Kinase A (PKA) signaling act downstream of AdoR and that Ras/MAPK mediates the major effect of AdoR on ISC proliferation. Extracellular adenosine, the ligand for AdoR, is a small metabolite that can be released by various cell types and degraded in the extracellular space by secreted adenosine deaminase. Interestingly, down-regulation of () from enterocytes is necessary for extracellular adenosine to activate AdoR and induce ISC overproliferation. As expression and its enzymatic activity decrease following tissue damage, our study provides important insights into how the enzymatic regulation of extracellular adenosine levels under tissue-damage conditions facilitates ISC proliferation. %B Proc Natl Acad Sci U S A %V 117 %P 464-471 %8 2020 01 07 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/31852821?dopt=Abstract %R 10.1073/pnas.1900103117 %0 Journal Article %J Genetics %D 2020 %T Large-Scale Transgenic Resource Collections for Loss- and Gain-of-Function Studies %A Zirin, Jonathan %A Hu, Yanhui %A Liu, Luping %A Yang-Zhou, Donghui %A Colbeth, Ryan %A Yan, Dong %A Ewen-Campen, Ben %A Tao, Rong %A Vogt, Eric %A VanNest, Sara %A Cavers, Cooper %A Villalta, Christians %A Comjean, Aram %A Sun, Jin %A Wang, Xia %A Jia, Yu %A Zhu, Ruibao %A Peng, Ping %A Yu, Jinchao %A Shen, Da %A Qiu, Yuhao %A Ayisi, Limmond %A Ragoowansi, Henna %A Fenton, Ethan %A Efrem, Senait %A Parks, Annette %A Saito, Kuniaki %A Kondo, Shu %A Perkins, Liz %A Mohr, Stephanie E %A Ni, Jianquan %A Perrimon, Norbert %X The Transgenic RNAi Project (TRiP), a functional genomics platform at Harvard Medical School, was initiated in 2008 to generate and distribute a genome-scale collection of RNA interference (RNAi) fly stocks. To date, it has generated >15,000 RNAi fly stocks. As this covers most genes, we have largely transitioned to development of new resources based on CRISPR technology. Here, we present an update on our libraries of publicly available RNAi and CRISPR fly stocks, and focus on the TRiP-CRISPR overexpression (TRiP-OE) and TRiP-CRISPR knockout (TRiP-KO) collections. TRiP-OE stocks express single guide RNAs targeting upstream of a gene transcription start site. Gene activation is triggered by coexpression of catalytically dead Cas9 fused to an activator domain, either VP64-p65-Rta or Synergistic Activation Mediator. TRiP-KO stocks express one or two single guide RNAs targeting the coding sequence of a gene or genes. Cutting is triggered by coexpression of Cas9, allowing for generation of indels in both germline and somatic tissue. To date, we have generated >5000 TRiP-OE or TRiP-KO stocks for the community. These resources provide versatile, transformative tools for gene activation, gene repression, and genome engineering. %B Genetics %V 214 %P 755-767 %8 2020 04 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/32071193?dopt=Abstract %R 10.1534/genetics.119.302964 %0 Journal Article %J Curr Protoc Mol Biol %D 2020 %T Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes %A Bosch, Justin A %A Knight, Shannon %A Kanca, Oguz %A Zirin, Jonathan %A Yang-Zhou, Donghui %A Hu, Yanhui %A Rodiger, Jonathan %A Amador, Gabriel %A Bellen, Hugo J %A Perrimon, Norbert %A Mohr, Stephanie E %K Animals %K Cells, Cultured %K Clustered Regularly Interspaced Short Palindromic Repeats %K CRISPR-Cas Systems %K DNA, Single-Stranded %K Drosophila %K Gene Editing %K Gene Knock-In Techniques %K Genes, Insect %K Green Fluorescent Proteins %K Open Reading Frames %K Plasmids %K RNA, Guide %K Transfection %X The CRISPR-Cas9 system makes it possible to cause double-strand breaks in specific regions, inducing repair. In the presence of a donor construct, repair can involve insertion or 'knock-in' of an exogenous cassette. One common application of knock-in technology is to generate cell lines expressing fluorescently tagged endogenous proteins. The standard approach relies on production of a donor plasmid with ∼500 to 1000 bp of homology on either side of an insertion cassette that contains the fluorescent protein open reading frame (ORF). We present two alternative methods for knock-in of fluorescent protein ORFs into Cas9-expressing Drosophila S2R+ cultured cells, the single-stranded DNA (ssDNA) Drop-In method and the CRISPaint universal donor method. Both methods eliminate the need to clone a large plasmid donor for each target. We discuss the advantages and limitations of the standard, ssDNA Drop-In, and CRISPaint methods for fluorescent protein tagging in Drosophila cultured cells. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Knock-in into Cas9-positive S2R+ cells using the ssDNA Drop-In approach Basic Protocol 2: Knock-in into Cas9-positive S2R+ cells by homology-independent insertion of universal donor plasmids that provide mNeonGreen (CRISPaint method) Support Protocol 1: sgRNA design and cloning Support Protocol 2: ssDNA donor synthesis Support Protocol 3: Transfection using Effectene Support Protocol 4: Electroporation of S2R+-MT::Cas9 Drosophila cells Support Protocol 5: Single-cell isolation of fluorescent cells using FACS. %B Curr Protoc Mol Biol %V 130 %P e112 %8 2020 03 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/31869524?dopt=Abstract %R 10.1002/cpmb.112 %0 Journal Article %J Genetics %D 2020 %T Gene Knock-Ins in Using Homology-Independent Insertion of Universal Donor Plasmids %A Bosch, Justin A %A Colbeth, Ryan %A Zirin, Jonathan %A Perrimon, Norbert %X Targeted genomic knock-ins are a valuable tool to probe gene function. However, knock-in methods involving homology-directed repair (HDR) can be laborious. Here, we adapt the mammalian CRISPaint [clustered regularly interspaced short palindromic repeat (CRISPR)-assisted insertion tagging] homology-independent knock-in method for , which uses CRISPR/Cas9 and nonhomologous end joining to insert "universal" donor plasmids into the genome. Using this method in cultured S2R+ cells, we efficiently tagged four endogenous proteins with the bright fluorescent protein mNeonGreen, thereby demonstrating that an existing collection of CRISPaint universal donor plasmids is compatible with insect cells. In addition, we inserted the transgenesis marker into seven genes in the fly germ line, producing heritable loss-of-function alleles that were isolated by simple fluorescence screening. Unlike in cultured cells, insertions/deletions always occurred at the genomic insertion site, which prevents predictably matching the insert coding frame to the target gene. Despite this effect, we were able to isolate insertions in four genes that serve as expression reporters. Therefore, homology-independent insertion in is a fast and simple alternative to HDR that will enable researchers to dissect gene function. %B Genetics %V 214 %P 75-89 %8 2020 01 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/31685521?dopt=Abstract %R 10.1534/genetics.119.302819 %0 Journal Article %J Nucleic Acids Res %D 2020 %T Alliance of Genome Resources Portal: unified model organism research platform %A Alliance-of-Genome-Resources-Consortium %X The Alliance of Genome Resources (Alliance) is a consortium of the major model organism databases and the Gene Ontology that is guided by the vision of facilitating exploration of related genes in human and well-studied model organisms by providing a highly integrated and comprehensive platform that enables researchers to leverage the extensive body of genetic and genomic studies in these organisms. Initiated in 2016, the Alliance is building a central portal (www.alliancegenome.org) for access to data for the primary model organisms along with gene ontology data and human data. All data types represented in the Alliance portal (e.g. genomic data and phenotype descriptions) have common data models and workflows for curation. All data are open and freely available via a variety of mechanisms. Long-term plans for the Alliance project include a focus on coverage of additional model organisms including those without dedicated curation communities, and the inclusion of new data types with a particular focus on providing data and tools for the non-model-organism researcher that support enhanced discovery about human health and disease. Here we review current progress and present immediate plans for this new bioinformatics resource. %B Nucleic Acids Res %8 2019 Sep 25 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31552413?dopt=Abstract %R 10.1093/nar/gkz813 %0 Journal Article %J Elife %D 2019 %T Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation %A Ryoji Amamoto %A Garcia, Mauricio D %A West, Emma R %A Choi, Jiho %A Lapan, Sylvain W %A Lane, Elizabeth A %A Perrimon, Norbert %A Cepko, Constance L %X Recent transcriptional profiling technologies are uncovering previously-undefined cell populations and molecular markers at an unprecedented pace. While single cell RNA (scRNA) sequencing is an attractive approach for unbiased transcriptional profiling of all cell types, a complementary method to isolate and sequence specific cell populations from heterogeneous tissue remains challenging. Here, we developed Probe-Seq, which allows deep transcriptional profiling of specific cell types isolated using RNA as the defining feature. Dissociated cells are labeled using fluorescent in situ hybridization (FISH) for RNA, and then isolated by fluorescent activated cell sorting (FACS). We used Probe-Seq to purify and profile specific cell types from mouse, human, and chick retinas, as well as from midguts. Probe-Seq is compatible with frozen nuclei, making cell types within archival tissue immediately accessible. As it can be multiplexed, combinations of markers can be used to create specificity. Multiplexing also allows for the isolation of multiple cell types from one cell preparation. Probe-Seq should enable RNA profiling of specific cell types from any organism. %B Elife %V 8 %8 2019 12 09 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31815670?dopt=Abstract %R 10.7554/eLife.51452 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2019 %T The role of translationally controlled tumor protein in proliferation of intestinal stem cells %A Kwon, Young V %A Zhao, Bingqing %A Xu, Chiwei %A Lee, Jiae %A Chen, Chiao-Lin %A Vinayagam, Arunachalam %A Edgar, Bruce A %A Perrimon, Norbert %X Translationally controlled tumor protein (TCTP) is a highly conserved protein functioning in multiple cellular processes, ranging from growth to immune responses. To explore the role of TCTP in tissue maintenance and regeneration, we employed the adult midgut, where multiple signaling pathways interact to precisely regulate stem cell division for tissue homeostasis. Tctp levels were significantly increased in stem cells and enteroblasts upon tissue damage or activation of the Hippo pathway that promotes regeneration of intestinal epithelium. Stem cells with reduced Tctp levels failed to proliferate during normal tissue homeostasis and regeneration. Mechanistically, Tctp forms a complex with multiple proteins involved in translation and genetically interacts with ribosomal subunits. In addition, Tctp increases both Akt1 protein abundance and phosphorylation in vivo. Altogether, Tctp regulates stem cell proliferation by interacting with key growth regulatory signaling pathways and the translation process in vivo. %B Proc Natl Acad Sci U S A %8 2019 Dec 16 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31843907?dopt=Abstract %R 10.1073/pnas.1910850116 %0 Journal Article %J Genetics %D 2019 %T The Alliance of Genome Resources: Building a Modern Data Ecosystem for Model Organism Databases %A AGRC %X Model organisms are essential experimental platforms for discovering gene functions, defining protein and genetic networks, uncovering functional consequences of human genome variation, and for modeling human disease. For decades, researchers who use model organisms have relied on Model Organism Databases (MODs) and the Gene Ontology Consortium (GOC) for expertly curated annotations, and for access to integrated genomic and biological information obtained from the scientific literature and public data archives. Through the development and enforcement of data and semantic standards, these genome resources provide rapid access to the collected knowledge of model organisms in human readable and computation-ready formats that would otherwise require countless hours for individual researchers to assemble on their own. Since their inception, the MODs for the predominant biomedical model organisms [ (laboratory mouse), , , , , and ] along with the GOC have operated as a network of independent, highly collaborative genome resources. In 2016, these six MODs and the GOC joined forces as the Alliance of Genome Resources (the Alliance). By implementing shared programmatic access methods and data-specific web pages with a unified "look and feel," the Alliance is tackling barriers that have limited the ability of researchers to easily compare common data types and annotations across model organisms. To adapt to the rapidly changing landscape for evaluating and funding core data resources, the Alliance is building a modern, extensible, and operationally efficient "knowledge commons" for model organisms using shared, modular infrastructure. %B Genetics %V 213 %P 1189-1196 %8 2019 Dec %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/31796553?dopt=Abstract %R 10.1534/genetics.119.302523 %0 Journal Article %J Current Protocols in Molecular Biology %D 2019 %T Pooled CRISPR Screens in Drosophila Cells %A Viswanatha, R %A Brathwaite, R %A Hu, Y. %A Li, Z. %A Rodiger, J %A Merckaert, P %A Chung, V %A Mohr, SE %A Perrimon, N %XHigh-throughput screens in Drosophila melanogaster cell lines have led to discovery of conserved gene functions related to signal transduction, host pathogen interactions, ion transport, and more. CRISPR/Cas9 technology has opened the door to new types of large-scale cell-based screens. Whereas array format screens require liquid handling automation and assay miniaturization, pooled-format screens, in which reagents are introduced at random and in bulk, can be done in a standard lab setting. We provide a detailed protocol for conducting and evaluating genome-wide CRISPR single guide RNA (sgRNA) pooled screens in Drosophila S2R+ cultured cells. Specifically, we provide step-by-step instructions for library design and production, optimization of cytotoxin-based selection assays, genome-scale screening, and data analysis. This type of project takes ~3 months to complete. Results can be used in follow-up studies performed in vivo in Drosophila, mammalian cells, and/or other systems.
%B Current Protocols in Molecular Biology %V 129 %G eng %0 Journal Article %J Elife %D 2019 %T An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms %A Kanca, Oguz %A Zirin, Jonathan %A Garcia-Marques, Jorge %A Knight, Shannon Marie %A Yang-Zhou, Donghui %A Amador, Gabriel %A Chung, Hyunglok %A Zuo, Zhongyuan %A Ma, Liwen %A He, Yuchun %A Lin, Wen-Wen %A Ying Fang %A Ge, Ming %A Yamamoto, Shinya %A Schulze, Karen L %A Hu, Yanhui %A Spradling, Allan C %A Mohr, Stephanie E %A Perrimon, Norbert %A Bellen, Hugo J %X We previously reported a CRISPR-mediated knock-in strategy into introns of genes, generating an - transgenic library for multiple uses (Lee et al., 2018b). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely . The approach is fast, cheap, and scalable. %B Elife %V 8 %8 2019 Nov 01 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31674908?dopt=Abstract %R 10.7554/eLife.51539 %0 Journal Article %J Sci Signal %D 2019 %T HIF-independent synthetic lethality between CDK4/6 inhibition and VHL loss across species %A Nicholson, Hilary E %A Tariq, Zeshan %A Housden, Benjamin E %A Jennings, Rebecca B %A Stransky, Laura A %A Perrimon, Norbert %A Signoretti, Sabina %A Kaelin, William G %X Inactivation of the tumor suppressor gene is the signature initiating event in clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, and causes the accumulation of hypoxia-inducible factor 2α (HIF-2α). HIF-2α inhibitors are effective in some ccRCC cases, but both de novo and acquired resistance have been observed in the laboratory and in the clinic. Here, we identified synthetic lethality between decreased activity of cyclin-dependent kinases 4 and 6 (CDK4/6) and inactivation in two species (human and ) and across diverse human ccRCC cell lines in culture and xenografts. Although HIF-2α transcriptionally induced the CDK4/6 partner cyclin D1, HIF-2α was not required for the increased CDK4/6 requirement of ccRCC cells. Accordingly, the antiproliferative effects of CDK4/6 inhibition were synergistic with HIF-2α inhibition in HIF-2α-dependent ccRCC cells and not antagonistic with HIF-2α inhibition in HIF-2α-independent cells. These findings support testing CDK4/6 inhibitors as treatments for ccRCC, alone and in combination with HIF-2α inhibitors. %B Sci Signal %V 12 %8 2019 Oct 01 %G eng %N 601 %1 http://www.ncbi.nlm.nih.gov/pubmed/31575731?dopt=Abstract %R 10.1126/scisignal.aay0482 %0 Journal Article %J Dis Model Mech %D 2019 %T Drosophila melanogaster: a simple system for understanding complexity %A Mohr, Stephanie E %A Perrimon, Norbert %X Understanding human gene function is fundamental to understanding and treating diseases. Research using the model organism benefits from a wealth of molecular genetic resources and information useful for efficient experimentation. Moreover, offers a balance as a relatively simple organism that nonetheless exhibits complex multicellular activities. Recent examples demonstrate the power and continued promise of research to further our understanding of conserved gene functions. %B Dis Model Mech %V 12 %8 2019 Sep 27 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/31562251?dopt=Abstract %R 10.1242/dmm.041871 %0 Journal Article %J Adv Exp Med Biol %D 2019 %T Drosophila as a Model for Tumor-Induced Organ Wasting %A Saavedra, Pedro %A Perrimon, Norbert %K Animals %K Cachexia %K Disease Models, Animal %K Drosophila %K Humans %K Muscle, Skeletal %K Neoplasms %K Quality of Life %X In humans, cancer-associated cachexia is a complex syndrome that reduces the overall quality of life and survival of cancer patients, particularly for those undergoing chemotherapy. The most easily observable sign of cachexia is organ wasting, the dramatic loss of skeletal muscle and adipose tissue mass. Estimates suggest that 80% of patients in advanced stages of cancer show signs of the syndrome and about 20% of cancer patients die directly of cachexia. Because there is no treatment or drug available to ameliorate organ wasting induced by cancer, cachexia is a relevant clinical problem. However, it is unclear how cachexia is mediated, what factors drive interactions between tumors and host tissues, and which markers of cachexia might be used to allow early detection before the observable signs of organ wasting. In this chapter, we review the current mammalian models of cachexia and the need to use new models of study. We also explain recent developments in Drosophila as a model for studying organ wasting induced by tumors and how fly studies can help unravel important mechanisms that drive cachexia. In particular, we discuss what lessons have been learned from tumor models recently reported to induce systemic organ wasting in Drosophila. %B Adv Exp Med Biol %V 1167 %P 191-205 %8 2019 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31520356?dopt=Abstract %R 10.1007/978-3-030-23629-8_11 %0 Journal Article %J Aging Cell %D 2019 %T Methionine metabolism and methyltransferases in the regulation of aging and lifespan extension across species %A Parkhitko, Andrey A %A Jouandin, Patrick %A Mohr, Stephanie E %A Perrimon, Norbert %X Methionine restriction (MetR) extends lifespan across different species and exerts beneficial effects on metabolic health and inflammatory responses. In contrast, certain cancer cells exhibit methionine auxotrophy that can be exploited for therapeutic treatment, as decreasing dietary methionine selectively suppresses tumor growth. Thus, MetR represents an intervention that can extend lifespan with a complementary effect of delaying tumor growth. Beyond its function in protein synthesis, methionine feeds into complex metabolic pathways including the methionine cycle, the transsulfuration pathway, and polyamine biosynthesis. Manipulation of each of these branches extends lifespan; however, the interplay between MetR and these branches during regulation of lifespan is not well understood. In addition, a potential mechanism linking the activity of methionine metabolism and lifespan is regulation of production of the methyl donor S-adenosylmethionine, which, after transferring its methyl group, is converted to S-adenosylhomocysteine. Methylation regulates a wide range of processes, including those thought to be responsible for lifespan extension by MetR. Although the exact mechanisms of lifespan extension by MetR or methionine metabolism reprogramming are unknown, it may act via reducing the rate of translation, modifying gene expression, inducing a hormetic response, modulating autophagy, or inducing mitochondrial function, antioxidant defense, or other metabolic processes. Here, we review the mechanisms of lifespan extension by MetR and different branches of methionine metabolism in different species and the potential for exploiting the regulation of methyltransferases to delay aging. %B Aging Cell %P e13034 %8 2019 Aug 28 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31460700?dopt=Abstract %R 10.1111/acel.13034 %0 Journal Article %J Dev Cell %D 2019 %T The Multidimensional Organization of Interorgan Communication Networks %A Droujinine, Ilia A %A Perrimon, Norbert %X Secreted molecules coordinate organ function. In a recent issue of Cell, Hudry et al. (2019) uncover a Drosophila testis-midgut interaction via cytokine and citrate signaling that regulates intestinal metabolism, spermatogenesis, and food intake. This impressive study is a striking example of the role of spatial organization in sex-specific interorgan communication. %B Dev Cell %V 50 %P 395-396 %8 2019 Aug 19 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/31430450?dopt=Abstract %R 10.1016/j.devcel.2019.07.029 %0 Journal Article %J J Cell Biol %D 2019 %T Apical polarity proteins recruit the RhoGEF Cysts to promote junctional myosin assembly %A Silver, Jordan T %A Wirtz-Peitz, Frederik %A Simões, Sérgio %A Pellikka, Milena %A Yan, Dong %A Binari, Richard %A Nishimura, Takashi %A Li, Yan %A Harris, Tony J C %A Perrimon, Norbert %A Tepass, Ulrich %X The spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo, we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resembles the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins, suggesting that Cysts is an integral component of the apical polarity protein network. We demonstrate that Cysts is recruited to the apico-lateral membrane through interactions with the Crumbs complex and Bazooka/Par3. Cysts activates Rho1 at adherens junctions and stabilizes junctional myosin. Junctional myosin depletion is similar in Cysts- and Crumbs-compromised embryos. Together, our findings indicate that Cysts is a downstream effector of the Crumbs complex and links apical polarity proteins to Rho1 and myosin activation at adherens junctions, supporting junctional integrity and epithelial polarity. %B J Cell Biol %8 2019 Aug 13 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31409654?dopt=Abstract %R 10.1083/jcb.201807106 %0 Journal Article %J Wiley Interdiscip Rev Dev Biol %D 2019 %T Regulation of insulin and adipokinetic hormone/glucagon production in flies %A Ahmad, Muhammad %A He, Li %A Perrimon, Norbert %X Metabolic homeostasis is under strict regulation of humoral factors across various taxa. In particular, insulin and glucagon, referred to in Drosophila as Drosophila insulin-like peptides (DILPs) and adipokinetic hormone (AKH), respectively, are key hormones that regulate metabolism in most metazoa. While much is known about the regulation of DILPs, the mechanisms regulating AKH/glucagon production is still poorly understood. In this review, we describe the various factors that regulate the production of DILPs and AKH and emphasize the need for future studies to decipher how energy homeostasis is governed in Drosophila. This article is categorized under: Invertebrate Organogenesis > Flies Signaling Pathways > Global Signaling Mechanisms. %B Wiley Interdiscip Rev Dev Biol %P e360 %8 2019 Aug 04 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31379062?dopt=Abstract %R 10.1002/wdev.360 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2019 %T Interspecies analysis of MYC targets identifies tRNA synthetases as mediators of growth and survival in MYC-overexpressing cells %A Zirin, Jonathan %A Ni, Xiaochun %A Sack, Laura M %A Yang-Zhou, Donghui %A Hu, Yanhui %A Brathwaite, Roderick %A Bulyk, Martha L %A Elledge, Stephen J %A Perrimon, Norbert %X Aberrant MYC oncogene activation is one of the most prevalent characteristics of cancer. By overlapping datasets of genes that are insulin-responsive and also regulate nucleolus size, we enriched for Myc target genes required for cellular biosynthesis. Among these, we identified the aminoacyl tRNA synthetases (aaRSs) as essential mediators of Myc growth control in and found that their pharmacologic inhibition is sufficient to kill MYC-overexpressing human cells, indicating that aaRS inhibitors might be used to selectively target MYC-driven cancers. We suggest a general principle in which oncogenic increases in cellular biosynthesis sensitize cells to disruption of protein homeostasis. %B Proc Natl Acad Sci U S A %8 2019 Jul 01 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31262815?dopt=Abstract %R 10.1073/pnas.1821863116 %0 Journal Article %J Cell Metab %D 2019 %T An Evolutionarily Conserved uORF Regulates PGC1α and Oxidative Metabolism in Mice, Flies, and Bluefin Tuna %A Dumesic, Phillip A %A Egan, Daniel F %A Gut, Philipp %A Tran, Mei T %A Parisi, Alice %A Chatterjee, Nirmalya %A Jedrychowski, Mark %A Paschini, Margherita %A Kazak, Lawrence %A Wilensky, Sarah E %A Dou, Florence %A Bogoslavski, Dina %A Cartier, Jeffrey A %A Perrimon, Norbert %A Kajimura, Shingo %A Parikh, Samir M %A Spiegelman, Bruce M %X Mitochondrial abundance and function are tightly controlled during metabolic adaptation but dysregulated in pathological states such as diabetes, neurodegeneration, cancer, and kidney disease. We show here that translation of PGC1α, a key governor of mitochondrial biogenesis and oxidative metabolism, is negatively regulated by an upstream open reading frame (uORF) in the 5' untranslated region of its gene (PPARGC1A). We find that uORF-mediated translational repression is a feature of PPARGC1A orthologs from human to fly. Strikingly, whereas multiple inhibitory uORFs are broadly present in fish PPARGC1A orthologs, they are completely absent in the Atlantic bluefin tuna, an animal with exceptionally high mitochondrial content. In mice, an engineered mutation disrupting the PPARGC1A uORF increases PGC1α protein levels and oxidative metabolism and confers protection from acute kidney injury. These studies identify a translational regulatory element governing oxidative metabolism and highlight its potential contribution to the evolution of organismal mitochondrial function. %B Cell Metab %8 2019 Apr 30 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31105043?dopt=Abstract %R 10.1016/j.cmet.2019.04.013 %0 Journal Article %J Elife %D 2019 %T In vivo study of gene expression with an enhanced dual-color fluorescent transcriptional timer %A He, Li %A Binari, Richard %A Huang, Jiuhong %A Falo-Sanjuan, Julia %A Perrimon, Norbert %X Fluorescent transcriptional reporters are widely used as signaling reporters and biomarkers to monitor pathway activities and determine cell type identities. However, a large amount of dynamic information is lost due to the long half-life of the fluorescent proteins. To better detect dynamics, fluorescent transcriptional reporters can be destabilized to shorten their half-lives. However, applications of this approach are limited due to significant reduction of signal intensities. To overcome this limitation, we enhanced translation of a destabilized fluorescent protein and demonstrate the advantages of this approach by characterizing spatio-temporal changes of transcriptional activities in . In addition, by combining a fast-folding destabilized fluorescent protein and a slow-folding long-lived fluorescent protein, we generated a dual-color transcriptional timer that provides spatio-temporal information about signaling pathway activities. Finally, we demonstrate the use of this transcriptional timer to identify new genes with dynamic expression patterns. %B Elife %V 8 %8 2019 May 29 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/31140975?dopt=Abstract %R 10.7554/eLife.46181 %0 Journal Article %J Nat Commun %D 2019 %T Conserved phosphorylation hotspots in eukaryotic protein domain families %A Strumillo, Marta J %A Oplová, Michaela %A Viéitez, Cristina %A Ochoa, David %A Shahraz, Mohammed %A Busby, Bede P %A Sopko, Richelle %A Studer, Romain A %A Perrimon, Norbert %A Panse, Vikram G %A Beltrao, Pedro %X Protein phosphorylation is the best characterized post-translational modification that regulates almost all cellular processes through diverse mechanisms such as changing protein conformations, interactions, and localization. While the inventory for phosphorylation sites across different species has rapidly expanded, their functional role remains poorly investigated. Here, we combine 537,321 phosphosites from 40 eukaryotic species to identify highly conserved phosphorylation hotspot regions within domain families. Mapping these regions onto structural data reveals that they are often found at interfaces, near catalytic residues and tend to harbor functionally important phosphosites. Notably, functional studies of a phospho-deficient mutant in the C-terminal hotspot region within the ribosomal S11 domain in the yeast ribosomal protein uS11 shows impaired growth and defective cytoplasmic 20S pre-rRNA processing at 16 °C and 20 °C. Altogether, our study identifies phosphorylation hotspots for 162 protein domains suggestive of an ancient role for the control of diverse eukaryotic domain families. %B Nat Commun %V 10 %P 1977 %8 2019 04 29 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/31036831?dopt=Abstract %R 10.1038/s41467-019-09952-x %0 Journal Article %J BMC Biol %D 2019 %T A role for actomyosin contractility in Notch signaling %A Hunter, Ginger L %A He, Li %A Perrimon, Norbert %A Charras, Guillaume %A Giniger, Edward %A Baum, Buzz %X BACKGROUND: Notch-Delta signaling functions across a wide array of animal systems to break symmetry in a sheet of undifferentiated cells and generate cells with different fates, a process known as lateral inhibition. Unlike many other signaling systems, however, since both the ligand and receptor are transmembrane proteins, the activation of Notch by Delta depends strictly on cell-cell contact. Furthermore, the binding of the ligand to the receptor may not be sufficient to induce signaling, since recent work in cell culture suggests that ligand-induced Notch signaling also requires a mechanical pulling force. This tension exposes a cleavage site in Notch that, when cut, activates signaling. Although it is not known if mechanical tension contributes to signaling in vivo, others have suggested that this is how endocytosis of the receptor-ligand complex contributes to the cleavage and activation of Notch. In a similar way, since Notch-mediated lateral inhibition at a distance in the dorsal thorax of the pupal fly is mediated via actin-rich protrusions, it is possible that cytoskeletal forces generated by networks of filamentous actin and non-muscle myosin during cycles of protrusion extension and retraction also contribute to Notch signaling. RESULTS: To test this hypothesis, we carried out a detailed analysis of the role of myosin II-dependent tension in Notch signaling in the developing fly and in cell culture. Using dynamic fluorescence-based reporters of Notch, we found that myosin II is important for signaling in signal sending and receiving cells in both systems-as expected if myosin II-dependent tension across the Notch-Delta complex contributes to Notch activation. While myosin II was found to contribute most to signaling at a distance, it was also required for maximal signaling between adjacent cells that share lateral contacts and for signaling between cells in culture. CONCLUSIONS: Together these results reveal a previously unappreciated role for non-muscle myosin II contractility in Notch signaling, providing further support for the idea that force contributes to the cleavage and activation of Notch in the context of ligand-dependent signaling, and a new paradigm for actomyosin-based mechanosensation. %B BMC Biol %V 17 %P 12 %8 2019 02 11 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/30744634?dopt=Abstract %R 10.1186/s12915-019-0625-9 %0 Journal Article %J Exp Cell Res %D 2019 %T Mechanosensitive channels and their functions in stem cell differentiation %A He, Li %A Ahmad, Muhammad %A Perrimon, Norbert %X Stem cells continuously perceive and respond to various environmental signals during development, tissue homeostasis, and pathological conditions. Mechanical force, one of the fundamental signals in the physical world, plays a vital role in the regulation of multiple functions of stem cells. The importance of cell adhesion to the extracellular matrix (ECM), cell-cell junctions, and a mechanoresponsive cell cytoskeleton has been under intensive study in the fields of stem cell biology and mechanobiology. However, the involvement of mechanosensitive (MS) ion channels in the mechanical regulation of stem cell activity has just begun to be realized. Here, we review the diversity and importance of mechanosensitive channels (MSCs), and discuss recently discovered functions of MSCs in stem cell regulation, especially in the determination of cell fate. %B Exp Cell Res %V 374 %P 259-265 %8 2019 Jan 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/30500393?dopt=Abstract %R 10.1016/j.yexcr.2018.11.016 %0 Journal Article %J Cell Rep %D 2019 %T The Septate Junction Protein Tsp2A Restricts Intestinal Stem Cell Activity via Endocytic Regulation of aPKC and Hippo Signaling %A Xu, Chiwei %A Tang, Hong-Wen %A Hung, Ruei-Jiun %A Hu, Yanhui %A Ni, Xiaochun %A Housden, Benjamin E %A Perrimon, Norbert %X Hippo signaling and the activity of its transcriptional coactivator, Yorkie (Yki), are conserved and crucial regulators of tissue homeostasis. In the Drosophila midgut, after tissue damage, Yki activity increases to stimulate stem cell proliferation, but how Yki activity is turned off once the tissue is repaired is unknown. From an RNAi screen, we identified the septate junction (SJ) protein tetraspanin 2A (Tsp2A) as a tumor suppressor. Tsp2A undergoes internalization to facilitate the endocytic degradation of atypical protein kinase C (aPKC), a negative regulator of Hippo signaling. In the Drosophila midgut epithelium, adherens junctions (AJs) and SJs are prominent in intestinal stem cells or enteroblasts (ISCs or EBs) and enterocytes (ECs), respectively. We show that when ISCs differentiate toward ECs, Tsp2A is produced, participates in SJ assembly, and turns off aPKC and Yki-JAK-Stat activity. Altogether, our study uncovers a mechanism allowing the midgut to restore Hippo signaling and restrict proliferation once tissue repair is accomplished. %B Cell Rep %V 26 %P 670-688.e6 %8 2019 Jan 15 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/30650359?dopt=Abstract %R 10.1016/j.celrep.2018.12.079 %0 Journal Article %J Dev Cell %D 2019 %T Tumor-Derived Ligands Trigger Tumor Growth and Host Wasting via Differential MEK Activation %A Song, Wei %A Kir, Serkan %A Hong, Shangyu %A Hu, Yanhui %A Wang, Xiaohui %A Binari, Richard %A Tang, Hong-Wen %A Chung, Verena %A Banks, Alexander S %A Spiegelman, Bruce %A Perrimon, Norbert %X Interactions between tumors and host tissues play essential roles in tumor-induced systemic wasting and cancer cachexia, including muscle wasting and lipid loss. However, the pathogenic molecular mechanisms of wasting are still poorly understood. Using a fly model of tumor-induced organ wasting, we observed aberrant MEK activation in both tumors and host tissues of flies bearing gut-yki tumors. We found that host MEK activation results in muscle wasting and lipid loss, while tumor MEK activation is required for tumor growth. Strikingly, host MEK suppression alone is sufficient to abolish the wasting phenotypes without affecting tumor growth. We further uncovered that yki tumors produce the vein (vn) ligand to trigger autonomous Egfr/MEK-induced tumor growth and produce the PDGF- and VEGF-related factor 1 (Pvf1) ligand to non-autonomously activate host Pvr/MEK signaling and wasting. Altogether, our results demonstrate the essential roles and molecular mechanisms of differential MEK activation in tumor-induced host wasting. %B Dev Cell %V 48 %P 277-286.e6 %8 2019 Jan 28 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/30639055?dopt=Abstract %R 10.1016/j.devcel.2018.12.003 %0 Journal Article %J G3 (Bethesda) %D 2019 %T iProteinDB: An Integrative Database of Post-translational Modifications %A Hu, Yanhui %A Sopko, Richelle %A Chung, Verena %A Foos, Marianna %A Studer, Romain A %A Landry, Sean D %A Liu, Daniel %A Rabinow, Leonard %A Gnad, Florian %A Beltrao, Pedro %A Perrimon, Norbert %X Post-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing their stability, interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, most commonly serine, threonine and tyrosine in metazoans. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that any given phosphorylation site might be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for At iProteinDB, scientists can view the PTM landscape for any protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related species. Further, iProteinDB enables comparison of PTM data from to that of orthologous proteins from other model organisms, including human, mouse, rat, , and %B G3 (Bethesda) %V 9 %P 1-11 %8 2018 Nov 05 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/30397019?dopt=Abstract %R 10.1534/g3.118.200637 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2018 %T Drosophila intestinal stem and progenitor cells are major sources and regulators of homeostatic niche signals %A Doupé, David P %A Marshall, Owen J %A Dayton, Hannah %A Brand, Andrea H %A Perrimon, Norbert %X Epithelial homeostasis requires the precise balance of epithelial stem/progenitor proliferation and differentiation. While many signaling pathways that regulate epithelial stem cells have been identified, it is probable that other regulators remain unidentified. Here, we use gene-expression profiling by targeted DamID to identify the stem/progenitor-specific transcription and signaling factors in the midgut. Many signaling pathway components, including ligands of most major pathways, exhibit stem/progenitor-specific expression and have regulatory regions bound by both intrinsic and extrinsic transcription factors. In addition to previously identified stem/progenitor-derived ligands, we show that both the insulin-like factor Ilp6 and TNF ligand eiger are specifically expressed in the stem/progenitors and regulate normal tissue homeostasis. We propose that intestinal stem cells not only integrate multiple signals but also contribute to and regulate the homeostatic signaling microenvironmental niche through the expression of autocrine and paracrine factors. %B Proc Natl Acad Sci U S A %V 115 %P 12218-12223 %8 2018 11 27 %G eng %N 48 %1 http://www.ncbi.nlm.nih.gov/pubmed/30404917?dopt=Abstract %R 10.1073/pnas.1719169115 %0 Journal Article %J Stem Cell Reports %D 2018 %T Intestinal Stem Cells Exhibit Conditional Circadian Clock Function %A Parasram, Kathyani %A Bernardon, Nathaniel %A Hammoud, Maha %A Chang, Hanna %A He, Li %A Perrimon, Norbert %A Karpowicz, Phillip %X The circadian clock is a molecular pacemaker that produces 24-hr physiological cycles known as circadian rhythms. How the clock regulates stem cells is an emerging area of research with many outstanding questions. We tested clock function in vivo at the single cell resolution in the Drosophila intestine, a tissue that is exquisitely sensitive to environmental cues and has circadian rhythms in regeneration. Our results indicate that circadian clocks function in intestinal stem cells and enterocytes but are downregulated during enteroendocrine cell differentiation. Drosophila intestinal cells are principally synchronized by the photoperiod, but intestinal stem cell clocks are highly responsive to signaling pathways that comprise their niche, and we find that the Wnt and Hippo signaling pathways positively regulate stem cell circadian clock function. These data reveal that intestinal stem cell circadian rhythms are regulated by cellular signaling and provide insight as to how clocks may be altered during physiological changes such as regeneration and aging. %B Stem Cell Reports %V 11 %P 1287-1301 %8 2018 Nov 13 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/30428387?dopt=Abstract %R 10.1016/j.stemcr.2018.10.010 %0 Journal Article %J Nucleic Acids Res %D 2018 %T FlyBase 2.0: the next generation %A Thurmond, Jim %A Goodman, Joshua L %A Strelets, Victor B %A Attrill, Helen %A Gramates, L Sian %A Marygold, Steven J %A Matthews, Beverley B %A Millburn, Gillian %A Antonazzo, Giulia %A Trovisco, Vitor %A Kaufman, Thomas C %A Calvi, Brian R %A FlyBase Consortium %X FlyBase (flybase.org) is a knowledge base that supports the community of researchers that use the fruit fly, Drosophila melanogaster, as a model organism. The FlyBase team curates and organizes a diverse array of genetic, molecular, genomic, and developmental information about Drosophila. At the beginning of 2018, 'FlyBase 2.0' was released with a significantly improved user interface and new tools. Among these important changes are a new organization of search results into interactive lists or tables (hitlists), enhanced reference lists, and new protein domain graphics. An important new data class called 'experimental tools' consolidates information on useful fly strains and other resources related to a specific gene, which significantly enhances the ability of the Drosophila researcher to design and carry out experiments. With the release of FlyBase 2.0, there has also been a restructuring of backend architecture and a continued development of application programming interfaces (APIs) for programmatic access to FlyBase data. In this review, we describe these major new features and functionalities of the FlyBase 2.0 site and how they support the use of Drosophila as a model organism for biological discovery and translational research. %B Nucleic Acids Res %8 2018 Oct 26 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/30364959?dopt=Abstract %R 10.1093/nar/gky1003 %0 Journal Article %J Dev Cell %D 2018 %T A Membrane Transporter Is Required for Steroid Hormone Uptake in Drosophila %A Okamoto, Naoki %A Viswanatha, Raghuvir %A Bittar, Riyan %A Li, Zhongchi %A Haga-Yamanaka, Sachiko %A Perrimon, Norbert %A Yamanaka, Naoki %X Steroid hormones are a group of lipophilic hormones that are believed to enter cells by simple diffusion to regulate diverse physiological processes through intracellular nuclear receptors. Here, we challenge this model in Drosophila by demonstrating that Ecdysone Importer (EcI), a membrane transporter identified from two independent genetic screens, is involved in cellular uptake of the steroid hormone ecdysone. EcI encodes an organic anion transporting polypeptide of the evolutionarily conserved solute carrier organic anion superfamily. In vivo, EcI loss of function causes phenotypes indistinguishable from ecdysone- or ecdysone receptor (EcR)-deficient animals, and EcI knockdown inhibits cellular uptake of ecdysone. Furthermore, EcI regulates ecdysone signaling in a cell-autonomous manner and is both necessary and sufficient for inducing ecdysone-dependent gene expression in culture cells expressing EcR. Altogether, our results challenge the simple diffusion model for cellular uptake of ecdysone and may have wide implications for basic and medical aspects of steroid hormone studies. %B Dev Cell %8 2018 Oct 01 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/30293839?dopt=Abstract %R 10.1016/j.devcel.2018.09.012 %0 Journal Article %J Bioessays %D 2018 %T Endocrine Regulation of Energy Balance by Drosophila TGF-β/Activins %A Song, Wei %A Ghosh, Arpan C %A Cheng, Daojun %A Perrimon, Norbert %X The Transforming growth factor beta (TGF-β) family of secreted proteins regulates a variety of key events in normal development and physiology. In mammals, this family, represented by 33 ligands, including TGF-β, activins, nodal, bone morphogenetic proteins (BMPs), and growth and differentiation factors (GDFs), regulate biological processes as diverse as cell proliferation, differentiation, apoptosis, metabolism, homeostasis, immune response, wound repair, and endocrine functions. In Drosophila, only 7 members of this family are present, with 4 TGF-β/BMP and 3 TGF-β/activin ligands. Studies in the fly have illustrated the role of TGF-β/BMP ligands during embryogenesis and organ patterning, while the TGF-β/activin ligands have been implicated in the control of wing growth and neuronal functions. In this review, we focus on the emerging roles of Drosophila TGF-β/activins in inter-organ communication via long-distance regulation, especially in systemic lipid and carbohydrate homeostasis, and discuss findings relevant to metabolic diseases in humans. %B Bioessays %8 2018 Sep 28 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/30264417?dopt=Abstract %R 10.1002/bies.201800044 %0 Journal Article %J Nat Biotechnol %D 2018 %T Efficient proximity labeling in living cells and organisms with TurboID %A Branon, Tess C %A Bosch, Justin A %A Sanchez, Ariana D %A Udeshi, Namrata D %A Svinkina, Tanya %A Carr, Steven A %A Feldman, Jessica L %A Perrimon, Norbert %A Ting, Alice Y %X Protein interaction networks and protein compartmentalization underlie all signaling and regulatory processes in cells. Enzyme-catalyzed proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, current PL methods require over 18 h of labeling time or utilize chemicals with limited cell permeability or high toxicity. We used yeast display-based directed evolution to engineer two promiscuous mutants of biotin ligase, TurboID and miniTurbo, which catalyze PL with much greater efficiency than BioID or BioID2, and enable 10-min PL in cells with non-toxic and easily deliverable biotin. Furthermore, TurboID extends biotin-based PL to flies and worms. %B Nat Biotechnol %8 2018 Aug 20 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/30125270?dopt=Abstract %R 10.1038/nbt.4201 %0 Journal Article %J Current Opinion in Systems Biology %D 2018 %T Understanding cellular signaling and systems biology with precision: A perspective from ultrastructure and organelle studies in the Drosophila midgut %A Xu, Chiwei %A Ericsson, Maria %A Perrimon, Norbert %XThe adult Drosophila midgut is a complex tissue with various cell types that interact closely to maintain tissue integrity and perform organ function. The gut consists of a pseudostratified epithelium, a latticework of circular and longitudinal visceral muscles that supports the epithelium, and a tracheal vascular system. The major cell types of the midgut epithelium are the absorptive enterocytes (ECs), characterized by a large nucleus and microvilli-covered luminal surface, the enteroendocrine cells (EEs) that produce various hormones, and the intestinal stem cells (ISCs) that produce ECs and EEs [1,2] . Interactions between these cell types are critical to maintaining tissue integrity and gut function. For example, ISCs proliferation and differentiation are controlled by a complex network integrating autocrine and paracrine signals [3,4] ; hormones derived from EEs regulate EC physiology; and EC-derived factors signal to ISCs following gut damage.
%B Current Opinion in Systems Biology %P 24-31 %G eng %U https://www.sciencedirect.com/science/article/pii/S2452310018300404?via%3Dihub %N 11 %0 Journal Article %J Elife %D 2018 %T Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in cells %A Viswanatha, Raghuvir %A Li, Zhongchi %A Hu, Yanhui %A Perrimon, Norbert %X Genome-wide screens in cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for , including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in invertebrate cells. %B Elife %V 7 %8 2018 07 27 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/30051818?dopt=Abstract %R 10.7554/eLife.36333 %0 Journal Article %J G3 (Bethesda) %D 2018 %T ovoD Co-selection: A Method for Enriching CRISPR/Cas9-Edited Alleles in Drosophila %A Ewen-Campen, Ben %A Perrimon, Norbert %X Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, In this system (" co-selection"), the only functional germ cells in injected females are those that have been edited at the locus, and thus all offspring of these flies have undergone editing of at least one locus. We demonstrate that co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that co-selection reduces the amount of screening necessary to isolate desired CRISPR events in %B G3 (Bethesda) %8 2018 Jun 22 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29934375?dopt=Abstract %R 10.1534/g3.118.200498 %0 Journal Article %J J Clin Invest %D 2018 %T Blocking p62/SQSTM1-dependent SMN degradation ameliorates Spinal Muscular Atrophy disease phenotypes %A Rodriguez-Muela, Natalia %A Parkhitko, Andrey %A Grass, Tobias %A Gibbs, Rebecca M %A Norabuena, Erika M %A Perrimon, Norbert %A Singh, Rajat %A Rubin, Lee L %X Spinal muscular atrophy (SMA), a degenerative motor neuron (MN) disease caused by loss of functional SMN protein due to SMN1 gene mutations, is a leading cause of infant mortality. Increasing SMN levels ameliorates the disease phenotype and is unanimously accepted as a therapeutic approach for SMA patients. The ubiquitin/proteasome system is known to regulate SMN protein levels; however whether autophagy controls SMN levels remains poorly explored. Here we show that SMN protein is degraded by autophagy. Pharmacological and genetic inhibition of autophagy increase SMN levels, while induction of autophagy decreases SMN. SMN degradation occurs via its interaction with the autophagy adapter p62/SQSTM1. We also show that SMA neurons display reduced autophagosome clearance, increased p62/ubiquitinated protein levels, and hyperactivated mTORC1 signaling. Importantly, reducing p62 levels markedly increases SMN and its binding partner gemin2, promotes MN survival and extends lifespan in fly and mouse SMA models revealing p62 as a new potential therapeutic target to treat SMA. %B J Clin Invest %8 2018 Apr 19 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29672276?dopt=Abstract %R 10.1172/JCI95231 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2018 %T Next-generation CRISPR/Cas9 transcriptional activation in using flySAM %A Jia, Yu %A Xu, Rong-Gang %A Ren, Xingjie %A Ewen-Campen, Ben %A Rajakumar, Rajendhran %A Zirin, Jonathan %A Yang-Zhou, Donghui %A Zhu, Ruibao %A Wang, Fang %A Mao, Decai %A Peng, Ping %A Qiao, Huan-Huan %A Wang, Xia %A Liu, Lu-Ping %A Xu, Bowen %A Ji, Jun-Yuan %A Liu, Qingfei %A Sun, Jin %A Perrimon, Norbert %A Ni, Jian-Quan %X CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic overexpression genetic analysis in We present flySAM, a potent tool for in vivo CRISPRa, which offers major improvements over existing strategies in terms of effectiveness, scalability, and ease of use. flySAM outperforms existing in vivo CRISPRa strategies and approximates phenotypes obtained using traditional Gal4-UAS overexpression. Moreover, because flySAM typically requires only a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental use of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic overexpression resource, TRiP-OE. %B Proc Natl Acad Sci U S A %8 2018 Apr 16 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29666231?dopt=Abstract %R 10.1073/pnas.1800677115 %0 Journal Article %J Mol Metab %D 2018 %T Phosphorylation of Beta-3 adrenergic receptor at serine 247 by ERK MAP kinase drives lipolysis in obese adipocytes %A Hong, Shangyu %A Song, Wei %A Zushin, Peter-James H %A Liu, Bingyang %A Jedrychowski, Mark P %A Mina, Amir I %A Deng, Zhaoming %A Cabarkapa, Dimitrije %A Hall, Jessica A %A Palmer, Colin J %A Aliakbarian, Hassan %A Szpyt, John %A Gygi, Steven P %A Tavakkoli, Ali %A Lynch, Lydia %A Perrimon, Norbert %A Banks, Alexander S %X OBJECTIVE: The inappropriate release of free fatty acids from obese adipose tissue stores has detrimental effects on metabolism, but key molecular mechanisms controlling FFA release from adipocytes remain undefined. Although obesity promotes systemic inflammation, we find activation of the inflammation-associated Mitogen Activated Protein kinase ERK occurs specifically in adipose tissues of obese mice, and provide evidence that adipocyte ERK activation may explain exaggerated adipose tissue lipolysis observed in obesity. METHODS AND RESULTS: We provide genetic and pharmacological evidence that inhibition of the MEK/ERK pathway in human adipose tissue, mice, and flies all effectively limit adipocyte lipolysis. In complementary findings, we show that genetic and obesity-mediated activation of ERK enhances lipolysis, whereas adipose tissue specific knock-out of ERK2, the exclusive ERK1/2 protein in adipocytes, dramatically impairs lipolysis in explanted mouse adipose tissue. In addition, acute inhibition of MEK/ERK signaling also decreases lipolysis in adipose tissue and improves insulin sensitivity in obese mice. Mice with decreased rates of adipose tissue lipolysis in vivo caused by either MEK or ATGL pharmacological inhibition were unable to liberate sufficient White Adipose Tissue (WAT) energy stores to fuel thermogenesis from brown fat during a cold temperature challenge. To identify a molecular mechanism controlling these actions, we performed unbiased phosphoproteomic analysis of obese adipose tissue at different time points following acute pharmacological MEK/ERK inhibition. MEK/ERK inhibition decreased levels of adrenergic signaling and caused de-phosphorylation of the β3-adrenergic receptor (β3AR) on serine 247. To define the functional implications of this phosphorylation, we showed that CRISPR/Cas9 engineered cells expressing wild type β3AR exhibited β3AR phosphorylation by ERK2 and enhanced lipolysis, but this was not seen when serine 247 of β3AR was mutated to alanine. CONCLUSION: Taken together, these data suggest that ERK activation in adipocytes and subsequent phosphorylation of the β3AR on S247 are critical regulatory steps in the enhanced adipocyte lipolysis of obesity. %B Mol Metab %8 2018 Mar 29 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29661693?dopt=Abstract %R 10.1016/j.molmet.2018.03.012 %0 Journal Article %J Cell Metab %D 2018 %T The TORC1-Regulated CPA Complex Rewires an RNA Processing Network to Drive Autophagy and Metabolic Reprogramming %A Tang, Hong-Wen %A Hu, Yanhui %A Chen, Chiao-Lin %A Xia, Baolong %A Zirin, Jonathan %A Yuan, Min %A Asara, John M %A Rabinow, Leonard %A Perrimon, Norbert %X Nutrient deprivation induces autophagy through inhibiting TORC1 activity. We describe a novel mechanism in Drosophila by which TORC1 regulates RNA processing of Atg transcripts and alters ATG protein levels and activities via the cleavage and polyadenylation (CPA) complex. We show that TORC1 signaling inhibits CDK8 and DOA kinases, which directly phosphorylate CPSF6, a component of the CPA complex. These phosphorylation events regulate CPSF6 localization, RNA binding, and starvation-induced alternative RNA processing of transcripts involved in autophagy, nutrient, and energy metabolism, thereby controlling autophagosome formation and metabolism. Similarly, we find that mammalian CDK8 and CLK2, a DOA ortholog, phosphorylate CPSF6 to regulate autophagy and metabolic changes upon starvation, revealing an evolutionarily conserved mechanism linking TORC1 signaling with RNA processing, autophagy, and metabolism. %B Cell Metab %8 2018 Mar 16 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29606597?dopt=Abstract %R 10.1016/j.cmet.2018.02.023 %0 Journal Article %J Elife %D 2018 %T A gene-specific T2A-GAL4 library for Drosophila %A Lee, Pei-Tseng %A Zirin, Jonathan %A Kanca, Oguz %A Lin, Wen-Wen %A Schulze, Karen L %A Li-Kroeger, David %A Tao, Rong %A Devereaux, Colby %A Hu, Yanhui %A Chung, Verena %A Ying Fang %A He, Yuchun %A Pan, Hongling %A Ge, Ming %A Zuo, Zhongyuan %A Housden, Benjamin E %A Mohr, Stephanie E %A Yamamoto, Shinya %A Levis, Robert W %A Spradling, Allan C %A Perrimon, Norbert %A Bellen, Hugo J %X We generated a library of ~1,000stocks in which we inserted a construct in the intron of genes allowing expression ofunder control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36(70%) of lethal insertions tested are rescued with a singlecDNA construct. Third, loss-of-function phenotypes associated with manyinsertions can be reverted by excision with. Fourth,drivenreports tissue and cell type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced withor any DNA. These stocks comprise a powerful resource for assessing gene function. %B Elife %V 7 %8 2018 Mar 22 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29565247?dopt=Abstract %R 10.7554/eLife.35574 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2018 %T Krüppel homolog 1 represses insect ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes %A Zhang, Tianlei %A Song, Wei %A Li, Zheng %A Qian, Wenliang %A Wei, Ling %A Yang, Yan %A Wang, Weina %A Zhou, Xuan %A Meng, Meng %A Peng, Jian %A Xia, Qingyou %A Perrimon, Norbert %A Cheng, Daojun %X In insects, juvenile hormone (JH) and the steroid hormone ecdysone have opposing effects on regulation of the larval-pupal transition. Although increasing evidence suggests that JH represses ecdysone biosynthesis during larval development, the mechanism underlying this repression is not well understood. Here, we demonstrate that the expression of the Krüppel homolog 1 (Kr-h1), a gene encoding a transcription factor that mediates JH signaling, in ecdysone-producing organ prothoracic gland (PG) represses ecdysone biosynthesis by directly inhibiting the transcription of steroidogenic enzymes in bothandApplication of a JH mimic on ex vivo cultured PGs fromandlarvae inducesexpression and inhibits the transcription of steroidogenic enzymes. In addition, PG-specific knockdown ofpromotes-while overexpression hampers-ecdysone production and pupariation. We further find that Kr-h1 inhibits the transcription of steroidogenic enzymes by directly binding to their promoters to induce promoter DNA methylation. Finally, we show that Kr-h1 does not affect DNA replication inPG cells and that the reduction of PG size mediated byoverexpression can be rescued by feeding ecdysone. Taken together, our data indicate direct and conserved Kr-h1 repression of insect ecdysone biosynthesis in response to JH stimulation, providing insights into mechanisms underlying the antagonistic roles of JH and ecdysone. %B Proc Natl Acad Sci U S A %8 2018 Mar 22 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29567866?dopt=Abstract %R 10.1073/pnas.1800435115 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2018 %T Xio is a component of thesex determination pathway and RNA-methyladenosine methyltransferase complex %A Guo, Jian %A Tang, Hong-Wen %A Li, Jing %A Perrimon, Norbert %A Yan, Dong %X -methyladenosine (mA), the most abundant chemical modification in eukaryotic mRNA, has been implicated insex determination by modifying() pre-mRNA and facilitating its alternative splicing. Here, we identify a sex determination gene,, and rename itaccording to its loss-of-function female-to-male transformation phenotype.encodes a conserved ubiquitous nuclear protein of unknown function. We show that Xio colocalizes and interacts with all previously known mA writer complex subunits (METTL3, METTL14, Fl(2)d/WTAP, Vir/KIAA1429, and Nito/Rbm15) and that loss ofis associated with phenotypes that resemble other mA factors, such as sexual transformations,splicing defect, held-out wings, flightless flies, and reduction of mA levels. Thus, Xio encodes a member of the mA methyltransferase complex involved in mRNA modification. Since its ortholog ZC3H13 (or KIAA0853) also associates with several mA writer factors, the function of Xio in the mA pathway is likely evolutionarily conserved. %B Proc Natl Acad Sci U S A %8 2018 Mar 19 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29555755?dopt=Abstract %R 10.1073/pnas.1720945115 %0 Journal Article %J Nature %D 2018 %T Mechanical regulation of stem-cell differentiation by the stretch-activated Piezo channel %A He, Li %A Si, Guangwei %A Huang, Jiuhong %A Aravinthan D. T. Samuel %A Perrimon, Norbert %X Somatic stem cells constantly adjust their self-renewal and lineage commitment by integrating various environmental cues to maintain tissue homeostasis. Although numerous chemical and biological signals have been identified that regulate stem-cell behaviour, whether stem cells can directly sense mechanical signals in vivo remains unclear. Here we show that mechanical stress regulates stem-cell differentiation in the adult Drosophila midgut through the stretch-activated ion channel Piezo. We find that Piezo is specifically expressed in previously unidentified enteroendocrine precursor cells, which have reduced proliferation ability and are destined to become enteroendocrine cells. Loss of Piezo activity reduces the generation of enteroendocrine cells in the adult midgut. In addition, ectopic expression of Piezo in all stem cells triggers both cell proliferation and enteroendocrine cell differentiation. Both the Piezo mutant and overexpression phenotypes can be rescued by manipulation of cytosolic Calevels, and increases in cytosolic Caresemble the Piezo overexpression phenotype, suggesting that Piezo functions through Casignalling. Further studies suggest that Casignalling promotes stem-cell proliferation and differentiation through separate pathways. Finally, Piezo is required for both mechanical activation of stem cells in a gut expansion assay and the increase of cytosolic Cain response to direct mechanical stimulus in a gut compression assay. Thus, our study demonstrates the existence of a specific group of stem cells in the fly midgut that can directly sense mechanical signals through Piezo. %B Nature %8 2018 Feb 07 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29414942?dopt=Abstract %R 10.1038/nature25744 %0 Journal Article %J G3 (Bethesda) %D 2018 %T Zinc Detoxification: A Functional Genomics and Transcriptomics Analysis in Drosophila melanogaster Cultured Cells %A Mohr, Stephanie E %A Rudd, Kirstin %A Hu, Yanhui %A Song, Wei R %A Gilly, Quentin %A Buckner, Michael %A Housden, Benjamin E %A Kelley, Colleen %A Zirin, Jonathan %A Tao, Rong %A Amador, Gabriel %A Sierzputowska, Katarzyna %A Comjean, Aram %A Perrimon, Norbert %X Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wildtype or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and more than 1800 putative zinc-responsive genes. Analysis of the 'omics data points to the relevance of ion transporters, glutathione-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells and that rdog is transcriptionally up-regulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the 'omics datasets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes. %B G3 (Bethesda) %8 2017 Dec 09 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29223976?dopt=Abstract %R 10.1534/g3.117.300447 %0 Journal Article %J Nucleic Acids Res %D 2018 %T Molecular Interaction Search Tool (MIST): an integrated resource for mining gene and protein interaction data %A Hu, Yanhui %A Vinayagam, Arunachalam %A Nand, Ankita %A Comjean, Aram %A Chung, Verena %A Hao, Tong %A Mohr, Stephanie E %A Perrimon, Norbert %X Model organism and human databases are rich with information about genetic and physical interactions. These data can be used to interpret and guide the analysis of results from new studies and develop new hypotheses. Here, we report the development of the Molecular Interaction Search Tool (MIST; http://fgrtools.hms.harvard.edu/MIST/). The MIST database integrates biological interaction data from yeast, nematode, fly, zebrafish, frog, rat and mouse model systems, as well as human. For individual or short gene lists, the MIST user interface can be used to identify interacting partners based on protein-protein and genetic interaction (GI) data from the species of interest as well as inferred interactions, known as interologs, and to view a corresponding network. The data, interologs and search tools at MIST are also useful for analyzing 'omics datasets. In addition to describing the integrated database, we also demonstrate how MIST can be used to identify an appropriate cut-off value that balances false positive and negative discovery, and present use-cases for additional types of analysis. Altogether, the MIST database and search tools support visualization and navigation of existing protein and GI data, as well as comparison of new and existing data. %B Nucleic Acids Res %8 2017 Nov 16 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29155944?dopt=Abstract %R 10.1093/nar/gkx1116 %0 Journal Article %J Bio Protoc %D 2017 %T Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Cells %A Housden, Benjamin E %A Nicholson, Hilary E %A Perrimon, Norbert %X A synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In , combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden , 2015). %B Bio Protoc %V 7 %8 2017 Feb 05 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/28523286?dopt=Abstract %R 10.21769/BioProtoc.2119 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2017 %T Improved detection of synthetic lethal interactions in Drosophila cells using variable dose analysis (VDA) %A Housden, Benjamin E %A Li, Zhongchi %A Kelley, Colleen %A Wang, Yuanli %A Hu, Yanhui %A Valvezan, Alexander J %A Manning, Brendan D %A Perrimon, Norbert %X Synthetic sick or synthetic lethal (SS/L) screens are a powerful way to identify candidate drug targets to specifically kill tumor cells, but this approach generally suffers from low consistency between screens. We found that many SS/L interactions involve essential genes and are therefore detectable within a limited range of knockdown efficiency. Such interactions are often missed by overly efficient RNAi reagents. We therefore developed an assay that measures viability over a range of knockdown efficiency within a cell population. This method, called Variable Dose Analysis (VDA), is highly sensitive to viability phenotypes and reproducibly detects SS/L interactions. We applied the VDA method to search for SS/L interactions with TSC1 and TSC2, the two tumor suppressors underlying tuberous sclerosis complex (TSC), and generated a SS/L network for TSC. Using this network, we identified four Food and Drug Administration-approved drugs that selectively affect viability of TSC-deficient cells, representing promising candidates for repurposing to treat TSC-related tumors. %B Proc Natl Acad Sci U S A %V 114 %P E10755-E10762 %8 2017 Dec 12 %G eng %N 50 %1 http://www.ncbi.nlm.nih.gov/pubmed/29183982?dopt=Abstract %R 10.1073/pnas.1713362114 %0 Journal Article %J Nature %D 2017 %T Data management: A global coalition to sustain core data %A Anderson, WP %A Global Life Science Data Resources Working Group %K Cooperative Behavior %K Information Storage and Retrieval %X Collaborators: Cochrane G, Di Francesco V, Donohue T, Durinx C, Game A, Green E, Gojobori T, Goodhand P, Hamosh A, Hermjakob H, Kanehisa M, Kiley R, McEntyre J, McKibbin R, Miyano S, Pauly B, Perrimon N, Ragan MA, Richards G, Teo YY, Westerfield M, Westhof E, Lasko PF. %B Nature %V 543 %P 179 %8 2017 03 08 %G eng %N 7644 %1 http://www.ncbi.nlm.nih.gov/pubmed/28277502?dopt=Abstract %R 10.1038/543179a %0 Journal Article %J Cancer Cell %D 2017 %T mTORC1 Couples Nucleotide Synthesis to Nucleotide Demand Resulting in a Targetable Metabolic Vulnerability %A Valvezan, Alexander J %A Turner, Marc %A Belaid, Amine %A Lam, Hilaire C %A Miller, Spencer K %A McNamara, Molly C %A Baglini, Christian %A Housden, Benjamin E %A Perrimon, Norbert %A Kwiatkowski, David J %A Asara, John M %A Henske, Elizabeth P %A Manning, Brendan D %X The mechanistic target of rapamycin complex 1 (mTORC1) supports proliferation through parallel induction of key anabolic processes, including protein, lipid, and nucleotide synthesis. We hypothesized that these processes are coupled to maintain anabolic balance in cells with mTORC1 activation, a common event in human cancers. Loss of the tuberous sclerosis complex (TSC) tumor suppressors results in activation of mTORC1 and development of the tumor syndrome TSC. We find that pharmacological inhibitors of guanylate nucleotide synthesis have selective deleterious effects on TSC-deficient cells, including in mouse tumor models. This effect stems from replication stress and DNA damage caused by mTORC1-driven rRNA synthesis, which renders nucleotide pools limiting. These findings reveal a metabolic vulnerability downstream of mTORC1 triggered by anabolic imbalance. %B Cancer Cell %8 2017 Oct 13 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/29056426?dopt=Abstract %R 10.1016/j.ccell.2017.09.013 %0 Journal Article %J Dev Cell %D 2017 %T Accessing the Phenotype Gap: Enabling Systematic Investigation of Paralog Functional Complexity with CRISPR %A Ewen-Campen, Ben %A Mohr, Stephanie E %A Hu, Yanhui %A Perrimon, Norbert %X Single-gene knockout experiments can fail to reveal function in the context of redundancy, which is frequently observed among duplicated genes (paralogs) with overlapping functions. We discuss the complexity associated with studying paralogs and outline how recent advances in CRISPR will help address the "phenotype gap" and impact biomedical research. %B Dev Cell %V 43 %P 6-9 %8 2017 Oct 09 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/29017030?dopt=Abstract %R 10.1016/j.devcel.2017.09.020 %0 Journal Article %J Dev Cell %D 2017 %T A Mechanism Coupling Systemic Energy Sensing to Adipokine Secretion %A Rajan, Akhila %A Housden, Benjamin E %A Wirtz-Peitz, Frederik %A Holderbaum, Laura %A Perrimon, Norbert %X Adipocytes sense systemic nutrient status and systemically communicate this information by releasing adipokines. The mechanisms that couple nutritional state to adipokine release are unknown. Here, we investigated how Unpaired 2 (Upd2), a structural and functional ortholog of the primary human adipokine leptin, is released from Drosophila fat cells. We find that Golgi reassembly stacking protein (GRASP), an unconventional secretion pathway component, is required for Upd2 secretion. In nutrient-rich fat cells, GRASP clusters in close proximity to the apical side of lipid droplets (LDs). During nutrient deprivation, glucagon-mediated increase in calcium (Ca(2+)) levels, via calmodulin kinase II (CaMKII) phosphorylation, inhibits proximal GRASP localization to LDs. Using a heterologous cell system, we show that human leptin secretion is also regulated by Ca(2+) and CaMKII. In summary, we describe a mechanism by which increased cytosolic Ca(2+) negatively regulates adipokine secretion and have uncovered an evolutionarily conserved molecular link between intracellular Ca(2+) levels and energy homeostasis. %B Dev Cell %V 43 %P 83-98.e6 %8 2017 Oct 09 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/29017032?dopt=Abstract %R 10.1016/j.devcel.2017.09.007 %0 Journal Article %J Cell Rep %D 2017 %T Proteomic and Metabolomic Characterization of a Mammalian Cellular Transition from Quiescence to Proliferation %A Lee, Ho-Joon %A Jedrychowski, Mark P %A Vinayagam, Arunachalam %A Wu, Ning %A Shyh-Chang, Ng %A Hu, Yanhui %A Min-Wen, Chua %A Moore, Jodene K %A Asara, John M %A Lyssiotis, Costas A %A Perrimon, Norbert %A Gygi, Steven P %A Cantley, Lewis C %A Kirschner, Marc W %X There exist similarities and differences in metabolism and physiology between normal proliferative cells and tumor cells. Once a cell enters the cell cycle, metabolic machinery is engaged to facilitate various processes. The kinetics and regulation of these metabolic changes have not been properly evaluated. To correlate the orchestration of these processes with the cell cycle, we analyzed the transition from quiescence to proliferation of a non-malignant murine pro-B lymphocyte cell line in response to IL-3. Using multiplex mass-spectrometry-based proteomics, we show that the transition to proliferation shares features generally attributed to cancer cells: upregulation of glycolysis, lipid metabolism, amino-acid synthesis, and nucleotide synthesis and downregulation of oxidative phosphorylation and the urea cycle. Furthermore, metabolomic profiling of this transition reveals similarities to cancer-related metabolic pathways. In particular, we find that methionine is consumed at a higher rate than that of other essential amino acids, with a potential link to maintenance of the epigenome. %B Cell Rep %V 20 %P 721-736 %8 2017 Jul 18 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/28723573?dopt=Abstract %R 10.1016/j.celrep.2017.06.074 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2017 %T Optimized strategy for in vivo Cas9-activation in Drosophila %A Ewen-Campen, Ben %A Yang-Zhou, Donghui %A Fernandes, Vitória R %A González, Delfina P %A Liu, Lu-Ping %A Tao, Rong %A Ren, Xingjie %A Sun, Jin %A Hu, Yanhui %A Zirin, Jonathan %A Mohr, Stephanie E %A Ni, Jian-Quan %A Perrimon, Norbert %X While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo. %B Proc Natl Acad Sci U S A %V 114 %P 9409-9414 %8 2017 Aug 29 %G eng %N 35 %1 http://www.ncbi.nlm.nih.gov/pubmed/28808002?dopt=Abstract %R 10.1073/pnas.1707635114 %0 Journal Article %J G3 (Bethesda) %D 2017 %T Gene2Function: An Integrated Online Resource for Gene Function Discovery %A Hu, Yanhui %A Comjean, Aram %A Mohr, Stephanie E %A FlyBase Consortium, The %A Perrimon, Norbert %X One of the most powerful ways to develop hypotheses regarding biological functions of conserved genes in a given species, such as in humans, is to first look at what is known about function in another species. Model organism databases (MODs) and other resources are rich with functional information but difficult to mine. Gene2Function (G2F) addresses a broad need by integrating information about conserved genes in a single online resource. %B G3 (Bethesda) %8 2017 Jun 29 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28663344?dopt=Abstract %R 10.1534/g3.117.043885 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2017 %T Activin signaling mediates muscle-to-adipose communication in a mitochondria dysfunction-associated obesity model %A Song, Wei %A Owusu-Ansah, Edward %A Hu, Yanhui %A Cheng, Daojun %A Ni, Xiaochun %A Zirin, Jonathan %A Perrimon, Norbert %X Mitochondrial dysfunction has been associated with obesity and metabolic disorders. However, whether mitochondrial perturbation in a single tissue influences mitochondrial function and metabolic status of another distal tissue remains largely unknown. We analyzed the nonautonomous role of muscular mitochondrial dysfunction in Drosophila Surprisingly, impaired muscle mitochondrial function via complex I perturbation results in simultaneous mitochondrial dysfunction in the fat body (the fly adipose tissue) and subsequent triglyceride accumulation, the major characteristic of obesity. RNA-sequencing (RNA-seq) analysis, in the context of muscle mitochondrial dysfunction, revealed that target genes of the TGF-β signaling pathway were induced in the fat body. Strikingly, expression of the TGF-β family ligand, Activin-β (Actβ), was dramatically increased in the muscles by NF-κB/Relish (Rel) signaling in response to mitochondrial perturbation, and decreasing Actβ expression in mitochondrial-perturbed muscles rescued both the fat body mitochondrial dysfunction and obesity phenotypes. Thus, perturbation of muscle mitochondrial activity regulates mitochondrial function in the fat body nonautonomously via modulation of Activin signaling. %B Proc Natl Acad Sci U S A %8 2017 Jul 24 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28739899?dopt=Abstract %R 10.1073/pnas.1708037114 %0 Journal Article %J BMC Biol %D 2017 %T Open questions: completing the parts list and finding the integrating signals %A Teleman, Aurelio A %A Perrimon, Norbert %X One of the great revelations of post-genomic biology has been the extent to which essential functions and mechanisms are conserved across vast phylogenetic distances. Because of this, we can look to the fruit fly for answers to pressing open questions on the unknown functions of genes and the mechanisms of their physiological integration. %B BMC Biol %V 15 %P 47 %8 2017 Jun 08 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/28595578?dopt=Abstract %R 10.1186/s12915-017-0388-0 %0 Journal Article %J Elife %D 2017 %T Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca(2+) signaling in the Drosophila midgut %A Xu, Chiwei %A Luo, Junjie %A He, Li %A Montell, Craig %A Perrimon, Norbert %X Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, we identified two regulators of cytosolic Ca(2+) levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca(2+) signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. Our findings provide a link between redox regulation and Ca(2+) signaling and reveal a novel mechanism by which ISCs detect stress signals. %B Elife %V 6 %8 2017 May 31 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28561738?dopt=Abstract %R 10.7554/eLife.22441 %0 Journal Article %J Genetics %D 2017 %T An Evolutionarily Conserved Role of Presenilin in Neuronal Protection in the Aging Drosophila Brain %A Kang, Jongkyun %A Shin, Sarah %A Perrimon, Norbert %A Shen, Jie %X
Mutations in the Presenilin genes are the major genetic cause of Alzheimer's disease. Presenilin and Nicastrin are essential components of γ-secretase, a multi-subunit protease that cleaves Type I transmembrane proteins. Genetic studies in mice previously demonstrated that conditional inactivation of Presenilin or Nicastrin in excitatory neurons of the postnatal forebrain results in memory deficits, synaptic impairment and age-dependent neurodegeneration. The roles of Drosophila Presenilin (Psn) and Nicastrin (Nct) in the adult fly brain, however, are unknown. To knockdown (KD) Psn or Nct selectively in neurons of the adult brain, we generated multiple shRNA lines. Using a ubiquitous driver, these shRNA lines resulted in 80-90% reduction of mRNA and pupal lethality, a phenotype that is shared with Psn and Nct mutants carrying nonsense mutations. Furthermore, expression of these shRNAs in the wing disc caused notching wing phenotypes, which are also shared with Psn and Nct mutants. Similar to Nct, neuron-specific Psn KD using two independent shRNA lines led to early mortality and rough eye phenotypes, which were rescued by a fly Psn transgene. Interestingly, conditional KD (cKD) of Psn or Nct in adult neurons using the elav-Gal4 and tubulin-Gal80(ts) system caused shortened lifespan, climbing defects, increases in apoptosis and age-dependent neurodegeneration. Together, these findings demonstrate that similar to their mammalian counterparts, Drosophila Psn and Nct are required for neuronal survival during aging and normal lifespan, highlighting an evolutionarily conserved role of Presenilin in neuronal protection in the aging brain.
%B Genetics %8 2017 May 11 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28495961?dopt=Abstract %R 10.1534/genetics.116.196881 %0 Journal Article %J Am J Hum Genet %D 2017 %T MARRVEL: Integration of Human and Model Organism Genetic Resources to Facilitate Functional Annotation of the Human Genome %A Wang, Julia %A Al-Ouran, Rami %A Hu, Yanhui %A Kim, Seon-Young %A Wan, Ying-Wooi %A Wangler, Michael F %A Yamamoto, Shinya %A Chao, Hsiao-Tuan %A Comjean, Aram %A Mohr, Stephanie E %A UDN %A Perrimon, Norbert %A Liu, Zhandong %A Bellen, Hugo J %XOne major challenge encountered with interpreting human genetic variants is the limited understanding of the functional impact of genetic alterations on biological processes. Furthermore, there remains an unmet demand for an efficient survey of the wealth of information on human homologs in model organisms across numerous databases. To efficiently assess the large volume of publically available information, it is important to provide a concise summary of the most relevant information in a rapid user-friendly format. To this end, we created MARRVEL (model organism aggregated resources for rare variant exploration). MARRVEL is a publicly available website that integrates information from six human genetic databases and seven model organism databases. For any given variant or gene, MARRVEL displays information from OMIM, ExAC, ClinVar, Geno2MP, DGV, and DECIPHER. Importantly, it curates model organism-specific databases to concurrently display a concise summary regarding the human gene homologs in budding and fission yeast, worm, fly, fish, mouse, and rat on a single webpage. Experiment-based information on tissue expression, protein subcellular localization, biological process, and molecular function for the human gene and homologs in the seven model organisms are arranged into a concise output. Hence, rather than visiting multiple separate databases for variant and gene analysis, users can obtain important information by searching once through MARRVEL. Altogether, MARRVEL dramatically improves efficiency and accessibility to data collection and facilitates analysis of human genes and variants by cross-disciplinary integration of 18 million records available in public databases to facilitate clinical diagnosis and basic research.
%B Am J Hum Genet %8 2017 May 03 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28502612?dopt=Abstract %R 10.1016/j.ajhg.2017.04.010 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2017 %T Development of an optimized synthetic Notch receptor as an in vivo cell-cell contact sensor %A He, Li %A Huang, Jiuhong %A Perrimon, Norbert %XDetection and manipulation of direct cell-cell contact in complex tissues is a fundamental and challenging problem in many biological studies. Here, we report an optimized Notch-based synthetic receptor (synNQ) useful to study direct cell-cell interactions in Drosophila With the synNQ system, cells expressing a synthetic receptor, which contains Notch activation machinery and a downstream transcriptional activator, QF, are activated by a synthetic GFP ligand expressed by contacting neighbor cells. To avoid cis-inhibition, mutually exclusive expression of the synthetic ligand and receptor is achieved using the "flippase-out" system. Expression of the synthetic GFP ligand is controlled by the Gal4/UAS system for easy and broad applications. Using synNQ, we successfully visualized cell-cell interactions within and between most fly tissues, revealing previously undocumented cell-cell contacts. Importantly, in addition to detection of cells in contact with one another, synNQ allows for genetic manipulation in all cells in contact with a targeted cell population, which we demonstrate in the context of cell competition in developing wing disks. Altogether, the synNQ genetic system will enable a broad range of studies of cell contact in developmental biology.
%B Proc Natl Acad Sci U S A %8 2017 May 10 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28490499?dopt=Abstract %R 10.1073/pnas.1703205114 %0 Journal Article %J Wiley Interdiscip Rev Dev Biol %D 2017 %T Proximity-dependent labeling methods for proteomic profiling in living cells %A Chen, Chiao-Lin %A Perrimon, Norbert %XCharacterizing the proteome composition of organelles and subcellular regions of living cells can facilitate the understanding of cellular organization as well as protein interactome networks. Proximity labeling-based methods coupled with mass spectrometry (MS) offer a high-throughput approach for systematic analysis of spatially restricted proteomes. Proximity labeling utilizes enzymes that generate reactive radicals to covalently tag neighboring proteins with biotin. The biotinylated endogenous proteins can then be isolated for further analysis by MS. To analyze protein-protein interactions or identify components that localize to discrete subcellular compartments, spatial expression is achieved by fusing the enzyme to specific proteins or signal peptides that target to particular subcellular regions. Although these technologies have only been introduced recently, they have already provided deep insights into a wide range of biological processes. Here, we describe and compare current methods of proximity labeling as well as their applications. As each method has its own unique features, the goal of this review is to describe how different proximity labeling methods can be used to answer different biological questions. For further resources related to this article, please visit the WIREs website.
%B Wiley Interdiscip Rev Dev Biol %8 2017 Apr 07 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28387482?dopt=Abstract %R 10.1002/wdev.272 %0 Journal Article %J Dev Cell %D 2017 %T Thermogenesis by THADA %A Chatterjee, Nirmalya %A Perrimon, Norbert %XTHADA has been associated with cold adaptation and diabetes in humans, but the cellular and molecular basis of its function has been unknown. Moraru and colleagues (2017) report in this issue of Developmental Cell that it triggers thermogenesis by uncoupling ATP hydrolysis from calcium transport into the endoplasmic reticulum.
%B Dev Cell %V 41 %P 1-2 %8 2017 Apr 10 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/28399393?dopt=Abstract %R 10.1016/j.devcel.2017.03.021 %0 Journal Article %J Bio-Protocol %D 2017 %T Synthetic Lethality Screens Using RNAi in Combination with CRISPR-based Knockout in Drosophila Cells %A Housden, Benjamin E. %A Nicholson, Hilary E. %A Perrimon, Norbert %XA synthetic lethal interaction is a type of genetic interaction where the disruption of either of two genes individually has little effect but their combined disruption is lethal. Knowledge of synthetic lethal interactions can allow for elucidation of network structure and identification of candidate drug targets for human diseases such as cancer. In Drosophila, combinatorial gene disruption has been achieved previously by combining multiple RNAi reagents. Here we describe a protocol for high-throughput combinatorial gene disruption by combining CRISPR and RNAi. This approach previously resulted in the identification of highly reproducible and conserved synthetic lethal interactions (Housden et al., 2015).
%B Bio-Protocol %V 7 %G eng %U http://www.bio-protocol.org/e2119 %N 3 %0 Journal Article %J Cell Metab %D 2017 %T Midgut-Derived Activin Regulates Glucagon-like Action in the Fat Body and Glycemic Control %A Song, Wei %A Cheng, Daojun %A Hong, Shangyu %A Sappe, Benoit %A Hu, Yanhui %A Wei, Neil %A Zhu, Changqi %A O'Connor, Michael B %A Pissios, Pavlos %A Perrimon, Norbert %XWhile high-caloric diet impairs insulin response to cause hyperglycemia, whether and how counter-regulatory hormones are modulated by high-caloric diet is largely unknown. We find that enhanced response of Drosophila adipokinetic hormone (AKH, the glucagon homolog) in the fat body is essential for hyperglycemia associated with a chronic high-sugar diet. We show that the activin type I receptor Baboon (Babo) autonomously increases AKH signaling without affecting insulin signaling in the fat body via, at least, increase of Akh receptor (AkhR) expression. Further, we demonstrate that Activin-β (Actβ), an activin ligand predominantly produced in the enteroendocrine cells (EEs) of the midgut, is upregulated by chronic high-sugar diet and signals through Babo to promote AKH action in the fat body, leading to hyperglycemia. Importantly, activin signaling in mouse primary hepatocytes also increases glucagon response and glucagon-induced glucose production, indicating a conserved role for activin in enhancing AKH/glucagon signaling and glycemic control.
%B Cell Metab %V 25 %P 386-399 %8 2017 Feb 07 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/28178568?dopt=Abstract %R 10.1016/j.cmet.2017.01.002 %0 Journal Article %J BMC Bioinformatics %D 2017 %T The Drosophila Gene Expression Tool (DGET) for expression analyses %A Hu, Yanhui %A Comjean, Aram %A Perrimon, Norbert %A Mohr, Stephanie E %XBACKGROUND: Next-generation sequencing technologies have greatly increased our ability to identify gene expression levels, including at specific developmental stages and in specific tissues. Gene expression data can help researchers understand the diverse functions of genes and gene networks, as well as help in the design of specific and efficient functional studies, such as by helping researchers choose the most appropriate tissue for a study of a group of genes, or conversely, by limiting a long list of gene candidates to the subset that are normally expressed at a given stage or in a given tissue. RESULTS: We report DGET, a Drosophila Gene Expression Tool ( www.flyrnai.org/tools/dget/web/ ), which stores and facilitates search of RNA-Seq based expression profiles available from the modENCODE consortium and other public data sets. Using DGET, researchers are able to look up gene expression profiles, filter results based on threshold expression values, and compare expression data across different developmental stages, tissues and treatments. In addition, at DGET a researcher can analyze tissue or stage-specific enrichment for an inputted list of genes (e.g., 'hits' from a screen) and search for additional genes with similar expression patterns. We performed a number of analyses to demonstrate the quality and robustness of the resource. In particular, we show that evolutionary conserved genes expressed at high or moderate levels in both fly and human tend to be expressed in similar tissues. Using DGET, we compared whole tissue profile and sub-region/cell-type specific datasets and estimated a potential source of false positives in one dataset. We also demonstrated the usefulness of DGET for synexpression studies by querying genes with expression profile similar to the mesodermal master regulator Twist. CONCLUSION: Altogether, DGET provides a flexible tool for expression data retrieval and analysis with short or long lists of Drosophila genes, which can help scientists to design stage- or tissue-specific in vivo studies and do other subsequent analyses.
%B BMC Bioinformatics %V 18 %P 98 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/28187709?dopt=Abstract %R 10.1186/s12859-017-1509-z %0 Journal Article %J Sci Rep %D 2017 %T eUnaG: a new ligand-inducible fluorescent reporter to detect drug transporter activity in live cells %A Yeh, Johannes T-H %A Nam, Kwangho %A Yeh, Joshua T-H %A Perrimon, Norbert %X The absorption, distribution, metabolism and excretion (ADME) of metabolites and toxic organic solutes are orchestrated by the ATP-binding cassette (ABC) transporters and the organic solute carrier family (SLC) proteins. A large number of ABC and SLC transpoters exist; however, only a small number have been well characterized. To facilitate the analysis of these transporters, which is important for drug safety and physiological studies, we developed a sensitive genetically encoded bilirubin (BR)-inducible fluorescence sensor (eUnaG) to detect transporter-coupled influx/efflux of organic compounds. This sensor can be used in live cells to measure transporter activity, as excretion of BR depends on ABC and SLC transporters. Applying eUnaG in functional RNAi screens, we characterize l(2)03659 as a Drosophila multidrug resistant-associated ABC transporter. %B Sci Rep %V 7 %P 41619 %8 2017 Feb 08 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/28176814?dopt=Abstract %R 10.1038/srep41619 %0 Journal Article %J Dev Cell %D 2017 %T miR-263a Regulates ENaC to Maintain Osmotic and Intestinal Stem Cell Homeostasis in Drosophila. %A Kim, Kevin %A Hung, Ruei-Jiun %A Perrimon, Norbert %XProper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. We found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na(+) levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Finally, we provide evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC.
%B Dev Cell %V 40 %P 23-36 %8 2017 Jan 09 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/28017617?dopt=Abstract %R 10.1016/j.devcel.2016.11.023 %0 Journal Article %J Nucleic Acid Research %D 2017 %T FlyRNAi.org—the database of the Drosophila RNAi Screening Center and Transgenic RNAi Project: 2017 update %A Hu, Y. %A Comjean, A %A Roesel, C %A Vinayagam, A %A Flockhart, I %A Zirin, J %A Perkins, L A %A Perrimon, N %A Mohr, SE %XThe FlyRNAi database of the Drosophila RNAi Screening Center (DRSC) and Transgenic RNAi Project (TRiP) at Harvard Medical School and associated DRSC/TRiP Functional Genomics Resources website (http://fgr.hms.harvard.edu) serve as a reagent production tracking system, screen data repository, and portal to the community. Through this portal, we make available protocols, online tools, and other resources useful to researchers at all stages of high-throughput functional genomics screening, from assay design and reagent identification to data analysis and interpretation. In this update, we describe recent changes and additions to our website, database and suite of online tools. Recent changes reflect a shift in our focus from a single technology (RNAi) and model species (Drosophila) to the application of additional technologies (e.g. CRISPR) and support of integrated, cross-species approaches to uncovering gene function using functional genomics and other approaches.
%B Nucleic Acid Research %8 23 Oct 2016 %G eng %0 Journal Article %J Nat Rev Genet %D 2017 %T Loss-of-function genetic tools for animal models: cross-species and cross-platform differences. %A Housden, Benjamin E %A Muhar, Matthias %A Gemberling, Matthew %A Gersbach, Charles A %A Stainier, Didier Y R %A Seydoux, Geraldine %A Mohr, Stephanie E %A Zuber, Johannes %A Perrimon, Norbert %X Our understanding of the genetic mechanisms that underlie biological processes has relied extensively on loss-of-function (LOF) analyses. LOF methods target DNA, RNA or protein to reduce or to ablate gene function. By analysing the phenotypes that are caused by these perturbations the wild-type function of genes can be elucidated. Although all LOF methods reduce gene activity, the choice of approach (for example, mutagenesis, CRISPR-based gene editing, RNA interference, morpholinos or pharmacological inhibition) can have a major effect on phenotypic outcomes. Interpretation of the LOF phenotype must take into account the biological process that is targeted by each method. The practicality and efficiency of LOF methods also vary considerably between model systems. We describe parameters for choosing the optimal combination of method and system, and for interpreting phenotypes within the constraints of each method. %B Nat Rev Genet %V 18 %P 24-40 %8 2017 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/27795562?dopt=Abstract %R 10.1038/nrg.2016.118 %0 Journal Article %J Dis Model Mech %D 2016 %T Fruit flies on the front line: the translational impact of Drosophila. %A Perrimon, Norbert %A Bonini, Nancy M %A Dhillon, Paraminder %XDrosophila melanogaster has been adopted as one of the most-used model systems since it was first introduced by Thomas Morgan for the study of heredity in the early 20th century. Its experimental tractability and similarity of its biological pathways to those of humans have placed the model at the forefront of research into human development and disease. With the ongoing accumulation of genetic tools and assays, the fly community has at its fingertips the resources to generate diverse Drosophila disease models for the study of genes and pathways involved in a wide range of disorders. In recent years, the fly has also been used successfully for drug screening. In this Editorial, we introduce a Special Collection of reviews, interviews and original research articles that highlight some of the many ways that Drosophila has made, and continues to make, an impact on basic biological insights and translational science.
%B Dis Model Mech %V 9 %P 229-31 %8 2016 Mar %G ENG %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/26935101?dopt=Abstract %R 10.1242/dmm.024810 %0 Journal Article %J Genes Dev %D 2016 %T Oncogenic transformation of Drosophila somatic cells induces a functional piRNA pathway. %A Fagegaltier, Delphine %A Falciatori, Ilaria %A Czech, Benjamin %A Castel, Stephane %A Perrimon, Norbert %A Simcox, Amanda %A Hannon, Gregory J %XGermline genes often become re-expressed in soma-derived human cancers as "cancer/testis antigens" (CTAs), and piRNA (PIWI-interacting RNA) pathway proteins are found among CTAs. However, whether and how the piRNA pathway contributes to oncogenesis in human neoplasms remain poorly understood. We found that oncogenic Ras combined with loss of the Hippo tumor suppressor pathway reactivates a primary piRNA pathway in Drosophila somatic cells coincident with oncogenic transformation. In these cells, Piwi becomes loaded with piRNAs derived from annotated generative loci, which are normally restricted to either the germline or the somatic follicle cells. Negating the pathway leads to increases in the expression of a wide variety of transposons and also altered expression of some protein-coding genes. This correlates with a reduction in the proliferation of the transformed cells in culture, suggesting that, at least in this context, the piRNA pathway may play a functional role in cancer.
%B Genes Dev %V 30 %P 1623-35 %8 2016 Jul 15 %G ENG %N 14 %1 http://www.ncbi.nlm.nih.gov/pubmed/27474441?dopt=Abstract %R 10.1101/gad.284927.116 %0 Journal Article %J Annu Rev Genet %D 2016 %T Interorgan Communication Pathways in Physiology: Focus on Drosophila. %A Droujinine, Ilia A %A Perrimon, Norbert %XStudies in mammals and Drosophila have demonstrated the existence and significance of secreted factors involved in communication between distal organs. In this review, primarily focusing on Drosophila, we examine the known interorgan communication factors and their functions, physiological inducers, and integration in regulating physiology. Moreover, we describe how organ-sensing screens in Drosophila can systematically identify novel conserved interorgan communication factors. Finally, we discuss how interorgan communication enabled and evolved as a result of specialization of organs. Together, we anticipate that future studies will establish a model for metazoan interorgan communication network (ICN) and how it is deregulated in disease. Expected final online publication date for the Annual Review of Genetics Volume 50 is November 23, 2016. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
%B Annu Rev Genet %8 2016 Oct 10 %G ENG %1 http://www.ncbi.nlm.nih.gov/pubmed/27732790?dopt=Abstract %R 10.1146/annurev-genet-121415-122024 %0 Journal Article %J Cold Spring Harb Protoc %D 2016 %T Cas9-Mediated Genome Engineering in Drosophila melanogaster. %A Housden, Benjamin E %A Perrimon, Norbert %XThe recent development of the CRISPR-Cas9 system for genome engineering has revolutionized our ability to modify the endogenous DNA sequence of many organisms, including Drosophila This system allows alteration of DNA sequences in situ with single base-pair precision and is now being used for a wide variety of applications. To use the CRISPR system effectively, various design parameters must be considered, including single guide RNA target site selection and identification of successful editing events. Here, we review recent advances in CRISPR methodology in Drosophila and introduce protocols for some of the more difficult aspects of CRISPR implementation: designing and generating CRISPR reagents and detecting indel mutations by high-resolution melt analysis.
%B Cold Spring Harb Protoc %V 2016 %P pdb.top086843 %8 2016 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/27587786?dopt=Abstract %R 10.1101/pdb.top086843 %0 Journal Article %J Cold Spring Harb Protoc %D 2016 %T Design and Generation of Donor Constructs for Genome Engineering in Drosophila. %A Housden, Benjamin E %A Perrimon, Norbert %XThe generation of precise alterations to the genome using CRISPR requires the combination of CRISPR and a donor construct containing homology to the target site. A double-strand break is first generated at the target locus using CRISPR. It is then repaired using the endogenous homologous recombination (HR) pathway. When a donor construct is provided, it can be used as a template for HR repair and can therefore be exploited to introduce alterations in the genomic sequence with single base-pair precision. Here we describe a protocol for the generation of donor constructs using Golden Gate assembly and discuss some key considerations for donor construct design for use in Drosophila.
%B Cold Spring Harb Protoc %V 2016 %P pdb.prot090787 %8 2016 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/27587780?dopt=Abstract %R 10.1101/pdb.prot090787 %0 Journal Article %J Cold Spring Harb Protoc %D 2016 %T Design and Generation of Drosophila Single Guide RNA Expression Constructs. %A Housden, Benjamin E %A Hu, Yanhui %A Perrimon, Norbert %XThe recent advances in CRISPR-based genome engineering have enabled a plethora of new experiments to study a wide range of biological questions. The major attraction of this system over previous methods is its high efficiency and simplicity of use. For example, whereas previous genome engineering technologies required the generation of new proteins to target each new locus, CRISPR requires only the expression of a different single guide RNA (sgRNA). This sgRNA binds to the Cas9 endonuclease protein and directs the generation of a double-strand break to a highly specific genomic site determined by the sgRNA sequence. In addition, the relative simplicity of the Drosophila genome is a particular advantage, as possible sgRNA off-target sites can easily be avoided. Here, we provide a step-by-step protocol for designing sgRNA target sites using the Drosophila RNAi Screening Center (DRSC) Find CRISPRs tool (version 2). We also describe the generation of sgRNA expression plasmids for the use in cultured Drosophila cells or in vivo. Finally, we discuss specific design requirements for various genome engineering applications.
%B Cold Spring Harb Protoc %V 2016 %P pdb.prot090779 %8 2016 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/27587779?dopt=Abstract %R 10.1101/pdb.prot090779 %0 Journal Article %J Cold Spring Harb Protoc %D 2016 %T Detection of Indel Mutations in Drosophila by High-Resolution Melt Analysis (HRMA). %A Housden, Benjamin E %A Perrimon, Norbert %XAlthough CRISPR technology allows specific genome alterations to be created with relative ease, detection of these events can be problematic. For example, CRISPR-induced double-strand breaks are often repaired imprecisely to generate unpredictable short indel mutations. Detection of these events requires the use of molecular screening techniques such as endonuclease assays, restriction profiling, or high-resolution melt analysis (HRMA). Here, we provide detailed protocols for HRMA-based mutation screening in Drosophila and analysis of the resulting data using the online tool HRMAnalyzer.
%B Cold Spring Harb Protoc %V 2016 %P pdb.prot090795 %8 2016 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/27587781?dopt=Abstract %R 10.1101/pdb.prot090795 %0 Journal Article %J Pediatr Neurol %D 2016 %T Advances and Future Directions for Tuberous Sclerosis Complex Research: Recommendations From the 2015 Strategic Planning Conference. %A Sahin, Mustafa %A Henske, Elizabeth P %A Manning, Brendan D %A Ess, Kevin C %A Bissler, John J %A Klann, Eric %A Kwiatkowski, David J %A Roberds, Steven L %A Silva, Alcino J %A Hillaire-Clarke, Coryse St %A Young, Lisa R %A Zervas, Mark %A Mamounas, Laura A %A Tuberous Sclerosis Complex Working Group to Update the Research Plan %XOn March 10 to March 12, 2015, the National Institute of Neurological Disorders and Stroke and the Tuberous Sclerosis Alliance sponsored a workshop in Bethesda, Maryland, to assess progress and new opportunities for research in tuberous sclerosis complex with the goal of updating the 2003 Research Plan for Tuberous Sclerosis (http://www.ninds.nih.gov/about_ninds/plans/tscler_research_plan.htm). In addition to the National Institute of Neurological Disorders and Stroke and Tuberous Sclerosis Alliance, participants in the strategic planning effort and workshop included representatives from six other Institutes of the National Institutes of Health, the Department of Defense Tuberous Sclerosis Complex Research Program, and a broad cross-section of basic scientists and clinicians with expertise in tuberous sclerosis complex along with representatives from the pharmaceutical industry. Here we summarize the outcomes from the extensive premeeting deliberations and final workshop recommendations, including (1) progress in the field since publication of the initial 2003 research plan for tuberous sclerosis complex, (2) the key gaps, needs, and challenges that hinder progress in tuberous sclerosis complex research, and (3) a new set of research priorities along with specific recommendations for addressing the major challenges in each priority area. The new research plan is organized around both short-term and long-term goals with the expectation that progress toward specific objectives can be achieved within a five to ten year time frame.
%B Pediatr Neurol %V 60 %P 1-12 %8 2016 Jul %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/27267556?dopt=Abstract %R 10.1016/j.pediatrneurol.2016.03.015 %0 Journal Article %J Elife %D 2016 %T Seipin is required for converting nascent to mature lipid droplets. %A Wang, Huajin %A Becuwe, Michel %A Housden, Benjamin E %A Chitraju, Chandramohan %A Porras, Ashley J %A Graham, Morven M %A Liu, Xinran N %A Thiam, Abdou Rachid %A Savage, David B %A Agarwal, Anil K %A Garg, Abhimanyu %A Olarte, Maria-Jesus %A Lin, Qingqing %A Fröhlich, Florian %A Hannibal-Bach, Hans Kristian %A Upadhyayula, Srigokul %A Perrimon, Norbert %A Kirchhausen, Tomas %A Ejsing, Christer S %A Walther, Tobias C %A Farese, Robert V %XHow proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.
%B Elife %V 5 %8 2016 Aug 26 %G ENG %1 http://www.ncbi.nlm.nih.gov/pubmed/27564575?dopt=Abstract %R 10.7554/eLife.16582 %0 Journal Article %J Nat Biotechnol %D 2016 %T Comparing CRISPR and RNAi-based screening technologies. %A Housden, Benjamin E %A Perrimon, Norbert %B Nat Biotechnol %V 34 %P 621-3 %8 2016 Jun 9 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/27281421?dopt=Abstract %R 10.1038/nbt.3599 %0 Journal Article %J Cell %D 2016 %T "ISN't Thirst Sweet?" Says the Fly. %A Petsakou, Afroditi %A Perrimon, Norbert %XHow food and water intake is reciprocally regulated to maintain homeostasis is unclear. New findings by Jourjine and colleagues identify four neurons in the Drosophila brain that receive both water and sugar abundance signals and oppositely regulate hunger and thirst.
%B Cell %V 166 %P 796-7 %8 2016 Aug 11 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/27518557?dopt=Abstract %R 10.1016/j.cell.2016.07.038 %0 Journal Article %J Cell Reports %D 2016 %T An integrative analysis of the InR/PI3K/Akt network identifies the dynamic response to Insulin signaling %A Vinayagam, A %A Kulkarni, MM %A Sopko, R %A Sun, X %A Hu, Y. %A Nand, A %A Villalta, C %A Moghimi, M %A Mohr, SE %A Hong, P %A Asara, A %A Perrimon, N %XSUMMARY
Insulin regulates an essential conserved signaling pathway affecting growth, proliferation, and metabolism. To expand our understanding of the insulin pathway, we combine biochemical, genetic, and computational approaches to build a comprehensive Drosophila InR/PI3K/Akt network. First, we map the dynamic protein-protein interaction network surrounding the insulin core pathway using bait-prey interactions connecting 566 proteins. Combining RNAi screening and phospho-specific antibodies, we find that 47% of interacting proteins affect pathway activity, and, using quantitative phosphoproteomics, we demonstrate that 10% of interacting proteins are regulated by insulin stimulation at the level of phosphorylation. Next, we integrate these orthogonal datasets to characterize the structure and dynamics of the insulin network at the level of protein complexes and validate our method by identifying regulatory roles for the Protein Phosphatase 2A (PP2A) and Reptin-Pontin chromatin-remodeling complexes as negative and positive regulators of ribosome biogenesis, respectively. Altogether, our study represents a comprehensive resource for the study of the evolutionary conserved insulin network.
%B Cell Reports %V 16 %P 3062–3074 %8 13 Sept, 2016 %G eng %N 11 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2016 %T Mapping signaling pathway cross-talk in Drosophila cells. %A Ammeux, Noemie %A Housden, Benjamin E %A Georgiadis, Andrew %A Hu, Yanhui %A Perrimon, Norbert %XDuring development and homeostasis, cells integrate multiple signals originating either from neighboring cells or systemically. In turn, responding cells can produce signals that act in an autocrine, paracrine, or endocrine manner. Although the nature of the signals and pathways used in cell-cell communication are well characterized, we lack, in most cases, an integrative view of signaling describing the spatial and temporal interactions between pathways (e.g., whether the signals are processed sequentially or concomitantly when two pathways are required for a specific outcome). To address the extent of cross-talk between the major metazoan signaling pathways, we characterized immediate transcriptional responses to either single- or multiple pathway stimulations in homogeneous Drosophila cell lines. Our study, focusing on seven core pathways, epidermal growth factor receptor (EGFR), bone morphogenetic protein (BMP), Jun kinase (JNK), JAK/STAT, Notch, Insulin, and Wnt, revealed that many ligands and receptors are primary targets of signaling pathways, highlighting that transcriptional regulation of genes encoding pathway components is a major level of signaling cross-talk. In addition, we found that ligands and receptors can integrate multiple pathway activities and adjust their transcriptional responses accordingly.
%B Proc Natl Acad Sci U S A %8 2016 Aug 15 %G ENG %1 http://www.ncbi.nlm.nih.gov/pubmed/27528688?dopt=Abstract %R 10.1073/pnas.1610432113 %0 Journal Article %J Genes Dev %D 2016 %T Tissue-specific down-regulation of S-adenosyl-homocysteine via suppression of dAhcyL1/dAhcyL2 extends health span and life span in Drosophila. %A Parkhitko, Andrey A %A Binari, Richard %A Zhang, Nannan %A Asara, John M %A Demontis, Fabio %A Perrimon, Norbert %XAging is a risk factor for many human pathologies and is characterized by extensive metabolic changes. Using targeted high-throughput metabolite profiling in Drosophila melanogaster at different ages, we demonstrate that methionine metabolism changes strikingly during aging. Methionine generates the methyl donor S-adenosyl-methionine (SAM), which is converted via methylation to S-adenosyl-homocysteine (SAH), which accumulates during aging. A targeted RNAi screen against methionine pathway components revealed significant life span extension in response to down-regulation of two noncanonical Drosophila homologs of the SAH hydrolase Ahcy (S-adenosyl-L-homocysteine hydrolase [SAHH[), CG9977/dAhcyL1 and Ahcy89E/CG8956/dAhcyL2, which act as dominant-negative regulators of canonical AHCY. Importantly, tissue-specific down-regulation of dAhcyL1/L2 in the brain and intestine extends health and life span. Furthermore, metabolomic analysis of dAhcyL1-deficient flies revealed its effect on age-dependent metabolic reprogramming and H3K4 methylation. Altogether, reprogramming of methionine metabolism in young flies and suppression of age-dependent SAH accumulation lead to increased life span. These studies highlight the role of noncanonical Ahcy enzymes as determinants of healthy aging and longevity.
%B Genes Dev %V 30 %P 1409-22 %8 2016 Jun 15 %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/27313316?dopt=Abstract %R 10.1101/gad.282277.116 %0 Journal Article %J Curr Top Dev Biol %D 2016 %T Toward a Systems Understanding of Signaling Pathway Function. %A Doupé, David P %A Perrimon, Norbert %XA small number of developmental signaling pathways are used repeatedly throughout development in many different contexts. How these pathways interact with each other and the specific cell context to generate a wide range of appropriate responses remains an important question. The application of genomic and proteomic approaches and imaging at high spatiotemporal resolution are providing answers to this question and revealing new levels of complexity. Here, we discuss pathways as complex networks and examples of how signaling outcomes can be influenced by the temporal nature of the signal, its spatial regulation, and the cell context.
%B Curr Top Dev Biol %V 117 %P 221-36 %8 2016 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26969980?dopt=Abstract %R 10.1016/bs.ctdb.2015.11.005 %0 Book Section %B Encyclopedia of Cell Biology %D 2016 %T Systematic methods to interrogate genetic perturbations and map phosphorylation-dependent signaling %A Sopko, Richelle %A Perrimon, Norbert %B Encyclopedia of Cell Biology %I Elsevier Inc. %V 4 %P 227-233 %G eng %0 Journal Article %J Nat Methods %D 2016 %T Comparison of Cas9 activators in multiple species. %A Chavez, Alejandro %A Tuttle, Marcelle %A Pruitt, Benjamin W %A Ewen-Campen, Ben %A Chari, Raj %A Ter-Ovanesyan, Dmitry %A Haque, Sabina J %A Cecchi, Ryan J %A Kowal, Emma J K %A Buchthal, Joanna %A Housden, Benjamin E %A Perrimon, Norbert %A Collins, James J %A Church, George %XSeveral programmable transcription factors exist based on the versatile Cas9 protein, yet their relative potency and effectiveness across various cell types and species remain unexplored. Here, we compare Cas9 activator systems and examine their ability to induce robust gene expression in several human, mouse, and fly cell lines. We also explore the potential for improved activation through the combination of the most potent activator systems, and we assess the role of cooperativity in maximizing gene expression.
%B Nat Methods %V 13 %P 563-7 %8 2016 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/27214048?dopt=Abstract %R 10.1038/nmeth.3871 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2016 %T Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets. %A Vinayagam, Arunachalam %A Gibson, Travis E %A Lee, Ho-Joon %A Yilmazel, Bahar %A Roesel, Charles %A Hu, Yanhui %A Kwon, Young %A Sharma, Amitabh %A Liu, Yang-Yu %A Perrimon, Norbert %A Barabási, Albert-László %XThe protein-protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as "indispensable," "neutral," or "dispensable," which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network's control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets.
%B Proc Natl Acad Sci U S A %V 113 %P 4976-81 %8 2016 May 3 %G eng %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/27091990?dopt=Abstract %R 10.1073/pnas.1603992113 %0 Journal Article %J Development %D 2016 %T Coordinated control of Notch/Delta signalling and cell cycle progression drives lateral inhibition-mediated tissue patterning. %A Hunter, Ginger L %A Hadjivasiliou, Zena %A Bonin, Hope %A He, Li %A Perrimon, Norbert %A Charras, Guillaume %A Baum, Buzz %XCoordinating cell differentiation with cell growth and division is crucial for the successful development, homeostasis and regeneration of multicellular tissues. Here, we use bristle patterning in the fly notum as a model system to explore the regulatory and functional coupling of cell cycle progression and cell fate decision-making. The pattern of bristles and intervening epithelial cells (ECs) becomes established through Notch-mediated lateral inhibition during G2 phase of the cell cycle, as neighbouring cells physically interact with each other via lateral contacts and/or basal protrusions. Since Notch signalling controls cell division timing downstream of Cdc25, ECs in lateral contact with a Delta-expressing cell experience higher levels of Notch signalling and divide first, followed by more distant neighbours, and lastly Delta-expressing cells. Conversely, mitotic entry and cell division makes ECs refractory to lateral inhibition signalling, fixing their fate. Using a combination of experiments and computational modelling, we show that this reciprocal relationship between Notch signalling and cell cycle progression acts like a developmental clock, providing a delimited window of time during which cells decide their fate, ensuring efficient and orderly bristle patterning.
%B Development %V 143 %P 2305-10 %8 2016 Jul 1 %G eng %N 13 %1 http://www.ncbi.nlm.nih.gov/pubmed/27226324?dopt=Abstract %R 10.1242/dev.134213 %0 Journal Article %J FEBS J %D 2016 %T CRISPR guide RNA design for research applications. %A Mohr, Stephanie E %A Hu, Yanhui %A Ewen-Campen, Benjamin %A Housden, Benjamin E %A Viswanatha, Raghuvir %A Perrimon, Norbert %XThe rapid rise of CRISPR as a technology for genome engineering and related research applications has created a need for algorithms and associated online tools that facilitate design of on-target and effective guide RNAs (gRNAs). Here, we review the state-of-the-art in CRISPR gRNA design for research applications of the CRISPR-Cas9 system, including knockout, activation and inhibition. Notably, achieving good gRNA design is not solely dependent on innovations in CRISPR technology. Good design and design tools also rely on availability of high-quality genome sequence and gene annotations, as well as on availability of accumulated data regarding off-targets and effectiveness metrics. This article is protected by copyright. All rights reserved.
%B FEBS J %8 2016 Jun 8 %G ENG %1 http://www.ncbi.nlm.nih.gov/pubmed/27276584?dopt=Abstract %R 10.1111/febs.13777 %0 Journal Article %J Sci Rep %D 2016 %T A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2. %A Breitkopf, Susanne B %A Yang, Xuemei %A Begley, Michael J %A Kulkarni, Meghana %A Chiu, Yu-Hsin %A Turke, Alexa B %A Lauriol, Jessica %A Yuan, Min %A Qi, Jie %A Engelman, Jeffrey A %A Hong, Pengyu %A Kontaridis, Maria I %A Cantley, Lewis C %A Perrimon, Norbert %A Asara, John M %XUsing a series of immunoprecipitation (IP)-tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.
%B Sci Rep %V 6 %P 20471 %8 2016 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26839216?dopt=Abstract %R 10.1038/srep20471 %0 Journal Article %J PLoS Genet %D 2016 %T miR-190 Enhances HIF-Dependent Responses to Hypoxia in Drosophila by Inhibiting the Prolyl-4-hydroxylase Fatiga. %A De Lella Ezcurra, Ana Laura %A Bertolin, Agustina Paola %A Kim, Kevin %A Katz, Maximiliano Javier %A Gándara, Lautaro %A Misra, Tvisha %A Luschnig, Stefan %A Perrimon, Norbert %A Melani, Mariana %A Wappner, Pablo %XCellular and systemic responses to low oxygen levels are principally mediated by Hypoxia Inducible Factors (HIFs), a family of evolutionary conserved heterodimeric transcription factors, whose alpha- and beta-subunits belong to the bHLH-PAS family. In normoxia, HIFα is hydroxylated by specific prolyl-4-hydroxylases, targeting it for proteasomal degradation, while in hypoxia the activity of these hydroxylases decreases due to low oxygen availability, leading to HIFα accumulation and expression of HIF target genes. To identify microRNAs required for maximal HIF activity, we conducted an overexpression screen in Drosophila melanogaster, evaluating the induction of a HIF transcriptional reporter. miR-190 overexpression enhanced HIF-dependent biological responses, including terminal sprouting of the tracheal system, while in miR-190 loss of function embryos the hypoxic response was impaired. In hypoxic conditions, miR-190 expression was upregulated and required for induction of HIF target genes by directly inhibiting the HIF prolyl-4-hydroxylase Fatiga. Thus, miR-190 is a novel regulator of the hypoxia response that represses the oxygen sensor Fatiga, leading to HIFα stabilization and enhancement of hypoxic responses.
%B PLoS Genet %V 12 %P e1006073 %8 2016 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/27223464?dopt=Abstract %R 10.1371/journal.pgen.1006073 %0 Journal Article %J Elife %D 2016 %T The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling. %A Harris, Kathryn P %A Zhang, Yao V %A Piccioli, Zachary D %A Perrimon, Norbert %A Littleton, J Troy %XPostsynaptic cells can induce synaptic plasticity through the release of activity-dependent retrograde signals. We previously described a Ca(2+)-dependent retrograde signaling pathway mediated by postsynaptic Synaptotagmin 4 (Syt4). To identify proteins involved in postsynaptic exocytosis, we conducted a screen for candidates that disrupted trafficking of a pHluorin-tagged Syt4 at Drosophila neuromuscular junctions (NMJs). Here we characterize one candidate, the postsynaptic t-SNARE Syntaxin 4 (Syx4). Analysis of Syx4 mutants reveals that Syx4 mediates retrograde signaling, modulating the membrane levels of Syt4 and the transsynaptic adhesion protein Neuroligin 1 (Nlg1). Syx4-dependent trafficking regulates synaptic development, including controlling synaptic bouton number and the ability to bud new varicosities in response to acute neuronal stimulation. Genetic interaction experiments demonstrate Syx4, Syt4, and Nlg1 regulate synaptic growth and plasticity through both shared and parallel signaling pathways. Our findings suggest a conserved postsynaptic SNARE machinery controls multiple aspects of retrograde signaling and cargo trafficking within the postsynaptic compartment.
%B Elife %V 5 %8 2016 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/27223326?dopt=Abstract %R 10.7554/eLife.13881 %0 Journal Article %J Dev Biol %D 2016 %T Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila. %A Ma, Meifang %A Zhao, Hang %A Zhao, Hanfei %A Binari, Richard %A Perrimon, Norbert %A Li, Zhouhua %XAdult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. We find that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of caspase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis.
%B Dev Biol %V 411 %P 207-16 %8 2016 Mar 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/26845534?dopt=Abstract %R 10.1016/j.ydbio.2016.01.040 %0 Journal Article %J Annu Rev Cell Dev Biol %D 2015 %T Stress signaling between organs in metazoa. %A Owusu-Ansah, Edward %A Perrimon, Norbert %XMany organisms have developed a robust ability to adapt and survive in the face of environmental perturbations that threaten the integrity of their genome, proteome, or metabolome. Studies in multiple model organisms have shown that, in general, when exposed to stress, cells activate a complex prosurvival signaling network that includes immune and DNA damage response genes, chaperones, antioxidant enzymes, structural proteins, metabolic enzymes, and noncoding RNAs. The manner of activation runs the gamut from transcriptional induction of genes to increased stability of transcripts to posttranslational modification of important biosynthetic proteins within the stressed tissue. Superimposed on these largely autonomous effects are nonautonomous responses in which the stressed tissue secretes peptides and other factors that stimulate tissues in different organs to embark on processes that ultimately help the organism as a whole cope with stress. This review focuses on the mechanisms by which tissues in one organ adapt to environmental challenges by regulating stress responses in tissues of different organs.
%B Annu Rev Cell Dev Biol %V 31 %P 497-522 %8 2015 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26393775?dopt=Abstract %R 10.1146/annurev-cellbio-100814-125523 %0 Journal Article %J Science %D 2015 %T BIOSAFETY. Safeguarding gene drive experiments in the laboratory. %A Akbari, Omar S %A Bellen, Hugo J %A Bier, Ethan %A Bullock, Simon L %A Burt, Austin %A Church, George M %A Cook, Kevin R %A Duchek, Peter %A Edwards, Owain R %A Esvelt, Kevin M %A Gantz, Valentino M %A Golic, Kent G %A Gratz, Scott J %A Harrison, Melissa M %A Hayes, Keith R %A James, Anthony A %A Kaufman, Thomas C %A Knoblich, Juergen %A Malik, Harmit S %A Matthews, Kathy A %A O'Connor-Giles, Kate M %A Parks, Annette L %A Perrimon, Norbert %A Port, Fillip %A Russell, Steven %A Ueda, Ryu %A Wildonger, Jill %K Animals %K Clustered Regularly Interspaced Short Palindromic Repeats %K Containment of Biohazards %K CRISPR-Cas Systems %K Endonucleases %K Genetic Engineering %K Genetic Research %K Genome %K Organisms, Genetically Modified %K Safety %B Science %V 349 %P 927-9 %8 2015 Aug 28 %G eng %N 6251 %1 http://www.ncbi.nlm.nih.gov/pubmed/26229113?dopt=Abstract %R 10.1126/science.aac7932 %0 Book Section %B Systems Genetics: Linking Genotypes and Phenotypes %D 2015 %T Inferring Genetic Architecture from Systems Genetics Studies in Drosophila Cells %A Sun, Xiaoyun %A Mohr, Stephanie %A Vinayagam, Arunachalam %A Hong, Pengyu %A Perrimon, Norbert %B Systems Genetics: Linking Genotypes and Phenotypes %I Cambridge University Press %P 133-156 %G eng %0 Journal Article %J Development %D 2015 %T The Atg1-Tor pathway regulates yolk catabolism in Drosophila embryos. %A Kuhn, Hallie %A Sopko, Richelle %A Coughlin, Margaret %A Perrimon, Norbert %A Mitchison, Tim %K Animals %K Blotting, Western %K Drosophila %K Drosophila Proteins %K Egg Yolk %K In Situ Nick-End Labeling %K Microscopy, Electron %K Protein-Serine-Threonine Kinases %K Real-Time Polymerase Chain Reaction %K Rosaniline Dyes %K Signal Transduction %K TOR Serine-Threonine Kinases %XYolk provides an important source of nutrients during the early development of oviparous organisms. It is composed mainly of vitellogenin proteins packed into membrane-bound compartments called yolk platelets. Catabolism of yolk is initiated by acidification of the yolk platelet, leading to the activation of Cathepsin-like proteinases, but it is unknown how this process is triggered. Yolk catabolism initiates at cellularization in Drosophila melanogaster embryos. Using maternal shRNA technology we found that yolk catabolism depends on the Tor pathway and on the autophagy-initiating kinase Atg1. Whereas Atg1 was required for a burst of spatially regulated autophagy during late cellularization, autophagy was not required for initiating yolk catabolism. We propose that the conserved Tor metabolic sensing pathway regulates yolk catabolism, similar to Tor-dependent metabolic regulation on the lysosome.
%B Development %V 142 %P 3869-78 %8 2015 Nov 15 %G eng %N 22 %1 http://www.ncbi.nlm.nih.gov/pubmed/26395483?dopt=Abstract %R 10.1242/dev.125419 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2015 %T Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. %A Leshchiner, Elizaveta S %A Parkhitko, Andrey %A Bird, Gregory H %A Luccarelli, James %A Bellairs, Joseph A %A Escudero, Silvia %A Opoku-Nsiah, Kwadwo %A Godes, Marina %A Perrimon, Norbert %A Walensky, Loren D %K Animals %K Blotting, Western %K Cell Line, Tumor %K Chromatography, Gel %K Drosophila melanogaster %K Drosophila Proteins %K Escherichia coli %K Fluorescence %K Gene Expression Regulation, Enzymologic %K Humans %K Magnetic Resonance Spectroscopy %K MAP Kinase Signaling System %K Microfluidics %K Mutation %K Peptides %K Protein Binding %K Protein Structure, Secondary %K Proto-Oncogene Proteins %K ras Proteins %K SOS1 Protein %XActivating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) underlie the pathogenesis and chemoresistance of ∼ 30% of all human tumors, yet the development of high-affinity inhibitors that target the broad range of KRAS mutants remains a formidable challenge. Here, we report the development and validation of stabilized alpha helices of son of sevenless 1 (SAH-SOS1) as prototype therapeutics that directly inhibit wild-type and mutant forms of KRAS. SAH-SOS1 peptides bound in a sequence-specific manner to KRAS and its mutants, and dose-responsively blocked nucleotide association. Importantly, this functional binding activity correlated with SAH-SOS1 cytotoxicity in cancer cells expressing wild-type or mutant forms of KRAS. The mechanism of action of SAH-SOS1 peptides was demonstrated by sequence-specific down-regulation of the ERK-MAP kinase phosphosignaling cascade in KRAS-driven cancer cells and in a Drosophila melanogaster model of Ras85D(V12) activation. These studies provide evidence for the potential utility of SAH-SOS1 peptides in neutralizing oncogenic KRAS in human cancer.
%B Proc Natl Acad Sci U S A %V 112 %P 1761-6 %8 2015 Feb 10 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/25624485?dopt=Abstract %R 10.1073/pnas.1413185112 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2015 %T Discovery of progenitor cell signatures by time-series synexpression analysis during Drosophila embryonic cell immortalization. %A Dequéant, Mary-Lee %A Fagegaltier, Delphine %A Hu, Yanhui %A Spirohn, Kerstin %A Simcox, Amanda %A Hannon, Gregory J %A Perrimon, Norbert %K Animals %K Cell Differentiation %K Cell Line, Transformed %K Cell Proliferation %K Drosophila %K Embryo, Nonmammalian %K Promoter Regions, Genetic %K Retinoblastoma Protein %K Stem Cells %K Transcription, Genetic %XThe use of time series profiling to identify groups of functionally related genes (synexpression groups) is a powerful approach for the discovery of gene function. Here we apply this strategy during Ras(V12) immortalization of Drosophila embryonic cells, a phenomenon not well characterized. Using high-resolution transcriptional time-series datasets, we generated a gene network based on temporal expression profile similarities. This analysis revealed that common immortalized cells are related to adult muscle precursors (AMPs), a stem cell-like population contributing to adult muscles and sharing properties with vertebrate satellite cells. Remarkably, the immortalized cells retained the capacity for myogenic differentiation when treated with the steroid hormone ecdysone. Further, we validated in vivo the transcription factor CG9650, the ortholog of mammalian Bcl11a/b, as a regulator of AMP proliferation predicted by our analysis. Our study demonstrates the power of time series synexpression analysis to characterize Drosophila embryonic progenitor lines and identify stem/progenitor cell regulators.
%B Proc Natl Acad Sci U S A %V 112 %P 12974-9 %8 2015 Oct 20 %G eng %N 42 %1 http://www.ncbi.nlm.nih.gov/pubmed/26438832?dopt=Abstract %R 10.1073/pnas.1517729112 %0 Journal Article %J J Genomics %D 2015 %T GLAD: an Online Database of Gene List Annotation for Drosophila. %A Hu, Yanhui %A Comjean, Aram %A Perkins, Lizabeth A %A Perrimon, Norbert %A Mohr, Stephanie E %XWe present a resource of high quality lists of functionally related Drosophila genes, e.g. based on protein domains (kinases, transcription factors, etc.) or cellular function (e.g. autophagy, signal transduction). To establish these lists, we relied on different inputs, including curation from databases or the literature and mapping from other species. Moreover, as an added curation and quality control step, we asked experts in relevant fields to review many of the lists. The resource is available online for scientists to search and view, and is editable based on community input. Annotation of gene groups is an ongoing effort and scientific need will typically drive decisions regarding which gene lists to pursue. We anticipate that the number of lists will increase over time; that the composition of some lists will grow and/or change over time as new information becomes available; and that the lists will benefit the scientific community, e.g. at experimental design and data analysis stages. Based on this, we present an easily updatable online database, available at www.flyrnai.org/glad, at which gene group lists can be viewed, searched and downloaded.
%B J Genomics %V 3 %P 75-81 %8 2015 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26157507?dopt=Abstract %R 10.7150/jgen.12863 %0 Journal Article %J Nat Methods %D 2015 %T Highly efficient Cas9-mediated transcriptional programming. %A Chavez, Alejandro %A Scheiman, Jonathan %A Vora, Suhani %A Pruitt, Benjamin W %A Tuttle, Marcelle %A P R Iyer, Eswar %A Lin, Shuailiang %A Kiani, Samira %A Guzman, Christopher D %A Wiegand, Daniel J %A Ter-Ovanesyan, Dmitry %A Braff, Jonathan L %A Davidsohn, Noah %A Housden, Benjamin E %A Perrimon, Norbert %A Weiss, Ron %A Aach, John %A Collins, James J %A Church, George M %K Cell Differentiation %K Endonucleases %K Genetic Techniques %K HEK293 Cells %K Humans %K Induced Pluripotent Stem Cells %K Neurons %K RNA, Guide %K Staphylococcus aureus %K Transcriptional Activation %XThe RNA-guided nuclease Cas9 can be reengineered as a programmable transcription factor. However, modest levels of gene activation have limited potential applications. We describe an improved transcriptional regulator obtained through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to nuclease-null Cas9. We demonstrate its utility in activating endogenous coding and noncoding genes, targeting several genes simultaneously and stimulating neuronal differentiation of human induced pluripotent stem cells (iPSCs).
%B Nat Methods %V 12 %P 326-8 %8 2015 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/25730490?dopt=Abstract %R 10.1038/nmeth.3312 %0 Journal Article %J Sci Signal %D 2015 %T Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. %A Housden, Benjamin E %A Valvezan, Alexander J %A Kelley, Colleen %A Sopko, Richelle %A Hu, Yanhui %A Roesel, Charles %A Lin, Shuailiang %A Buckner, Michael %A Tao, Rong %A Yilmazel, Bahar %A Mohr, Stephanie E %A Manning, Brendan D %A Perrimon, Norbert %K Animals %K Cell Cycle Proteins %K Cell Line %K CRISPR-Cas Systems %K Drosophila melanogaster %K Drosophila Proteins %K Gene Knockdown Techniques %K Humans %K RNA, Small Interfering %K Tuberous Sclerosis %XThe tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.
%B Sci Signal %V 8 %P rs9 %8 2015 Sep 8 %G eng %N 393 %1 http://www.ncbi.nlm.nih.gov/pubmed/26350902?dopt=Abstract %R 10.1126/scisignal.aab3729 %0 Journal Article %J Genetics %D 2015 %T In Vivo Transcriptional Activation Using CRISPR/Cas9 in Drosophila. %A Lin, Shuailiang %A Ewen-Campen, Ben %A Ni, Xiaochun %A Housden, Benjamin E %A Perrimon, Norbert %XA number of approaches for Cas9-mediated transcriptional activation have recently been developed, allowing target genes to be overexpressed from their endogenous genomic loci. However, these approaches have thus far been limited to cell culture, and this technique has not been demonstrated in vivo in any animal. The technique involving the fewest separate components, and therefore the most amenable to in vivo applications, is the dCas9-VPR system, where a nuclease-dead Cas9 is fused to a highly active chimeric activator domain. In this study, we characterize the dCas9-VPR system in Drosophila cells and in vivo. We show that this system can be used in cell culture to upregulate a range of target genes, singly and in multiplex, and that a single guide RNA upstream of the transcription start site can activate high levels of target transcription. We observe marked heterogeneity in guide RNA efficacy for any given gene, and we confirm that transcription is inhibited by guide RNAs binding downstream of the transcription start site. To demonstrate one application of this technique in cells, we used dCas9-VPR to identify target genes for Twist and Snail, two highly conserved transcription factors that cooperate during Drosophila mesoderm development. In addition, we simultaneously activated both Twist and Snail to identify synergistic responses to this physiologically relevant combination. Finally, we show that dCas9-VPR can activate target genes and cause dominant phenotypes in vivo, providing the first demonstration of dCas9 activation in a multicellular animal. Transcriptional activation using dCas9-VPR thus offers a simple and broadly applicable technique for a variety of overexpression studies.
%B Genetics %V 201 %P 433-42 %8 2015 Oct %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/26245833?dopt=Abstract %R 10.1534/genetics.115.181065 %0 Journal Article %J Dev Cell %D 2015 %T Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch. %A Gordon, Wendy R %A Zimmerman, Brandon %A He, Li %A Miles, Laura J %A Huang, Jiuhong %A Tiyanont, Kittichoat %A McArthur, Debbie G %A Aster, Jon C %A Perrimon, Norbert %A Loparo, Joseph J %A Blacklow, Stephen C %K ADAM Proteins %K Allosteric Regulation %K Animals %K Artificial Cells %K Biomechanical Phenomena %K Cell Line %K Endocytosis %K HEK293 Cells %K Humans %K Ligands %K Mechanotransduction, Cellular %K Models, Biological %K Proteolysis %K Receptors, Notch %K Signal Transduction %XLigands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the Notch1 regulatory switch results in sensitivity to metalloprotease cleavage, and bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction.
%B Dev Cell %V 33 %P 729-36 %8 2015 Jun 22 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/26051539?dopt=Abstract %R 10.1016/j.devcel.2015.05.004 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2015 %T Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase. %A Chen, Chiao-Lin %A Hu, Yanhui %A Udeshi, Namrata D %A Lau, Thomas Y %A Wirtz-Peitz, Frederik %A He, Li %A Ting, Alice Y %A Carr, Steven A %A Perrimon, Norbert %K Animals %K Ascorbate Peroxidases %K Computational Biology %K Databases, Protein %K Drosophila %K Genetic Engineering %K Proteomics %K Staining and Labeling %XCharacterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate "MitoMax," a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions.
%B Proc Natl Acad Sci U S A %V 112 %P 12093-8 %8 2015 Sep 29 %G eng %N 39 %1 http://www.ncbi.nlm.nih.gov/pubmed/26362788?dopt=Abstract %R 10.1073/pnas.1515623112 %0 Journal Article %J G3 (Bethesda) %D 2015 %T Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells. %A Mohr, Stephanie E %A Hu, Yanhui %A Rudd, Kirstin %A Buckner, Michael %A Gilly, Quentin %A Foster, Blake %A Sierzputowska, Katarzyna %A Comjean, Aram %A Ye, Bing %A Perrimon, Norbert %K Adenosine Triphosphate %K Animals %K Cells, Cultured %K Computational Biology %K Drosophila melanogaster %K Drosophila Proteins %K Gene Library %K Genomics %K High-Throughput Screening Assays %K RNA Interference %K RNA, Small Interfering %K RNA-Binding Proteins %XRNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online.
%B G3 (Bethesda) %V 5 %P 1919-24 %8 2015 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/26199285?dopt=Abstract %R 10.1534/g3.115.019364 %0 Journal Article %J PLoS Genet %D 2015 %T Regulators of autophagosome formation in Drosophila muscles. %A Zirin, Jonathan %A Nieuwenhuis, Joppe %A Samsonova, Anastasia %A Tao, Rong %A Perrimon, Norbert %K Animals %K Autophagy %K Cell Communication %K Chloroquine %K Drosophila melanogaster %K Glycogen %K GTPase-Activating Proteins %K Larva %K Muscle Cells %K Muscle, Skeletal %K Muscular Diseases %K Phagosomes %K Protein Interaction Maps %K rab3 GTP-Binding Proteins %K Sirolimus %K Ubiquitin %XGiven the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.
%B PLoS Genet %V 11 %P e1005006 %8 2015 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/25692684?dopt=Abstract %R 10.1371/journal.pgen.1005006 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2015 %T spenito is required for sex determination in Drosophila melanogaster. %A Yan, Dong %A Perrimon, Norbert %K Alternative Splicing %K Animals %K Cell Differentiation %K Cell Nucleus %K Crosses, Genetic %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Gene Expression Regulation, Developmental %K Genes, Insect %K Male %K Nuclear Proteins %K Ovary %K Phenotype %K RNA Interference %K RNA Precursors %K RNA Splicing %K RNA, Messenger %K RNA-Binding Proteins %K Sex Determination Processes %K Wings, Animal %XSex-lethal (Sxl) encodes the master regulator of the sex determination pathway in Drosophila and acts by controlling sex identity in both soma and germ line. In females Sxl maintains its own expression by controlling the alternative splicing of its own mRNA. Here, we identify a novel sex determination gene, spenito (nito) that encodes a SPEN family protein. Loss of nito activity results in stem cell tumors in the female germ line as well as female-to-male somatic transformations. We show that Nito is a ubiquitous nuclear protein that controls the alternative splicing of the Sxl mRNA by interacting with Sxl protein and pre-mRNA, suggesting that it is directly involved in Sxl auto-regulation. Given that SPEN family proteins are frequently mutated in cancers, our results suggest that these factors might be implicated in tumorigenesis through splicing regulation.
%B Proc Natl Acad Sci U S A %V 112 %P 11606-11 %8 2015 Sep 15 %G eng %N 37 %1 http://www.ncbi.nlm.nih.gov/pubmed/26324914?dopt=Abstract %R 10.1073/pnas.1515891112 %0 Journal Article %J Dev Cell %D 2015 %T Stable Force Balance between Epithelial Cells Arises from F-Actin Turnover. %A Jodoin, Jeanne N %A Coravos, Jonathan S %A Chanet, Soline %A Vasquez, Claudia G %A Tworoger, Michael %A Kingston, Elena R %A Perkins, Lizabeth A %A Perrimon, Norbert %A Martin, Adam C %K Actins %K Adherens Junctions %K Animals %K Cadherins %K Cytoskeleton %K Drosophila %K Epithelial Cells %K Epithelium %K Intercellular Junctions %XThe propagation of force in epithelial tissues requires that the contractile cytoskeletal machinery be stably connected between cells through E-cadherin-containing adherens junctions. In many epithelial tissues, the cells' contractile network is positioned at a distance from the junction. However, the mechanism or mechanisms that connect the contractile networks to the adherens junctions, and thus mechanically connect neighboring cells, are poorly understood. Here, we identified the role for F-actin turnover in regulating the contractile cytoskeletal network's attachment to adherens junctions. Perturbing F-actin turnover via gene depletion or acute drug treatments that slow F-actin turnover destabilized the attachment between the contractile actomyosin network and adherens junctions. Our work identifies a critical role for F-actin turnover in connecting actomyosin to intercellular junctions, defining a dynamic process required for the stability of force balance across intercellular contacts in tissues.
%B Dev Cell %V 35 %P 685-97 %8 2015 Dec 21 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/26688336?dopt=Abstract %R 10.1016/j.devcel.2015.11.018 %0 Journal Article %J Dev Cell %D 2015 %T Systemic organ wasting induced by localized expression of the secreted insulin/IGF antagonist ImpL2. %A Kwon, Young %A Song, Wei %A Droujinine, Ilia A %A Hu, Yanhui %A Asara, John M %A Perrimon, Norbert %K Animals %K Animals, Genetically Modified %K Biomarkers %K Blotting, Western %K Cells, Cultured %K Drosophila melanogaster %K Drosophila Proteins %K Fat Body %K Female %K Gastrointestinal Tract %K Gene Expression Profiling %K Hemolymph %K Hyperglycemia %K Insulin %K Insulin-Like Growth Factor I %K Male %K Metabolomics %K Muscle, Skeletal %K Nuclear Proteins %K Oligonucleotide Array Sequence Analysis %K Ovary %K Real-Time Polymerase Chain Reaction %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Messenger %K Stem Cells %K Trans-Activators %K Wasting Syndrome %XOrgan wasting, related to changes in nutrition and metabolic activity of cells and tissues, is observed under conditions of starvation and in the context of diseases, including cancers. We have developed a model for organ wasting in adult Drosophila, whereby overproliferation induced by activation of Yorkie, the Yap1 oncogene ortholog, in intestinal stem cells leads to wasting of the ovary, fat body, and muscle. These organ-wasting phenotypes are associated with a reduction in systemic insulin/IGF signaling due to increased expression of the secreted insulin/IGF antagonist ImpL2 from the overproliferating gut. Strikingly, expression of rate-limiting glycolytic enzymes and central components of the insulin/IGF pathway is upregulated with activation of Yorkie in the gut, which may provide a mechanism for this overproliferating tissue to evade the effect of ImpL2. Altogether, our study provides insights into the mechanisms underlying organ-wasting phenotypes in Drosophila and how overproliferating tissues adapt to global changes in metabolism.
%B Dev Cell %V 33 %P 36-46 %8 2015 Apr 6 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25850671?dopt=Abstract %R 10.1016/j.devcel.2015.02.012 %0 Journal Article %J PLoS Genet %D 2015 %T A systems-level interrogation identifies regulators of Drosophila blood cell number and survival. %A Sopko, Richelle %A Lin, You Bin %A Makhijani, Kalpana %A Alexander, Brandy %A Perrimon, Norbert %A Brückner, Katja %K Animals %K Apoptosis %K Cell Line %K Cell Survival %K Drosophila melanogaster %K Drosophila Proteins %K Genome-Wide Association Study %K Hemocytes %K Insulin %K Receptor Protein-Tyrosine Kinases %K Receptors, Steroid %K RNA Interference %K Signal Transduction %XIn multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.
%B PLoS Genet %V 11 %P e1005056 %8 2015 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/25749252?dopt=Abstract %R 10.1371/journal.pgen.1005056 %0 Journal Article %J Nat Commun %D 2015 %T A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs. %A Fulga, Tudor A %A McNeill, Elizabeth M %A Binari, Richard %A Yelick, Julia %A Blanche, Alexandra %A Booker, Matthew %A Steinkraus, Bruno R %A Schnall-Levin, Michael %A Zhao, Yong %A DeLuca, Todd %A Bejarano, Fernando %A Han, Zhe %A Lai, Eric C %A Wall, Dennis P %A Perrimon, Norbert %A Van Vactor, David %K Animals %K Animals, Genetically Modified %K Drosophila %K Female %K Gene Library %K Male %K MicroRNAs %K Muscles %XAlthough the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4-8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes.
%B Nat Commun %V 6 %P 7279 %8 2015 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/26081261?dopt=Abstract %R 10.1038/ncomms8279 %0 Journal Article %J Genetics %D 2015 %T The Transgenic RNAi Project at Harvard Medical School: Resources and Validation. %A Perkins, Lizabeth A %A Holderbaum, Laura %A Tao, Rong %A Hu, Yanhui %A Sopko, Richelle %A McCall, Kim %A Yang-Zhou, Donghui %A Flockhart, Ian %A Binari, Richard %A Shim, Hye-Seok %A Miller, Audrey %A Housden, Amy %A Foos, Marianna %A Randkelv, Sakara %A Kelley, Colleen %A Namgyal, Pema %A Villalta, Christians %A Liu, Lu-Ping %A Jiang, Xia %A Huan-Huan, Qiao %A Wang, Xia %A Fujiyama, Asao %A Toyoda, Atsushi %A Ayers, Kathleen %A Blum, Allison %A Czech, Benjamin %A Neumuller, Ralph %A Yan, Dong %A Cavallaro, Amanda %A Hibbard, Karen %A Hall, Don %A Cooley, Lynn %A Hannon, Gregory J %A Lehmann, Ruth %A Parks, Annette %A Mohr, Stephanie E %A Ueda, Ryu %A Kondo, Shu %A Ni, Jian-Quan %A Perrimon, Norbert %XTo facilitate large-scale functional studies in Drosophila, the Drosophila Transgenic RNAi Project (TRiP) at Harvard Medical School (HMS) was established along with several goals: developing efficient vectors for RNAi that work in all tissues, generating a genome-scale collection of RNAi stocks with input from the community, distributing the lines as they are generated through existing stock centers, validating as many lines as possible using RT-qPCR and phenotypic analyses, and developing tools and web resources for identifying RNAi lines and retrieving existing information on their quality. With these goals in mind, here we describe in detail the various tools we developed and the status of the collection, which is currently composed of 11,491 lines and covering 71% of Drosophila genes. Data on the characterization of the lines either by RT-qPCR or phenotype is available on a dedicated website, the RNAi Stock Validation and Phenotypes Project (RSVP, http://www.flyrnai.org/RSVP.html), and stocks are available from three stock centers, the Bloomington Drosophila Stock Center (United States), National Institute of Genetics (Japan), and TsingHua Fly Center (China).
%B Genetics %V 201 %P 843-52 %8 2015 Nov %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/26320097?dopt=Abstract %R 10.1534/genetics.115.180208 %0 Journal Article %J J Cell Physiol %D 2014 %T Drosophila as a model for context-dependent tumorigenesis. %A Tipping, Marla %A Perrimon, Norbert %K Animals %K Carcinogenesis %K Disease Models, Animal %K Drosophila melanogaster %K Genes, Tumor Suppressor %K Genomic Instability %K Humans %K Neoplasms %K Oncogenes %XDrosophila can exhibit classic hallmarks of cancer, such as evasion of apoptosis, sustained proliferation, metastasis, prolonged survival, genome instability, and metabolic reprogramming, when cancer-related genes are perturbed. In the last two decades, studies in flies have identified several tumor suppressor and oncogenes. However, the greatest strength of the fly lies in its ability to model cancer hallmarks in a variety of tissue types, which enables the study of context-dependent tumorigenesis. We review the organs and tissues that have been used to model tumor formation, and propose new strategies to maximize the potential of Drosophila in cancer research.
%B J Cell Physiol %V 229 %P 27-33 %8 2014 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23836429?dopt=Abstract %R 10.1002/jcp.24427 %0 Journal Article %J Cold Spring Harb Protoc %D 2014 %T Inducing RNAi in Drosophila cells by soaking with dsRNA. %A Zhou, Rui %A Mohr, Stephanie %A Hannon, Gregory J %A Perrimon, Norbert %K Animals %K Cell Line %K Drosophila %K Molecular Biology %K RNA Interference %K RNA, Double-Stranded %XRNA interference (RNAi) triggered by synthetic long double-stranded RNAs (dsRNAs) has been applied in many Drosophila cell lines to study the functions of individual genes or for genome-wide scans. One contributor to the popularity of this approach is that many fly cell lines spontaneously take up dsRNAs from media, obviating the need for assisted uptake methods such as transfection. In this protocol, RNAi is induced in Drosophila S2 cells by soaking with dsRNA. Cell lines other than S2 can also be used, although the ability of each line to passively take up dsRNA does vary. Therefore, the efficiency of passive uptake should be carefully verified for each line.
%B Cold Spring Harb Protoc %V 2014 %8 2014 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/24786505?dopt=Abstract %R 10.1101/pdb.prot080747 %0 Journal Article %J Dev Dyn %D 2014 %T Mechanisms of muscle growth and atrophy in mammals and Drosophila. %A Piccirillo, Rosanna %A Demontis, Fabio %A Perrimon, Norbert %A Goldberg, Alfred L %K Animals %K Drosophila %K Inflammation %K Mammals %K Models, Animal %K Models, Biological %K Muscle Development %K Muscular Atrophy %K Myostatin %K Neurodegenerative Diseases %K Oxidative Stress %K Proteolysis %K Signal Transduction %K Transcription Factors %XBACKGROUND: The loss of skeletal muscle mass (atrophy) that accompanies disuse and systemic diseases is highly debilitating. Although the pathogenesis of this condition has been primarily studied in mammals, Drosophila is emerging as an attractive system to investigate some of the mechanisms involved in muscle growth and atrophy. RESULTS: In this review, we highlight the outstanding unsolved questions that may benefit from a combination of studies in both flies and mammals. In particular, we discuss how different environmental stimuli and signaling pathways influence muscle mass and strength and how a variety of disease states can cause muscle wasting. CONCLUSIONS: Studies in Drosophila and mammals should help identify molecular targets for the treatment of muscle wasting in humans.
%B Dev Dyn %V 243 %P 201-15 %8 2014 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/24038488?dopt=Abstract %R 10.1002/dvdy.24036 %0 Journal Article %J Dis Model Mech %D 2014 %T Modeling metabolic homeostasis and nutrient sensing in Drosophila: implications for aging and metabolic diseases. %A Owusu-Ansah, Edward %A Perrimon, Norbert %K Aging %K Animals %K Drosophila melanogaster %K Feeding Behavior %K Homeostasis %K Metabolic Diseases %K Models, Biological %XOver the past decade, numerous reports have underscored the similarities between the metabolism of Drosophila and vertebrates, with the identification of evolutionarily conserved enzymes and analogous organs that regulate carbohydrate and lipid metabolism. It is now well established that the major metabolic, energy-sensing and endocrine signaling networks of vertebrate systems are also conserved in flies. Accordingly, studies in Drosophila are beginning to unravel how perturbed energy balance impinges on lifespan and on the ensuing diseases when energy homeostasis goes awry. Here, we highlight several emerging concepts that are at the nexus between obesity, nutrient sensing, metabolic homeostasis and aging. Specifically, we summarize the endocrine mechanisms that regulate carbohydrate and lipid metabolism, and provide an overview of the neuropeptides that regulate feeding behavior. We further describe the various efforts at modeling the effects of high-fat or -sugar diets in Drosophila and the signaling mechanisms involved in integrating organ function. Finally, we draw attention to some of the cardinal discoveries made with these disease models and how these could spur new research questions in vertebrate systems.
%B Dis Model Mech %V 7 %P 343-50 %8 2014 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/24609035?dopt=Abstract %R 10.1242/dmm.012989 %0 Journal Article %J Genetics %D 2014 %T Resources for functional genomics studies in Drosophila melanogaster. %A Mohr, Stephanie E %A Hu, Yanhui %A Kim, Kevin %A Housden, Benjamin E %A Perrimon, Norbert %K Animals %K Databases, Genetic %K Drosophila melanogaster %K Genetic Engineering %K Genome, Insect %K Genomics %K Humans %XDrosophila melanogaster has become a system of choice for functional genomic studies. Many resources, including online databases and software tools, are now available to support design or identification of relevant fly stocks and reagents or analysis and mining of existing functional genomic, transcriptomic, proteomic, etc. datasets. These include large community collections of fly stocks and plasmid clones, "meta" information sites like FlyBase and FlyMine, and an increasing number of more specialized reagents, databases, and online tools. Here, we introduce key resources useful to plan large-scale functional genomics studies in Drosophila and to analyze, integrate, and mine the results of those studies in ways that facilitate identification of highest-confidence results and generation of new hypotheses. We also discuss ways in which existing resources can be used and might be improved and suggest a few areas of future development that would further support large- and small-scale studies in Drosophila and facilitate use of Drosophila information by the research community more generally.
%B Genetics %V 197 %P 1-18 %8 2014 May %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/24653003?dopt=Abstract %R 10.1534/genetics.113.154344 %0 Journal Article %J Nat Rev Mol Cell Biol %D 2014 %T RNAi screening comes of age: improved techniques and complementary approaches. %A Mohr, Stephanie E %A Smith, Jennifer A %A Shamu, Caroline E %A Neumüller, Ralph A %A Perrimon, Norbert %K Animals %K Gene Regulatory Networks %K Genetic Testing %K Humans %K Inverted Repeat Sequences %K RNA Interference %K RNA, Small Interfering %XGene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.
%B Nat Rev Mol Cell Biol %V 15 %P 591-600 %8 2014 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/25145850?dopt=Abstract %R 10.1038/nrm3860 %0 Journal Article %J J Cell Biol %D 2014 %T A sharp end to sugary Wingless travels. %A Droujinine, Ilia A %A Yan, Dong %A Perrimon, Norbert %K Animals %K Cell Proliferation %K Drosophila melanogaster %K Female %K Male %K Matrix Metalloproteinase 2 %K Stem Cells %XDrosophila melanogaster follicle stem cells are controlled by Wingless (Wg) ligands secreted 50 µm away, raising the question of how long-distance Wg spreading occurs. In this issue of JCB, Wang and Page-McCaw (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201403084) demonstrate a potential mechanism by which the heparan sulfate proteoglycan Dally-like (Dlp) promotes Wg travel, whereas matrix Mmp2 (Metalloproteinase 2) impedes it by inactivating Dlp.
%B J Cell Biol %V 206 %P 819-21 %8 2014 Sep 29 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/25267292?dopt=Abstract %R 10.1083/jcb.201408115 %0 Journal Article %J Trends Biochem Sci %D 2014 %T Spatial and temporal organization of signaling pathways. %A Housden, Benjamin E %A Perrimon, Norbert %K Animals %K Cell Communication %K Cells, Cultured %K Drosophila %K Feedback %K Janus Kinases %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %K Transcription Factors %XThe development and maintenance of the many different cell types in metazoan organisms requires robust and diverse intercellular communication mechanisms. Relatively few such signaling pathways have been identified, leading to the question of how such a broad diversity of output is generated from relatively simple signals. Recent studies have revealed complex mechanisms integrating temporal and spatial information to generate diversity in signaling pathway output. We review some general principles of signaling pathways, focusing on transcriptional outputs in Drosophila. We consider the role of spatial and temporal aspects of different transduction pathways and then discuss how recently developed tools and approaches are helping to dissect the complex mechanisms linking pathway stimulation to output.
%B Trends Biochem Sci %V 39 %P 457-64 %8 2014 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/25155749?dopt=Abstract %R 10.1016/j.tibs.2014.07.008 %0 Journal Article %J Sci Signal %D 2014 %T Visualizing and manipulating temporal signaling dynamics with fluorescence-based tools. %A Doupé, David P %A Perrimon, Norbert %K Animals %K Caenorhabditis elegans %K Cell Tracking %K Cells, Cultured %K Fluorescence Resonance Energy Transfer %K Humans %K Microscopy, Fluorescence %K Molecular Dynamics Simulation %K Sensory Receptor Cells %K Signal Transduction %XThe use of genome-wide proteomic and RNA interference approaches has moved our understanding of signal transduction from linear pathways to highly integrated networks centered on core nodes. However, probing the dynamics of flow of information through such networks remains technically challenging. In particular, how the temporal dynamics of an individual pathway can elicit distinct outcomes in a single cell type and how multiple pathways may interact sequentially or synchronously to influence cell fate remain open questions in many contexts. The development of fluorescence-based reporters and optogenetic regulators of pathway activity enables the analysis of signaling in living cells and organisms with unprecedented spatiotemporal resolution and holds the promise of addressing these key questions. We present a brief overview of the evidence for the importance of temporal dynamics in cellular regulation, introduce these fluorescence-based tools, and highlight specific studies that leveraged these tools to probe the dynamics of information flow through signaling networks. In particular, we highlight two studies in Caenorhabditis elegans sensory neurons and cultured mammalian cells that demonstrate the importance of signal dynamics in determining cellular responses.
%B Sci Signal %V 7 %P re1 %8 2014 Apr 1 %G eng %N 319 %1 http://www.ncbi.nlm.nih.gov/pubmed/24692594?dopt=Abstract %R 10.1126/scisignal.2005077 %0 Journal Article %J Methods %D 2014 %T Drosophila developmental biology methods. %A Perrimon, Norbert %K Animals %K Developmental Biology %K Drosophila %B Methods %V 68 %P 1 %8 2014 Jun 15 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/24908372?dopt=Abstract %R 10.1016/j.ymeth.2014.05.010 %0 Journal Article %J Dev Cell %D 2014 %T Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos. %A Sopko, Richelle %A Foos, Marianna %A Vinayagam, Arunachalam %A Zhai, Bo %A Binari, Richard %A Hu, Yanhui %A Randklev, Sakara %A Perkins, Lizabeth A %A Gygi, Steven P %A Perrimon, Norbert %K Animals %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Gene Expression Regulation, Developmental %K Gene Knockdown Techniques %K Gene Regulatory Networks %K Phosphoprotein Phosphatases %K Protein Kinases %K Proteome %XConnecting phosphorylation events to kinases and phosphatases is key to understanding the molecular organization and signaling dynamics of networks. We have generated a validated set of transgenic RNA-interference reagents for knockdown and characterization of all protein kinases and phosphatases present during early Drosophila melanogaster development. These genetic tools enable collection of sufficient quantities of embryos depleted of single gene products for proteomics. As a demonstration of an application of the collection, we have used multiplexed isobaric labeling for quantitative proteomics to derive global phosphorylation signatures associated with kinase-depleted embryos to systematically link phosphosites with relevant kinases. We demonstrate how this strategy uncovers kinase consensus motifs and prioritizes phosphoproteins for kinase target validation. We validate this approach by providing auxiliary evidence for Wee kinase-directed regulation of the chromatin regulator Stonewall. Further, we show how correlative phosphorylation at the site level can indicate function, as exemplified by Sterile20-like kinase-dependent regulation of Stat92E.
%B Dev Cell %V 31 %P 114-27 %8 2014 Oct 13 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25284370?dopt=Abstract %R 10.1016/j.devcel.2014.07.027 %0 Journal Article %J Nature %D 2014 %T Comparative analysis of the transcriptome across distant species. %A Gerstein, Mark B %A Rozowsky, Joel %A Yan, Koon-Kiu %A Wang, Daifeng %A Cheng, Chao %A Brown, James B %A Davis, Carrie A %A Hillier, LaDeana %A Sisu, Cristina %A Li, Jingyi Jessica %A Pei, Baikang %A Harmanci, Arif O %A Duff, Michael O %A Djebali, Sarah %A Alexander, Roger P %A Alver, Burak H %A Auerbach, Raymond %A Bell, Kimberly %A Bickel, Peter J %A Boeck, Max E %A Boley, Nathan P %A Booth, Benjamin W %A Cherbas, Lucy %A Cherbas, Peter %A Di, Chao %A Dobin, Alex %A Drenkow, Jorg %A Ewing, Brent %A Fang, Gang %A Fastuca, Megan %A Feingold, Elise A %A Frankish, Adam %A Gao, Guanjun %A Good, Peter J %A Guigó, Roderic %A Hammonds, Ann %A Harrow, Jen %A Hoskins, Roger A %A Howald, Cédric %A Hu, Long %A Huang, Haiyan %A Hubbard, Tim J P %A Huynh, Chau %A Jha, Sonali %A Kasper, Dionna %A Kato, Masaomi %A Kaufman, Thomas C %A Kitchen, Robert R %A Ladewig, Erik %A Lagarde, Julien %A Lai, Eric %A Leng, Jing %A Lu, Zhi %A MacCoss, Michael %A May, Gemma %A McWhirter, Rebecca %A Merrihew, Gennifer %A Miller, David M %A Mortazavi, Ali %A Murad, Rabi %A Oliver, Brian %A Olson, Sara %A Park, Peter J %A Pazin, Michael J %A Perrimon, Norbert %A Pervouchine, Dmitri %A Reinke, Valerie %A Reymond, Alexandre %A Robinson, Garrett %A Samsonova, Anastasia %A Saunders, Gary I %A Schlesinger, Felix %A Sethi, Anurag %A Slack, Frank J %A Spencer, William C %A Stoiber, Marcus H %A Strasbourger, Pnina %A Tanzer, Andrea %A Thompson, Owen A %A Wan, Kenneth H %A Wang, Guilin %A Wang, Huaien %A Watkins, Kathie L %A Wen, Jiayu %A Wen, Kejia %A Xue, Chenghai %A Yang, Li %A Yip, Kevin %A Zaleski, Chris %A Zhang, Yan %A Zheng, Henry %A Brenner, Steven E %A Graveley, Brenton R %A Celniker, Susan E %A Gingeras, Thomas R %A Waterston, Robert %K Animals %K Caenorhabditis elegans %K Chromatin %K Cluster Analysis %K Drosophila melanogaster %K Gene Expression Profiling %K Gene Expression Regulation, Developmental %K Histones %K Humans %K Larva %K Models, Genetic %K Molecular Sequence Annotation %K Promoter Regions, Genetic %K Pupa %K RNA, Untranslated %K Sequence Analysis, RNA %K Transcriptome %XThe transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.
%B Nature %V 512 %P 445-8 %8 2014 Aug 28 %G eng %N 7515 %1 http://www.ncbi.nlm.nih.gov/pubmed/25164755?dopt=Abstract %R 10.1038/nature13424 %0 Journal Article %J Cell Rep %D 2014 %T Control of lipid metabolism by tachykinin in Drosophila. %A Song, Wei %A Veenstra, Jan A %A Perrimon, Norbert %K Animals %K Drosophila %K Enterocytes %K Homeostasis %K Lipid Metabolism %K Tachykinins %XThe intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. We demonstrate that tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila and that TKs repress lipogenesis in enterocytes (ECs) associated with TKR99D receptor and protein kinase A (PKA) signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions.
%B Cell Rep %V 9 %P 40-7 %8 2014 Oct 9 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25263556?dopt=Abstract %R 10.1016/j.celrep.2014.08.060 %0 Journal Article %J Nature %D 2014 %T Diversity and dynamics of the Drosophila transcriptome. %A Brown, James B %A Boley, Nathan %A Eisman, Robert %A May, Gemma E %A Stoiber, Marcus H %A Duff, Michael O %A Booth, Ben W %A Wen, Jiayu %A Park, Soo %A Suzuki, Ana Maria %A Wan, Kenneth H %A Yu, Charles %A Zhang, Dayu %A Carlson, Joseph W %A Cherbas, Lucy %A Eads, Brian D %A Miller, David %A Mockaitis, Keithanne %A Roberts, Johnny %A Davis, Carrie A %A Frise, Erwin %A Hammonds, Ann S %A Olson, Sara %A Shenker, Sol %A Sturgill, David %A Samsonova, Anastasia A %A Weiszmann, Richard %A Robinson, Garret %A Hernandez, Juan %A Andrews, Justen %A Bickel, Peter J %A Carninci, Piero %A Cherbas, Peter %A Gingeras, Thomas R %A Hoskins, Roger A %A Kaufman, Thomas C %A Lai, Eric C %A Oliver, Brian %A Perrimon, Norbert %A Graveley, Brenton R %A Celniker, Susan E %K Alternative Splicing %K Animals %K Drosophila melanogaster %K Female %K Gene Expression Profiling %K Male %K Molecular Sequence Annotation %K Nerve Tissue %K Organ Specificity %K Poly A %K Polyadenylation %K Promoter Regions, Genetic %K RNA, Long Noncoding %K RNA, Messenger %K Sex Characteristics %K Stress, Physiological %K Transcriptome %XAnimal transcriptomes are dynamic, with each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. Here we have identified new genes, transcripts and proteins using poly(A)+ RNA sequencing from Drosophila melanogaster in cultured cell lines, dissected organ systems and under environmental perturbations. We found that a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long non-coding RNAs (lncRNAs), some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized, with this complexity arising from combinatorial usage of promoters, splice sites and polyadenylation sites.
%B Nature %V 512 %P 393-9 %8 2014 Aug 28 %G eng %N 7515 %1 http://www.ncbi.nlm.nih.gov/pubmed/24670639?dopt=Abstract %R 10.1038/nature12962 %0 Journal Article %J Cell Rep %D 2014 %T Enteroendocrine cells support intestinal stem-cell-mediated homeostasis in Drosophila. %A Amcheslavsky, Alla %A Song, Wei %A Li, Qi %A Nie, Yingchao %A Bragatto, Ivan %A Ferrandon, Dominique %A Perrimon, Norbert %A Ip, Y Tony %K Animals %K Cell Differentiation %K Drosophila %K Enterocytes %K Enteroendocrine Cells %K Female %K Homeostasis %K Intestines %K Male %K Stem Cells %K Tachykinins %XIntestinal stem cells in the adult Drosophila midgut are regulated by growth factors produced from the surrounding niche cells including enterocytes and visceral muscle. The role of the other major cell type, the secretory enteroendocrine cells, in regulating intestinal stem cells remains unclear. We show here that newly eclosed scute loss-of-function mutant flies are completely devoid of enteroendocrine cells. These enteroendocrine cell-less flies have normal ingestion and fecundity but shorter lifespan. Moreover, in these newly eclosed mutant flies, the diet-stimulated midgut growth that depends on the insulin-like peptide 3 expression in the surrounding muscle is defective. The depletion of Tachykinin-producing enteroendocrine cells or knockdown of Tachykinin leads to a similar although less severe phenotype. These results establish that enteroendocrine cells serve as an important link between diet and visceral muscle expression of an insulin-like growth factor to stimulate intestinal stem cell proliferation and tissue growth.
%B Cell Rep %V 9 %P 32-9 %8 2014 Oct 9 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/25263551?dopt=Abstract %R 10.1016/j.celrep.2014.08.052 %0 Journal Article %J Hum Mol Genet %D 2014 %T Functional screening in Drosophila identifies Alzheimer's disease susceptibility genes and implicates Tau-mediated mechanisms. %A Shulman, Joshua M %A Imboywa, Selina %A Giagtzoglou, Nikolaos %A Powers, Martin P %A Hu, Yanhui %A Devenport, Danelle %A Chipendo, Portia %A Chibnik, Lori B %A Diamond, Allison %A Perrimon, Norbert %A Brown, Nicholas H %A De Jager, Philip L %A Feany, Mel B %K Alzheimer Disease %K Animals %K Animals, Genetically Modified %K Antigens, CD11b %K Disease Models, Animal %K Drosophila melanogaster %K Drosophila Proteins %K Gene Knockdown Techniques %K Genetic Association Studies %K Genetic Predisposition to Disease %K Humans %K Integrins %K RNA Interference %K tau Proteins %XUsing a Drosophila model of Alzheimer's disease (AD), we systematically evaluated 67 candidate genes based on AD-associated genomic loci (P < 10(-4)) from published human genome-wide association studies (GWAS). Genetic manipulation of 87 homologous fly genes was tested for modulation of neurotoxicity caused by human Tau, which forms neurofibrillary tangle pathology in AD. RNA interference (RNAi) targeting 9 genes enhanced Tau neurotoxicity, and in most cases reciprocal activation of gene expression suppressed Tau toxicity. Our screen implicates cindr, the fly ortholog of the human CD2AP AD susceptibility gene, as a modulator of Tau-mediated disease mechanisms. Importantly, we also identify the fly orthologs of FERMT2 and CELF1 as Tau modifiers, and these loci have been independently validated as AD susceptibility loci in the latest GWAS meta-analysis. Both CD2AP and FERMT2 have been previously implicated with roles in cell adhesion, and our screen additionally identifies a fly homolog of the human integrin adhesion receptors, ITGAM and ITGA9, as a modifier of Tau neurotoxicity. Our results highlight cell adhesion pathways as important in Tau toxicity and AD susceptibility and demonstrate the power of model organism genetic screens for the functional follow-up of human GWAS.
%B Hum Mol Genet %V 23 %P 870-7 %8 2014 Feb 15 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/24067533?dopt=Abstract %R 10.1093/hmg/ddt478 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2014 %T Genetic odyssey to generate marked clones in Drosophila mosaics. %A Griffin, Ruth %A Binari, Richard %A Perrimon, Norbert %K Animals %K Clone Cells %K Drosophila %K Genetic Techniques %K Mitosis %K Mosaicism %K Recombination, Genetic %XChimaeras, fanciful beasts that drew their force from being composed of parts of disparate animals, have stimulated our collective imagination for centuries. In modern terms, chimaeras are composite animals consisting of genetically distinct cell populations and are called "mosaics" if the different cell types have emerged from the same zygote. Phenotypic studies of chimeric animals formed from invertebrates, amphibians, birds, and mammals have provided many fundamental insights into biological processes, most notably in developmental biology. Many methods for generating both chimaeras and a range of markers for tracing their lineages have been developed over the years. Our laboratory has been intimately involved in the development of methods that facilitate the creation of genetic mosaics in Drosophila. Here, we review our contributions to the development of this field and discuss a number of approaches that will improve further the tool kit for generating mosaic animals.
%B Proc Natl Acad Sci U S A %V 111 %P 4756-63 %8 2014 Apr 1 %G eng %N 13 %1 http://www.ncbi.nlm.nih.gov/pubmed/24623854?dopt=Abstract %R 10.1073/pnas.1403218111 %0 Journal Article %J Nat Methods %D 2014 %T Integrating protein-protein interaction networks with phenotypes reveals signs of interactions. %A Vinayagam, Arunachalam %A Zirin, Jonathan %A Roesel, Charles %A Hu, Yanhui %A Yilmazel, Bahar %A Samsonova, Anastasia A %A Neumüller, Ralph A %A Mohr, Stephanie E %A Perrimon, Norbert %K Alcohol Oxidoreductases %K Aldehyde Reductase %K Animals %K Computational Biology %K Drosophila melanogaster %K Gene Expression Regulation %K Phenotype %K Proteasome Endopeptidase Complex %K Protein Interaction Mapping %K Protein Interaction Maps %K Proteins %K RNA Interference %K RNA, Double-Stranded %K Signal Transduction %K Systems Biology %XA major objective of systems biology is to organize molecular interactions as networks and to characterize information flow within networks. We describe a computational framework to integrate protein-protein interaction (PPI) networks and genetic screens to predict the 'signs' of interactions (i.e., activation-inhibition relationships). We constructed a Drosophila melanogaster signed PPI network consisting of 6,125 signed PPIs connecting 3,352 proteins that can be used to identify positive and negative regulators of signaling pathways and protein complexes. We identified an unexpected role for the metabolic enzymes enolase and aldo-keto reductase as positive and negative regulators of proteolysis, respectively. Characterization of the activation-inhibition relationships between physically interacting proteins within signaling pathways will affect our understanding of many biological functions, including signal transduction and mechanisms of disease.
%B Nat Methods %V 11 %P 94-9 %8 2014 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/24240319?dopt=Abstract %R 10.1038/nmeth.2733 %0 Journal Article %J Cell Rep %D 2014 %T Intertissue control of the nucleolus via a myokine-dependent longevity pathway. %A Demontis, Fabio %A Patel, Vishal K %A Swindell, William R %A Perrimon, Norbert %K Adipocytes %K Aging %K Amino Acid Sequence %K Animals %K Basic Helix-Loop-Helix Leucine Zipper Transcription Factors %K Cell Nucleolus %K Drosophila %K Drosophila Proteins %K Longevity %K Molecular Sequence Data %K Muscle, Skeletal %K Myostatin %K p38 Mitogen-Activated Protein Kinases %K RNA, Ribosomal %K Transforming Growth Factor beta %XRecent evidence indicates that skeletal muscle influences systemic aging, but little is known about the signaling pathways and muscle-released cytokines (myokines) responsible for this intertissue communication. Here, we show that muscle-specific overexpression of the transcription factor Mnt decreases age-related climbing defects and extends lifespan in Drosophila. Mnt overexpression in muscle autonomously decreases the expression of nucleolar components and systemically decreases rRNA levels and the size of the nucleolus in adipocytes. This nonautonomous control of the nucleolus, a regulator of ribosome biogenesis and lifespan, relies on Myoglianin, a myokine induced by Mnt and orthologous to human GDF11 and Myostatin. Myoglianin overexpression in muscle extends lifespan and decreases nucleolar size in adipocytes by activating p38 mitogen-activated protein kinase (MAPK), whereas Myoglianin RNAi in muscle has converse effects. Altogether, these findings highlight a key role for myokine signaling in the integration of signaling events in muscle and distant tissues during aging.
%B Cell Rep %V 7 %P 1481-94 %8 2014 Jun 12 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/24882005?dopt=Abstract %R 10.1016/j.celrep.2014.05.001 %0 Journal Article %J BMC Bioinformatics %D 2014 %T Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis. %A Yilmazel, Bahar %A Hu, Yanhui %A Sigoillot, Frederic %A Smith, Jennifer A %A Shamu, Caroline E %A Perrimon, Norbert %A Mohr, Stephanie E %K Animals %K Drosophila melanogaster %K High-Throughput Nucleotide Sequencing %K MicroRNAs %K RNA Interference %K RNA, Small Interfering %K Sequence Analysis, RNA %XBACKGROUND: RNA interference (RNAi) is an effective and important tool used to study gene function. For large-scale screens, RNAi is used to systematically down-regulate genes of interest and analyze their roles in a biological process. However, RNAi is associated with off-target effects (OTEs), including microRNA (miRNA)-like OTEs. The contribution of reagent-specific OTEs to RNAi screen data sets can be significant. In addition, the post-screen validation process is time and labor intensive. Thus, the availability of robust approaches to identify candidate off-targeted transcripts would be beneficial. RESULTS: Significant efforts have been made to eliminate false positive results attributable to sequence-specific OTEs associated with RNAi. These approaches have included improved algorithms for RNAi reagent design, incorporation of chemical modifications into siRNAs, and the use of various bioinformatics strategies to identify possible OTEs in screen results. Genome-wide Enrichment of Seed Sequence matches (GESS) was developed to identify potential off-targeted transcripts in large-scale screen data by seed-region analysis. Here, we introduce a user-friendly web application that provides researchers a relatively quick and easy way to perform GESS analysis on data from human or mouse cell-based screens using short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), as well as for Drosophila screens using shRNAs. Online GESS relies on up-to-date transcript sequence annotations for human and mouse genes extracted from NCBI Reference Sequence (RefSeq) and Drosophila genes from FlyBase. The tool also accommodates analysis with user-provided reference sequence files. CONCLUSION: Online GESS provides a straightforward user interface for genome-wide seed region analysis for human, mouse and Drosophila RNAi screen data. With the tool, users can either use a built-in database or provide a database of transcripts for analysis. This makes it possible to analyze RNAi data from any organism for which the user can provide transcript sequences.
%B BMC Bioinformatics %V 15 %P 192 %8 2014 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/24934636?dopt=Abstract %R 10.1186/1471-2105-15-192 %0 Journal Article %J Cell Rep %D 2014 %T A rapid genome-wide microRNA screen identifies miR-14 as a modulator of Hedgehog signaling. %A Kim, Kevin %A Vinayagam, Arunachalam %A Perrimon, Norbert %K 3' Untranslated Regions %K Animals %K Down-Regulation %K Drosophila %K Genome %K Hedgehog Proteins %K MicroRNAs %K Signal Transduction %XMicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to sequences within the 3' UTR of mRNAs. Because miRNAs bind to short sequences with partial complementarity, target identification is challenging. To complement the existing target prediction algorithms, we devised a systematic "reverse approach" screening platform that allows the empirical prediction of miRNA-target interactions. Using Drosophila cells, we screened the 3' untranslated regions (3' UTRs) of the Hedgehog pathway genes against a genome-wide miRNA library and identified both predicted and many nonpredicted miRNA-target interactions. We demonstrate that miR-14 is essential for maintaining the proper level of Hedgehog signaling activity by regulating its physiological target, hedgehog. Furthermore, elevated levels of miR-14 suppress Hedgehog signaling activity by cotargeting its apparent nonphysiological targets, patched and smoothened. Altogether, our systematic screening platform is a powerful approach to identifying both physiological and apparent nonphysiological targets of miRNAs, which are relevant in both normal and diseased tissues.
%B Cell Rep %V 7 %P 2066-77 %8 2014 Jun 26 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/24931604?dopt=Abstract %R 10.1016/j.celrep.2014.05.025 %0 Journal Article %J Dev Cell %D 2014 %T A regulatory network of Drosophila germline stem cell self-renewal. %A Yan, Dong %A Neumüller, Ralph A %A Buckner, Michael %A Ayers, Kathleen %A Li, Hua %A Hu, Yanhui %A Yang-Zhou, Donghui %A Pan, Lei %A Wang, Xiaoxi %A Kelley, Colleen %A Vinayagam, Arunachalam %A Binari, Richard %A Randklev, Sakara %A Perkins, Lizabeth A %A Xie, Ting %A Cooley, Lynn %A Perrimon, Norbert %K Animals %K Cell Differentiation %K Cell Division %K Cell Lineage %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Germ Cells %K Ovary %K RNA Interference %K Signal Transduction %K Stem Cells %XStem cells possess the capacity to generate two cells of distinct fate upon division: one cell retaining stem cell identity and the other cell destined to differentiate. These cell fates are established by cell-type-specific genetic networks. To comprehensively identify components of these networks, we performed a large-scale RNAi screen in Drosophila female germline stem cells (GSCs) covering ∼25% of the genome. The screen identified 366 genes that affect GSC maintenance, differentiation, or other processes involved in oogenesis. Comparison of GSC regulators with neural stem cell self-renewal factors identifies common and cell-type-specific self-renewal genes. Importantly, we identify the histone methyltransferase Set1 as a GSC-specific self-renewal factor. Loss of Set1 in neural stem cells does not affect cell fate decisions, suggesting a differential requirement of H3K4me3 in different stem cell lineages. Altogether, our study provides a resource that will help to further dissect the networks underlying stem cell self-renewal.
%B Dev Cell %V 28 %P 459-73 %8 2014 Feb 24 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/24576427?dopt=Abstract %R 10.1016/j.devcel.2014.01.020 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2014 %T Systematic screen of chemotherapeutics in Drosophila stem cell tumors. %A Markstein, Michele %A Dettorre, Samantha %A Cho, Julio %A Neumüller, Ralph A %A Craig-Müller, Sören %A Perrimon, Norbert %K Animals %K Antineoplastic Agents %K Cell Proliferation %K Disease Models, Animal %K Drosophila %K Drug Screening Assays, Antitumor %K Neoplasms, Experimental %K Neoplastic Stem Cells %K Tumor Microenvironment %XHere we report the development of an in vivo system to study the interaction of stem cells with drugs using a tumor model in the adult Drosophila intestine. Strikingly, we find that some Food and Drug Administration-approved chemotherapeutics that can inhibit the growth of Drosophila tumor stem cells can paradoxically promote the hyperproliferation of their wild-type counterparts. These results reveal an unanticipated side effect on stem cells that may contribute to tumor recurrence. We propose that the same side effect may occur in humans based on our finding that it is driven in Drosophila by the evolutionarily conserved Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. An immediate implication of our findings is that supplementing traditional chemotherapeutics with anti-inflammatories may reduce tumor recurrence.
%B Proc Natl Acad Sci U S A %V 111 %P 4530-5 %8 2014 Mar 25 %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/24616500?dopt=Abstract %R 10.1073/pnas.1401160111 %0 Book Section %B Methods Enzymol %D 2014 %T Cas9-based genome editing in Drosophila. %A Housden, Benjamin E %A Lin, Shuailiang %A Perrimon, Norbert %K Animals %K Cloning, Molecular %K Clustered Regularly Interspaced Short Palindromic Repeats %K CRISPR-Cas Systems %K Drosophila %K Gene Targeting %K Genetic Engineering %K Genetic Vectors %K Genome %K Homologous Recombination %K Mutagenesis %K RNA, Guide %XOur ability to modify the Drosophila genome has recently been revolutionized by the development of the CRISPR system. The simplicity and high efficiency of this system allows its widespread use for many different applications, greatly increasing the range of genome modification experiments that can be performed. Here, we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals.
%B Methods Enzymol %V 546 %P 415-39 %8 2014 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/25398351?dopt=Abstract %R 10.1016/B978-0-12-801185-0.00019-2 %0 Journal Article %J Dis Model Mech %D 2013 %T Mechanisms of skeletal muscle aging: insights from Drosophila and mammalian models. %A Demontis, Fabio %A Piccirillo, Rosanna %A Goldberg, Alfred L %A Perrimon, Norbert %K Aging %K Animals %K Drosophila melanogaster %K Homeostasis %K Mammals %K Mitochondria, Muscle %K Models, Animal %K Models, Biological %K Muscle, Skeletal %K Regeneration %K Stem Cells %XA characteristic feature of aged humans and other mammals is the debilitating, progressive loss of skeletal muscle function and mass that is known as sarcopenia. Age-related muscle dysfunction occurs to an even greater extent during the relatively short lifespan of the fruit fly Drosophila melanogaster. Studies in model organisms indicate that sarcopenia is driven by a combination of muscle tissue extrinsic and intrinsic factors, and that it fundamentally differs from the rapid atrophy of muscles observed following disuse and fasting. Extrinsic changes in innervation, stem cell function and endocrine regulation of muscle homeostasis contribute to muscle aging. In addition, organelle dysfunction and compromised protein homeostasis are among the primary intrinsic causes. Some of these age-related changes can in turn contribute to the induction of compensatory stress responses that have a protective role during muscle aging. In this Review, we outline how studies in Drosophila and mammalian model organisms can each provide distinct advantages to facilitate the understanding of this complex multifactorial condition and how they can be used to identify suitable therapies.
%B Dis Model Mech %V 6 %P 1339-52 %8 2013 Nov %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/24092876?dopt=Abstract %R 10.1242/dmm.012559 %0 Journal Article %J Frontiers in Cellular and Infection Microbiology %D 2013 %T Defining the interorgan communication network: Systemic coordination of organismal cellular processes under homeostasis and localized stress. %A Droujinine, Ilia A %A Perrimon, Norbert %B Frontiers in Cellular and Infection Microbiology %V 3 %P 1-5 %G eng %N 82 %0 Journal Article %J Cold Spring Harb Protoc %D 2013 %T Inducing RNAi in Drosophila cells by transfection with dsRNA. %A Zhou, Rui %A Mohr, Stephanie %A Hannon, Gregory J %A Perrimon, Norbert %K Animals %K Cell Line %K Drosophila %K Plasmids %K RNA Interference %K RNA, Double-Stranded %K Transfection %XIn Drosophila cells, RNA interference (RNAi) can be triggered by synthetic long double-stranded RNAs (dsRNAs). For many Drosophila cell lines and cell types, passive dsRNA uptake is inefficient. More complete silencing responses can often be obtained in Drosophila S2 cells using transfection, perhaps because higher levels of intracellular dsRNA are achieved. In this protocol, S2 cells are transfected with dsRNA using QIAGEN's Effectene reagent, which has proven to be reliable for many investigators. A plasmid DNA can also be included in the transfection mix to provide additional functionality. The plasmid DNA can encode, for example, a reporter of the activity of a pathway or specific transcription factor, or a marker that allows visualization of some cellular behavior or structure. It is also useful to include a plasmid that encodes a fluorescent protein simply to monitor transfection efficiency.
%B Cold Spring Harb Protoc %V 2013 %P 461-3 %8 2013 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/23637371?dopt=Abstract %R 10.1101/pdb.prot074351 %0 Journal Article %J BMC Biol %D 2013 %T Of flies and men: insights on organismal metabolism from fruit flies. %A Rajan, Akhila %A Perrimon, Norbert %K Animals %K Disease Models, Animal %K Drosophila melanogaster %K Humans %K Lipid Metabolism %K Longevity %K Mammals %K Stress, Physiological %XThe fruit fly Drosophila has contributed significantly to our general understanding of the basic principles of signaling, cell and developmental biology, and neurobiology. However, answers to questions pertaining to energy metabolism have been so far mostly addressed in more complex model organisms such as mice. We review in this article recent studies that show how the genetic tractability and simplicity of Drosophila are being used to identify novel regulatory mechanisms at the organismal level, and to query the co-ordination between energy metabolism and other processes such as neurodegeneration, circadian rhythms, immunity, and tumor biology.
%B BMC Biol %V 11 %P 38 %8 2013 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/23587196?dopt=Abstract %R 10.1186/1741-7007-11-38 %0 Journal Article %J Cancer Cell %D 2013 %T Complementary genomic screens identify SERCA as a therapeutic target in NOTCH1 mutated cancer. %A Roti, Giovanni %A Carlton, Anne %A Ross, Kenneth N %A Markstein, Michele %A Pajcini, Kostandin %A Su, Angela H %A Perrimon, Norbert %A Pear, Warren S %A Kung, Andrew L %A Blacklow, Stephen C %A Aster, Jon C %A Stegmaier, Kimberly %K Alleles %K Animals %K Calcium Channels %K Cell Line, Tumor %K Drosophila %K Drug Screening Assays, Antitumor %K Enzyme Inhibitors %K Female %K G1 Phase Cell Cycle Checkpoints %K Gene Library %K High-Throughput Screening Assays %K Humans %K Leukemia %K Mice %K Mice, SCID %K Mutation %K Neoplasm Transplantation %K Receptor, Notch1 %K Sarcoplasmic Reticulum Calcium-Transporting ATPases %K Signal Transduction %K Small Molecule Libraries %K Thapsigargin %K Transplantation, Heterologous %XNotch1 is a rational therapeutic target in several human cancers, but as a transcriptional regulator, it poses a drug discovery challenge. To identify Notch1 modulators, we performed two cell-based, high-throughput screens for small-molecule inhibitors and cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. Sarco/endoplasmic reticulum calcium ATPase (SERCA) channels emerged at the intersection of these complementary screens. SERCA inhibition preferentially impairs the maturation and activity of mutated Notch1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human leukemia cells. A small-molecule SERCA inhibitor has on-target activity in two mouse models of human leukemia and interferes with Notch signaling in Drosophila. These studies "credential" SERCA as a therapeutic target in cancers associated with NOTCH1 mutations.
%B Cancer Cell %V 23 %P 390-405 %8 2013 Mar 18 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/23434461?dopt=Abstract %R 10.1016/j.ccr.2013.01.015 %0 Book Section %B Handbook of Systems Biology %D 2013 %T Analyzing the Structure, Function and Information Flow in Signaling Networks using Quantitative Cellular Signatures %A Kulkarni, Meghana M %A Perrimon, Norbert %E Walhout, N %E Vidal, M %E Dekker, J %B Handbook of Systems Biology %I Academic Press %P 89-113 %G eng %0 Journal Article %J Cell Rep %D 2013 %T The circadian clock gates the intestinal stem cell regenerative state. %A Karpowicz, Phillip %A Zhang, Yong %A Hogenesch, John B %A Emery, Patrick %A Perrimon, Norbert %K Animals %K Circadian Clocks %K Circadian Rhythm %K Drosophila %K Drosophila Proteins %K Interphase %K Intestines %K Period Circadian Proteins %K Regeneration %K Signal Transduction %K Stem Cells %K Transcription, Genetic %XThe intestine has evolved under constant environmental stresses, because an animal may ingest harmful pathogens or chemicals at any time during its lifespan. Following damage, intestinal stem cells (ISCs) regenerate the intestine by proliferating to replace dying cells. ISCs from diverse animals are remarkably similar, and the Wnt, Notch, and Hippo signaling pathways, important regulators of mammalian ISCs, are conserved from flies to humans. Unexpectedly, we identified the transcription factor period, a component of the circadian clock, to be critical for regeneration, which itself follows a circadian rhythm. We discovered hundreds of transcripts that are regulated by the clock during intestinal regeneration, including components of stress response and regeneration pathways. Disruption of clock components leads to arrhythmic ISC divisions, revealing their underappreciated role in the healing process.
%B Cell Rep %V 3 %P 996-1004 %8 2013 Apr 25 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/23583176?dopt=Abstract %R 10.1016/j.celrep.2013.03.016 %0 Journal Article %J Sci Signal %D 2013 %T Conserved regulators of nucleolar size revealed by global phenotypic analyses. %A Neumüller, Ralph A %A Gross, Thomas %A Samsonova, Anastasia A %A Vinayagam, Arunachalam %A Buckner, Michael %A Founk, Karen %A Hu, Yanhui %A Sharifpoor, Sara %A Rosebrock, Adam P %A Andrews, Brenda %A Winston, Fred %A Perrimon, Norbert %K Cell Nucleolus %K DNA, Fungal %K DNA, Ribosomal %K Genes, Fungal %K Genes, rRNA %K Histones %K RNA Polymerase I %K RNA, Fungal %K RNA, Ribosomal %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Transcription, Genetic %XRegulation of cell growth is a fundamental process in development and disease that integrates a vast array of extra- and intracellular information. A central player in this process is RNA polymerase I (Pol I), which transcribes ribosomal RNA (rRNA) genes in the nucleolus. Rapidly growing cancer cells are characterized by increased Pol I-mediated transcription and, consequently, nucleolar hypertrophy. To map the genetic network underlying the regulation of nucleolar size and of Pol I-mediated transcription, we performed comparative, genome-wide loss-of-function analyses of nucleolar size in Saccharomyces cerevisiae and Drosophila melanogaster coupled with mass spectrometry-based analyses of the ribosomal DNA (rDNA) promoter. With this approach, we identified a set of conserved and nonconserved molecular complexes that control nucleolar size. Furthermore, we characterized a direct role of the histone information regulator (HIR) complex in repressing rRNA transcription in yeast. Our study provides a full-genome, cross-species analysis of a nuclear subcompartment and shows that this approach can identify conserved molecular modules.
%B Sci Signal %V 6 %P ra70 %8 2013 Aug 20 %G eng %N 289 %1 http://www.ncbi.nlm.nih.gov/pubmed/23962978?dopt=Abstract %R 10.1126/scisignal.2004145 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2013 %T Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila. %A Xiong, Xiao-Peng %A Kurthkoti, Krishna %A Chang, Kung-Yen %A Lichinchi, Gianluigi %A De, Nabanita %A Schneemann, Anette %A MacRae, Ian J %A Rana, Tariq M %A Perrimon, Norbert %A Zhou, Rui %K Animals %K Blotting, Northern %K Cell Line %K Drosophila %K Immunoprecipitation %K RNA Interference %K RNA Precursors %K RNA, Small Interfering %K snRNP Core Proteins %XRNAi is an evolutionarily conserved gene regulatory process that operates in a wide variety of organisms. During RNAi, long double-stranded RNA precursors are processed by Dicer proteins into ∼21-nt siRNAs. Subsequently, siRNAs are incorporated into the RNA-induced silencing complexes (RISCs) that contain Argonaute-family proteins and guide RISC to target RNAs via complementary base pairing, leading to posttranscriptional gene silencing. Select pre-mRNA splicing factors have been implicated in RNAi in fission yeast, worms, and flies, but the underlying molecular mechanisms are not well understood. Here, we show that SmD1, a core component of the Drosophila small nuclear ribonucleoprotein particle implicated in splicing, is required for RNAi and antiviral immunity in cultured cells and in vivo. SmD1 interacts with both Dicer-2 and dsRNA precursors and is indispensable for optimal siRNA biogenesis. Depletion of SmD1 impairs the assembly and function of the small interfering RISC without significantly affecting the expression of major canonical siRNA pathway components. Moreover, SmD1 physically and functionally associates with components of the small interfering RISC, including Argonaute 2, both in flies and in humans. Notably, RNAi defects resulting from SmD1 silencing can be uncoupled from defects in pre-mRNA splicing, and the RNAi and splicing machineries are physically and functionally distinct entities. Our results suggest that Drosophila SmD1 plays a direct role in RNAi-mediated gene silencing independently of its pre-mRNA splicing activity and indicate that the dual roles of splicing factors in posttranscriptional gene regulation may be evolutionarily widespread.
%B Proc Natl Acad Sci U S A %V 110 %P 16520-5 %8 2013 Oct 8 %G eng %N 41 %1 http://www.ncbi.nlm.nih.gov/pubmed/24067655?dopt=Abstract %R 10.1073/pnas.1315803110 %0 Journal Article %J Science %D 2013 %T The Hippo signaling pathway interactome. %A Kwon, Young %A Vinayagam, Arunachalam %A Sun, Xiaoyun %A Dephoure, Noah %A Gygi, Steven P %A Hong, Pengyu %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Intracellular Signaling Peptides and Proteins %K Nuclear Proteins %K Protein Interaction Maps %K Protein-Serine-Threonine Kinases %K Proteome %K RNA Interference %K Trans-Activators %XThe Hippo pathway controls metazoan organ growth by regulating cell proliferation and apoptosis. Many components have been identified, but our knowledge of the composition and structure of this pathway is still incomplete. Using existing pathway components as baits, we generated by mass spectrometry a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67% of the proteins by RNA interference regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively. We selected for further characterization a new member of the alpha-arrestin family, Leash, and show that it promotes degradation of Yki through the lysosomal pathway. Given the importance of the Hippo pathway in tumor development, the Hippo-PPIN will contribute to our understanding of this network in both normal growth and cancer.
%B Science %V 342 %P 737-40 %8 2013 Nov 8 %G eng %N 6159 %1 http://www.ncbi.nlm.nih.gov/pubmed/24114784?dopt=Abstract %R 10.1126/science.1243971 %0 Journal Article %J Sci Signal %D 2013 %T The homeobox transcription factor cut coordinates patterning and growth during Drosophila airway remodeling. %A Pitsouli, Chrysoula %A Perrimon, Norbert %K Animals %K Body Patterning %K Cell Cycle %K Drosophila %K Genes, Homeobox %K Signal Transduction %K Trachea %K Transcription Factors %XA fundamental question in developmental biology is how tissue growth and patterning are coordinately regulated to generate complex organs with characteristic shapes and sizes. We showed that in the developing primordium that produces the Drosophila adult trachea, the homeobox transcription factor Cut regulates both growth and patterning, and its effects depend on its abundance. Quantification of the abundance of Cut in the developing airway progenitors during late larval stage 3 revealed that the cells of the developing trachea had different amounts of Cut, with the most proliferative region having an intermediate amount of Cut and the region lacking Cut exhibiting differentiation. By manipulating Cut abundance, we showed that Cut functioned in different regions to regulate proliferation or patterning. Transcriptional profiling of progenitor populations with different amounts of Cut revealed the Wingless (known as Wnt in vertebrates) and Notch signaling pathways as positive and negative regulators of cut expression, respectively. Furthermore, we identified the gene encoding the receptor Breathless (Btl, known as fibroblast growth factor receptor in vertebrates) as a transcriptional target of Cut. Cut inhibited btl expression and tracheal differentiation to maintain the developing airway cells in a progenitor state. Thus, Cut functions in the integration of patterning and growth in a developing epithelial tissue.
%B Sci Signal %V 6 %P ra12 %8 2013 Feb 19 %G eng %N 263 %1 http://www.ncbi.nlm.nih.gov/pubmed/23423438?dopt=Abstract %R 10.1126/scisignal.2003424 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2013 %T Optimized gene editing technology for Drosophila melanogaster using germ line-specific Cas9. %A Ren, Xingjie %A Sun, Jin %A Housden, Benjamin E %A Hu, Yanhui %A Roesel, Charles %A Lin, Shuailiang %A Liu, Lu-Ping %A Yang, Zhihao %A Mao, Decai %A Sun, Lingzhu %A Wu, Qujie %A Ji, Jun-Yuan %A Xi, Jianzhong %A Mohr, Stephanie E %A Xu, Jiang %A Perrimon, Norbert %A Ni, Jian-Quan %K Animals %K Animals, Genetically Modified %K CRISPR-Cas Systems %K Databases, Genetic %K Drosophila melanogaster %K Drosophila Proteins %K Genetic Engineering %K Genomics %K Germ Cells %K Mutagenesis %K Promoter Regions, Genetic %K RNA-Binding Proteins %XThe ability to engineer genomes in a specific, systematic, and cost-effective way is critical for functional genomic studies. Recent advances using the CRISPR-associated single-guide RNA system (Cas9/sgRNA) illustrate the potential of this simple system for genome engineering in a number of organisms. Here we report an effective and inexpensive method for genome DNA editing in Drosophila melanogaster whereby plasmid DNAs encoding short sgRNAs under the control of the U6b promoter are injected into transgenic flies in which Cas9 is specifically expressed in the germ line via the nanos promoter. We evaluate the off-targets associated with the method and establish a Web-based resource, along with a searchable, genome-wide database of predicted sgRNAs appropriate for genome engineering in flies. Finally, we discuss the advantages of our method in comparison with other recently published approaches.
%B Proc Natl Acad Sci U S A %V 110 %P 19012-7 %8 2013 Nov 19 %G eng %N 47 %1 http://www.ncbi.nlm.nih.gov/pubmed/24191015?dopt=Abstract %R 10.1073/pnas.1318481110 %0 Journal Article %J Proteome Sci %D 2013 %T PPIRank - an advanced method for ranking protein-protein interations in TAP/MS data. %A Sun, Xiaoyun %A Hong, Pengyu %A Kulkarni, Meghana %A Kwon, Young %A Perrimon, Norbert %XBACKGROUND: Tandem affinity purification coupled with mass-spectrometry (TAP/MS) analysis is a popular method for the identification of novel endogenous protein-protein interactions (PPIs) in large-scale. Computational analysis of TAP/MS data is a critical step, particularly for high-throughput datasets, yet it remains challenging due to the noisy nature of TAP/MS data. RESULTS: We investigated several major TAP/MS data analysis methods for identifying PPIs, and developed an advanced method, which incorporates an improved statistical method to filter out false positives from the negative controls. Our method is named PPIRank that stands for PPI ranking in TAP/MS data. We compared PPIRank with several other existing methods in analyzing two pathway-specific TAP/MS PPI datasets from Drosophila. CONCLUSION: Experimental results show that PPIRank is more capable than other approaches in terms of identifying known interactions collected in the BioGRID PPI database. Specifically, PPIRank is able to capture more true interactions and simultaneously less false positives in both Insulin and Hippo pathways of Drosophila Melanogaster.
%B Proteome Sci %V 11 %P S16 %8 2013 Nov 7 %G eng %N Suppl 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/24565074?dopt=Abstract %R 10.1186/1477-5956-11-S1-S16 %0 Journal Article %J PLoS Biol %D 2013 %T Role of autophagy in glycogen breakdown and its relevance to chloroquine myopathy. %A Zirin, Jonathan %A Nieuwenhuis, Joppe %A Perrimon, Norbert %K Amino Acid Sequence %K Animals %K Autophagy %K Catalytic Domain %K Chloroquine %K Drosophila melanogaster %K Drosophila Proteins %K Glycogen %K Glycogen Synthase %K Glycogenolysis %K Lysosomes %K Molecular Sequence Data %K Muscles %K Muscular Diseases %K Mutation, Missense %K Phagosomes %XSeveral myopathies are associated with defects in autophagic and lysosomal degradation of glycogen, but it remains unclear how glycogen is targeted to the lysosome and what significance this process has for muscle cells. We have established a Drosophila melanogaster model to study glycogen autophagy in skeletal muscles, using chloroquine (CQ) to simulate a vacuolar myopathy that is completely dependent on the core autophagy genes. We show that autophagy is required for the most efficient degradation of glycogen in response to starvation. Furthermore, we show that CQ-induced myopathy can be improved by reduction of either autophagy or glycogen synthesis, the latter possibly due to a direct role of Glycogen Synthase in regulating autophagy through its interaction with Atg8.
%B PLoS Biol %V 11 %P e1001708 %8 2013 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/24265594?dopt=Abstract %R 10.1371/journal.pbio.1001708 %0 Journal Article %J Genetics %D 2013 %T Depleting gene activities in early Drosophila embryos with the "maternal-Gal4-shRNA" system. %A Staller, Max V %A Yan, Dong %A Randklev, Sakara %A Bragdon, Meghan D %A Wunderlich, Zeba B %A Tao, Rong %A Perkins, Lizabeth A %A Depace, Angela H %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Gene Dosage %K Gene Expression Regulation, Developmental %K Gene Knockdown Techniques %K Male %K Oogenesis %K Phenotype %K RNA Interference %K RNA, Small Interfering %K Transcription Factors %K Transcription, Genetic %XIn a developing Drosophila melanogaster embryo, mRNAs have a maternal origin, a zygotic origin, or both. During the maternal-zygotic transition, maternal products are degraded and gene expression comes under the control of the zygotic genome. To interrogate the function of mRNAs that are both maternally and zygotically expressed, it is common to examine the embryonic phenotypes derived from female germline mosaics. Recently, the development of RNAi vectors based on short hairpin RNAs (shRNAs) effective during oogenesis has provided an alternative to producing germline clones. Here, we evaluate the efficacies of: (1) maternally loaded shRNAs to knockdown zygotic transcripts and (2) maternally loaded Gal4 protein to drive zygotic shRNA expression. We show that, while Gal4-driven shRNAs in the female germline very effectively generate phenotypes for genes expressed maternally, maternally loaded shRNAs are not very effective at generating phenotypes for early zygotic genes. However, maternally loaded Gal4 protein is very efficient at generating phenotypes for zygotic genes expressed during mid-embryogenesis. We apply this powerful and simple method to unravel the embryonic functions of a number of pleiotropic genes.
%B Genetics %V 193 %P 51-61 %8 2013 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23105012?dopt=Abstract %R 10.1534/genetics.112.144915 %0 Journal Article %J Dev Biol %D 2013 %T Ecdysone signaling at metamorphosis triggers apoptosis of Drosophila abdominal muscles. %A Zirin, Jonathan %A Cheng, Daojun %A Dhanyasi, Nagaraju %A Cho, Julio %A Dura, Jean-Maurice %A Vijayraghavan, Krishnaswamy %A Perrimon, Norbert %K Abdomen %K Abdominal Muscles %K Animals %K Apoptosis %K Autophagy %K Caspases %K Drosophila melanogaster %K Drosophila Proteins %K Ecdysone %K Epistasis, Genetic %K Larva %K Metamorphosis, Biological %K Muscles %K Sarcomeres %K Signal Transduction %K Time Factors %XOne of the most dramatic examples of programmed cell death occurs during Drosophila metamorphosis, when most of the larval tissues are destroyed in a process termed histolysis. Much of our understanding of this process comes from analyses of salivary gland and midgut cell death. In contrast, relatively little is known about the degradation of the larval musculature. Here, we analyze the programmed destruction of the abdominal dorsal exterior oblique muscle (DEOM) which occurs during the first 24h of metamorphosis. We find that ecdysone signaling through Ecdysone receptor isoform B1 is required cell autonomously for the muscle death. Furthermore, we show that the orphan nuclear receptor FTZ-F1, opposed by another nuclear receptor, HR39, plays a critical role in the timing of DEOM histolysis. Finally, we show that unlike the histolysis of salivary gland and midgut, abdominal muscle death occurs by apoptosis, and does not require autophagy. Thus, there is no set rule as to the role of autophagy and apoptosis during Drosophila histolysis.
%B Dev Biol %V 383 %P 275-84 %8 2013 Nov 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/24051228?dopt=Abstract %R 10.1016/j.ydbio.2013.08.029 %0 Journal Article %J G3 (Bethesda) %D 2013 %T FlyPrimerBank: an online database for Drosophila melanogaster gene expression analysis and knockdown evaluation of RNAi reagents. %A Hu, Yanhui %A Sopko, Richelle %A Foos, Marianna %A Kelley, Colleen %A Flockhart, Ian %A Ammeux, Noemie %A Wang, Xiaowei %A Perkins, Lizabeth %A Perrimon, Norbert %A Mohr, Stephanie E %K Algorithms %K Animals %K Databases, Genetic %K DNA Primers %K Drosophila melanogaster %K Embryo, Nonmammalian %K Gene Expression %K Internet %K Real-Time Polymerase Chain Reaction %K RNA Interference %K RNA, Small Interfering %K User-Computer Interface %XThe evaluation of specific endogenous transcript levels is important for understanding transcriptional regulation. More specifically, it is useful for independent confirmation of results obtained by the use of microarray analysis or RNA-seq and for evaluating RNA interference (RNAi)-mediated gene knockdown. Designing specific and effective primers for high-quality, moderate-throughput evaluation of transcript levels, i.e., quantitative, real-time PCR (qPCR), is nontrivial. To meet community needs, predefined qPCR primer pairs for mammalian genes have been designed and sequences made available, e.g., via PrimerBank. In this work, we adapted and refined the algorithms used for the mammalian PrimerBank to design 45,417 primer pairs for 13,860 Drosophila melanogaster genes, with three or more primer pairs per gene. We experimentally validated primer pairs for ~300 randomly selected genes expressed in early Drosophila embryos, using SYBR Green-based qPCR and sequence analysis of products derived from conventional PCR. All relevant information, including primer sequences, isoform specificity, spatial transcript targeting, and any available validation results and/or user feedback, is available from an online database (www.flyrnai.org/flyprimerbank). At FlyPrimerBank, researchers can retrieve primer information for fly genes either one gene at a time or in batch mode. Importantly, we included the overlap of each predicted amplified sequence with RNAi reagents from several public resources, making it possible for researchers to choose primers suitable for knockdown evaluation of RNAi reagents (i.e., to avoid amplification of the RNAi reagent itself). We demonstrate the utility of this resource for validation of RNAi reagents in vivo.
%B G3 (Bethesda) %V 3 %P 1607-16 %8 2013 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/23893746?dopt=Abstract %R 10.1534/g3.113.007021 %0 Journal Article %J PLoS One %D 2013 %T Genetic determinants of phosphate response in Drosophila. %A Bergwitz, Clemens %A Wee, Mark J %A Sinha, Sumi %A Huang, Joanne %A DeRobertis, Charles %A Mensah, Lawrence B %A Cohen, Jonathan %A Friedman, Adam %A Kulkarni, Meghana %A Hu, Yanhui %A Vinayagam, Arunachalam %A Schnall-Levin, Michael %A Berger, Bonnie %A Perkins, Lizabeth A %A Mohr, Stephanie E %A Perrimon, Norbert %K Animals %K Cell Line %K Drosophila melanogaster %K Drosophila Proteins %K Hemocytes %K Hemolymph %K Longevity %K Malpighian Tubules %K MAP Kinase Signaling System %K Phosphates %K RNA Interference %XPhosphate is required for many important cellular processes and having too little phosphate or too much can cause disease and reduce life span in humans. However, the mechanisms underlying homeostatic control of extracellular phosphate levels and cellular effects of phosphate are poorly understood. Here, we establish Drosophila melanogaster as a model system for the study of phosphate effects. We found that Drosophila larval development depends on the availability of phosphate in the medium. Conversely, life span is reduced when adult flies are cultured on high phosphate medium or when hemolymph phosphate is increased in flies with impaired malpighian tubules. In addition, RNAi-mediated inhibition of MAPK-signaling by knockdown of Ras85D, phl/D-Raf or Dsor1/MEK affects larval development, adult life span and hemolymph phosphate, suggesting that some in vivo effects involve activation of this signaling pathway by phosphate. To identify novel genetic determinants of phosphate responses, we used Drosophila hemocyte-like cultured cells (S2R+) to perform a genome-wide RNAi screen using MAPK activation as the readout. We identified a number of candidate genes potentially important for the cellular response to phosphate. Evaluation of 51 genes in live flies revealed some that affect larval development, adult life span and hemolymph phosphate levels.
%B PLoS One %V 8 %P e56753 %8 2013 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/23520455?dopt=Abstract %R 10.1371/journal.pone.0056753 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2013 %T Genome-wide RNAi screen reveals a role for the ESCRT complex in rotavirus cell entry. %A Silva-Ayala, Daniela %A López, Tomás %A Gutiérrez, Michelle %A Perrimon, Norbert %A López, Susana %A Arias, Carlos F %K Animals %K Caco-2 Cells %K cdc42 GTP-Binding Protein %K Cercopithecus aethiops %K Endosomal Sorting Complexes Required for Transport %K Genome-Wide Association Study %K Humans %K Protein Transport %K rab GTP-Binding Proteins %K rab5 GTP-Binding Proteins %K rhoA GTP-Binding Protein %K RNA Interference %K Rotavirus %K Rotavirus Infections %K Transport Vesicles %K Vacuolar Proton-Translocating ATPases %K Vero Cells %K Vesicular Transport Proteins %XRotavirus (RV) is the major cause of childhood gastroenteritis worldwide. This study presents a functional genome-scale analysis of cellular proteins and pathways relevant for RV infection using RNAi. Among the 522 proteins selected in the screen for their ability to affect viral infectivity, an enriched group that participates in endocytic processes was identified. Within these proteins, subunits of the vacuolar ATPase, small GTPases, actinin 4, and, of special interest, components of the endosomal sorting complex required for transport (ESCRT) machinery were found. Here we provide evidence for a role of the ESCRT complex in the entry of simian and human RV strains in both monkey and human epithelial cells. In addition, the ESCRT-associated ATPase VPS4A and phospholipid lysobisphosphatidic acid, both crucial for the formation of intralumenal vesicles in multivesicular bodies, were also found to be required for cell entry. Interestingly, it seems that regardless of the molecules that rhesus RV and human RV strains use for cell-surface attachment and the distinct endocytic pathway used, all these viruses converge in early endosomes and use multivesicular bodies for cell entry. Furthermore, the small GTPases RHOA and CDC42, which regulate different types of clathrin-independent endocytosis, as well as early endosomal antigen 1 (EEA1), were found to be involved in this process. This work reports the direct involvement of the ESCRT machinery in the life cycle of a nonenveloped virus and highlights the complex mechanism that these viruses use to enter cells. It also illustrates the efficiency of high-throughput RNAi screenings as genetic tools for comprehensively studying the interaction between viruses and their host cells.
%B Proc Natl Acad Sci U S A %V 110 %P 10270-5 %8 2013 Jun 18 %G eng %N 25 %1 http://www.ncbi.nlm.nih.gov/pubmed/23733942?dopt=Abstract %R 10.1073/pnas.1304932110 %0 Journal Article %J Aging Cell %D 2013 %T The influence of skeletal muscle on systemic aging and lifespan. %A Demontis, Fabio %A Piccirillo, Rosanna %A Goldberg, Alfred L %A Perrimon, Norbert %K Animals %K Cell Communication %K Cytokines %K Exercise %K Humans %K Longevity %K Muscle, Skeletal %K Organ Specificity %XEpidemiological studies in humans suggest that skeletal muscle aging is a risk factor for the development of several age-related diseases such as metabolic syndrome, cancer, Alzheimer's and Parkinson's disease. Here, we review recent studies in mammals and Drosophila highlighting how nutrient- and stress-sensing in skeletal muscle can influence lifespan and overall aging of the organism. In addition to exercise and indirect effects of muscle metabolism, growing evidence suggests that muscle-derived growth factors and cytokines, known as myokines, modulate systemic physiology. Myokines may influence the progression of age-related diseases and contribute to the intertissue communication that underlies systemic aging.
%B Aging Cell %V 12 %P 943-9 %8 2013 Dec %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/23802635?dopt=Abstract %R 10.1111/acel.12126 %0 Journal Article %J Cell %D 2013 %T Muscle mitohormesis promotes longevity via systemic repression of insulin signaling. %A Owusu-Ansah, Edward %A Song, Wei %A Perrimon, Norbert %K Aging %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Insulin %K Insulin-Like Growth Factor Binding Proteins %K Larva %K Longevity %K Male %K Mitochondria %K Muscles %K Reactive Oxygen Species %K Signal Transduction %K Unfolded Protein Response %XMitochondrial dysfunction is usually associated with aging. To systematically characterize the compensatory stress signaling cascades triggered in response to muscle mitochondrial perturbation, we analyzed a Drosophila model of muscle mitochondrial injury. We find that mild muscle mitochondrial distress preserves mitochondrial function, impedes the age-dependent deterioration of muscle function and architecture, and prolongs lifespan. Strikingly, this effect is mediated by at least two prolongevity compensatory signaling modules: one involving a muscle-restricted redox-dependent induction of genes that regulate the mitochondrial unfolded protein response (UPR(mt)) and another involving the transcriptional induction of the Drosophila ortholog of insulin-like growth factor-binding protein 7, which systemically antagonizes insulin signaling and facilitates mitophagy. Given that several secreted IGF-binding proteins (IGFBPs) exist in mammals, our work raises the possibility that muscle mitochondrial injury in humans may similarly result in the secretion of IGFBPs, with important ramifications for diseases associated with aberrant insulin signaling.
%B Cell %V 155 %P 699-712 %8 2013 Oct 24 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/24243023?dopt=Abstract %R 10.1016/j.cell.2013.09.021 %0 Journal Article %J Sci Rep %D 2013 %T PAPTi: a peptide aptamer interference toolkit for perturbation of protein-protein interaction networks. %A Yeh, Johannes TH %A Binari, Richard %A Gocha, Tenzin %A Dasgupta, Ramanuj %A Perrimon, Norbert %K Adaptor Proteins, Signal Transducing %K Aptamers, Peptide %K beta Catenin %K HEK293 Cells %K Humans %K Phosphoproteins %K Protein Binding %K Protein Interaction Mapping %K Wnt Signaling Pathway %XSignaling proteins often form dynamic protein-protein interaction (PPI) complexes to achieve multi-functionality. Methods to abrogate a subset of PPI interfaces without depleting the full-length protein will be valuable for structure-function relationship annotations. Here, we describe the use of Peptide Aptamer Interference (PAPTi) approach for structure-function network studies. We identified peptide aptamers against Dishevelled (Dsh) and β-catenin (β-cat) to target the Wnt signaling pathway and demonstrate that these FN3-based MONOBODYs (FNDYs) can be used to perturb protein activities both in vitro and in vivo. Further, to investigate the crosstalk between the Wnt and Notch pathways, we isolated FNDYs against the Notch Ankyrin (ANK) region and demonstrate that perturbing the ANK domain of Notch increases the inhibitory activity of Notch towards Wnt signaling. Altogether, these studies demonstrate the power of the PAPTi approach to dissect specific PPI interactions within signaling networks.
%B Sci Rep %V 3 %P 1156 %8 2013 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/23362456?dopt=Abstract %R 10.1038/srep01156 %0 Journal Article %J Sci Signal %D 2013 %T Protein complex-based analysis framework for high-throughput data sets. %A Vinayagam, Arunachalam %A Hu, Yanhui %A Kulkarni, Meghana %A Roesel, Charles %A Sopko, Richelle %A Mohr, Stephanie E %A Perrimon, Norbert %K Animals %K Cell Cycle Proteins %K data mining %K Databases, Genetic %K Drosophila melanogaster %K Drosophila Proteins %K Gene Expression Profiling %K High Mobility Group Proteins %K High-Throughput Screening Assays %K Humans %K Insulin %K Internet %K Molecular Sequence Annotation %K Multiprotein Complexes %K Protein Interaction Maps %K Proteomics %K RNA Interference %K Saccharomyces cerevisiae %K Software %K Species Specificity %K Systems Biology %K Trans-Activators %XAnalysis of high-throughput data increasingly relies on pathway annotation and functional information derived from Gene Ontology. This approach has limitations, in particular for the analysis of network dynamics over time or under different experimental conditions, in which modules within a network rather than complete pathways might respond and change. We report an analysis framework based on protein complexes, which are at the core of network reorganization. We generated a protein complex resource for human, Drosophila, and yeast from the literature and databases of protein-protein interaction networks, with each species having thousands of complexes. We developed COMPLEAT (http://www.flyrnai.org/compleat), a tool for data mining and visualization for complex-based analysis of high-throughput data sets, as well as analysis and integration of heterogeneous proteomics and gene expression data sets. With COMPLEAT, we identified dynamically regulated protein complexes among genome-wide RNA interference data sets that used the abundance of phosphorylated extracellular signal-regulated kinase in cells stimulated with either insulin or epidermal growth factor as the output. The analysis predicted that the Brahma complex participated in the insulin response.
%B Sci Signal %V 6 %P rs5 %8 2013 Feb 26 %G eng %N 264 %1 http://www.ncbi.nlm.nih.gov/pubmed/23443684?dopt=Abstract %R 10.1126/scisignal.2003629 %0 Journal Article %J Cold Spring Harb Perspect Biol %D 2013 %T Receptor tyrosine kinases in Drosophila development. %A Sopko, Richelle %A Perrimon, Norbert %K Animals %K Axons %K Body Patterning %K Cell Differentiation %K Cell Movement %K Drosophila %K Metamorphosis, Biological %K Models, Biological %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %K Wound Healing %XTyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases.
%B Cold Spring Harb Perspect Biol %V 5 %8 2013 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/23732470?dopt=Abstract %R 10.1101/cshperspect.a009050 %0 Journal Article %J Nat Cell Biol %D 2013 %T A screen for morphological complexity identifies regulators of switch-like transitions between discrete cell shapes. %A Yin, Zheng %A Sadok, Amine %A Sailem, Heba %A McCarthy, Afshan %A Xia, Xiaofeng %A Li, Fuhai %A Garcia, Mar Arias %A Evans, Louise %A Barr, Alexis R %A Perrimon, Norbert %A Marshall, Christopher J %A Wong, Stephen T C %A Bakal, Chris %K Animals %K Cell Shape %K Drosophila melanogaster %K Genes, Tumor Suppressor %K Genetic Testing %K Humans %K Melanoma %K Mice %K Phenotype %K PTEN Phosphohydrolase %K RNA Interference %K Tumor Cells, Cultured %XThe way in which cells adopt different morphologies is not fully understood. Cell shape could be a continuous variable or restricted to a set of discrete forms. We developed quantitative methods to describe cell shape and show that Drosophila haemocytes in culture are a heterogeneous mixture of five discrete morphologies. In an RNAi screen of genes affecting the morphological complexity of heterogeneous cell populations, we found that most genes regulate the transition between discrete shapes rather than generating new morphologies. In particular, we identified a subset of genes, including the tumour suppressor PTEN, that decrease the heterogeneity of the population, leading to populations enriched in rounded or elongated forms. We show that these genes have a highly conserved function as regulators of cell shape in both mouse and human metastatic melanoma cells.
%B Nat Cell Biol %V 15 %P 860-71 %8 2013 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/23748611?dopt=Abstract %R 10.1038/ncb2764 %0 Journal Article %J Genetics %D 2013 %T UP-TORR: online tool for accurate and Up-to-Date annotation of RNAi Reagents. %A Hu, Yanhui %A Roesel, Charles %A Flockhart, Ian %A Perkins, Lizabeth %A Perrimon, Norbert %A Mohr, Stephanie E %K Animals %K Drosophila %K Indicators and Reagents %K Internet %K Molecular Sequence Annotation %K RNA Interference %K RNA, Small Interfering %K Software %XRNA interference (RNAi) is a widely adopted tool for loss-of-function studies but RNAi results only have biological relevance if the reagents are appropriately mapped to genes. Several groups have designed and generated RNAi reagent libraries for studies in cells or in vivo for Drosophila and other species. At first glance, matching RNAi reagents to genes appears to be a simple problem, as each reagent is typically designed to target a single gene. In practice, however, the reagent-gene relationship is complex. Although the sequences of oligonucleotides used to generate most types of RNAi reagents are static, the reference genome and gene annotations are regularly updated. Thus, at the time a researcher chooses an RNAi reagent or analyzes RNAi data, the most current interpretation of the RNAi reagent-gene relationship, as well as related information regarding specificity (e.g., predicted off-target effects), can be different from the original interpretation. Here, we describe a set of strategies and an accompanying online tool, UP-TORR (for Updated Targets of RNAi Reagents; www.flyrnai.org/up-torr), useful for accurate and up-to-date annotation of cell-based and in vivo RNAi reagents. Importantly, UP-TORR automatically synchronizes with gene annotations daily, retrieving the most current information available, and for Drosophila, also synchronizes with the major reagent collections. Thus, UP-TORR allows users to choose the most appropriate RNAi reagents at the onset of a study, as well as to perform the most appropriate analyses of results of RNAi-based studies.
%B Genetics %V 195 %P 37-45 %8 2013 Sep %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23792952?dopt=Abstract %R 10.1534/genetics.113.151340 %0 Journal Article %J J Biol Chem %D 2012 %T Drosophila heparan sulfate, a novel design. %A Kusche-Gullberg, Marion %A Nybakken, Kent %A Perrimon, Norbert %A Lindahl, Ulf %K Animals %K Anions %K Carbohydrate Sequence %K Chromatography, Gel %K Chromatography, High Pressure Liquid %K Disaccharides %K Drosophila %K Electrophoresis %K Gene Expression Regulation, Developmental %K Glycosaminoglycans %K Heparan Sulfate Proteoglycans %K Heparitin Sulfate %K Molecular Sequence Data %K Protein Structure, Tertiary %K Structure-Activity Relationship %XHeparan sulfate (HS) proteoglycans play critical roles in a wide variety of biological processes such as growth factor signaling, cell adhesion, wound healing, and tumor metastasis. Functionally important interactions between HS and a variety of proteins depend on specific structural features within the HS chains. The fruit fly (Drosophila melanogaster) is frequently applied as a model organism to study HS function in development. Previous structural studies of Drosophila HS have been restricted to disaccharide composition, without regard to the arrangement of saccharide domains typically found in vertebrate HS. Here, we biochemically characterized Drosophila HS by selective depolymerization with nitrous acid. Analysis of the generated saccharide products revealed a novel HS design, involving a peripheral, extended, presumably single, N-sulfated domain linked to an N-acetylated sequence contiguous with the linkage to core protein. The N-sulfated domain may be envisaged as a heparin structure of unusually low O-sulfate content.
%B J Biol Chem %V 287 %P 21950-6 %8 2012 Jun 22 %G eng %N 26 %1 http://www.ncbi.nlm.nih.gov/pubmed/22556423?dopt=Abstract %R 10.1074/jbc.M112.350389 %0 Journal Article %J Development %D 2012 %T A genome-wide transgenic resource for conditional expression of Drosophila microRNAs. %A Bejarano, Fernando %A Bortolamiol-Becet, Diane %A Dai, Qi %A Sun, Kailiang %A Saj, Abil %A Chou, Yu-Ting %A Raleigh, David R %A Kim, Kevin %A Ni, Jian-Quan %A Duan, Hong %A Yang, Jr-Shiuan %A Fulga, Tudor A %A Van Vactor, David %A Perrimon, Norbert %A Lai, Eric C %K Animals %K Databases, Genetic %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Gene Expression Profiling %K Gene Expression Regulation %K Genome %K Genome-Wide Association Study %K Male %K MicroRNAs %K Models, Biological %K Phenotype %K Receptors, Notch %K Signal Transduction %K Transgenes %K Wings, Animal %XmicroRNAs (miRNAs) are endogenous short RNAs that mediate vast networks of post-transcriptional gene regulation. Although computational searches and experimental profiling provide evidence for hundreds of functional targets for individual miRNAs, such data rarely provide clear insight into the phenotypic consequences of manipulating miRNAs in vivo. We describe a genome-wide collection of 165 Drosophila miRNA transgenes and find that a majority induced specific developmental defects, including phenocopies of mutants in myriad cell-signaling and patterning genes. Such connections allowed us to validate several likely targets for miRNA-induced phenotypes. Importantly, few of these phenotypes could be predicted from computationally predicted target lists, thus highlighting the value of whole-animal readouts of miRNA activities. Finally, we provide an example of the relevance of these data to miRNA loss-of-function conditions. Whereas misexpression of several K box miRNAs inhibited Notch pathway activity, reciprocal genetic interaction tests with miRNA sponges demonstrated endogenous roles of the K box miRNA family in restricting Notch signaling. In summary, we provide extensive evidence that misexpression of individual miRNAs often induces specific mutant phenotypes that can guide their functional study. By extension, these data suggest that the deregulation of individual miRNAs in other animals may frequently yield relatively specific phenotypes during disease conditions.
%B Development %V 139 %P 2821-31 %8 2012 Aug %G eng %N 15 %1 http://www.ncbi.nlm.nih.gov/pubmed/22745315?dopt=Abstract %R 10.1242/dev.079939 %0 Journal Article %J PLoS One %D 2012 %T Roles of major facilitator superfamily transporters in phosphate response in Drosophila. %A Bergwitz, Clemens %A Rasmussen, Matthew D %A DeRobertis, Charles %A Wee, Mark J %A Sinha, Sumi %A Chen, Hway H %A Huang, Joanne %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Mitogen-Activated Protein Kinases %K Phosphates %K Proton-Phosphate Symporters %K Saccharomyces cerevisiae Proteins %K Sodium-Phosphate Cotransporter Proteins, Type III %K Tissue Distribution %XThe major facilitator superfamily (MFS) transporter Pho84 and the type III transporter Pho89 are responsible for metabolic effects of inorganic phosphate in yeast. While the Pho89 ortholog Pit1 was also shown to be involved in phosphate-activated MAPK in mammalian cells, it is currently unknown, whether orthologs of Pho84 have a role in phosphate-sensing in metazoan species. We show here that the activation of MAPK by phosphate observed in mammals is conserved in Drosophila cells, and used this assay to characterize the roles of putative phosphate transporters. Surprisingly, while we found that RNAi-mediated knockdown of the fly Pho89 ortholog dPit had little effect on the activation of MAPK in Drosophila S2R+ cells by phosphate, two Pho84/SLC17A1-9 MFS orthologs (MFS10 and MFS13) specifically inhibited this response. Further, using a Xenopus oocyte assay, we show that MSF13 mediates uptake of [(33)P]-orthophosphate in a sodium-dependent fashion. Consistent with a role in phosphate physiology, MSF13 is expressed highest in the Drosophila crop, midgut, Malpighian tubule, and hindgut. Altogether, our findings provide the first evidence that Pho84 orthologs mediate cellular effects of phosphate in metazoan cells. Finally, while phosphate is essential for Drosophila larval development, loss of MFS13 activity is compatible with viability indicating redundancy at the levels of the transporters.
%B PLoS One %V 7 %P e31730 %8 2012 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/22359624?dopt=Abstract %R 10.1371/journal.pone.0031730 %0 Journal Article %J Genome Biol %D 2012 %T A computational framework for boosting confidence in high-throughput protein-protein interaction datasets. %A Hosur, Raghavendra %A Peng, Jian %A Vinayagam, Arunachalam %A Stelzl, Ulrich %A Xu, Jinbo %A Perrimon, Norbert %A Bienkowska, Jadwiga %A Berger, Bonnie %K Algorithms %K Computational Biology %K Databases, Protein %K High-Throughput Screening Assays %K Humans %K Mitogen-Activated Protein Kinases %K Mutation, Missense %K Polymorphism, Single Nucleotide %K Protein Conformation %K Protein Interaction Mapping %K Reproducibility of Results %K Software %XImproving the quality and coverage of the protein interactome is of tantamount importance for biomedical research, particularly given the various sources of uncertainty in high-throughput techniques. We introduce a structure-based framework, Coev2Net, for computing a single confidence score that addresses both false-positive and false-negative rates. Coev2Net is easily applied to thousands of binary protein interactions and has superior predictive performance over existing methods. We experimentally validate selected high-confidence predictions in the human MAPK network and show that predicted interfaces are enriched for cancer -related or damaging SNPs. Coev2Net can be downloaded at http://struct2net.csail.mit.edu.
%B Genome Biol %V 13 %P R76 %8 2012 %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/22937800?dopt=Abstract %R 10.1186/gb-2012-13-8-r76 %0 Journal Article %J Mol Cell %D 2012 %T Control of proinflammatory gene programs by regulated trimethylation and demethylation of histone H4K20. %A Stender, Joshua D %A Pascual, Gabriel %A Liu, Wen %A Kaikkonen, Minna U %A Do, Kevin %A Spann, Nathanael J %A Boutros, Michael %A Perrimon, Norbert %A Rosenfeld, Michael G %A Glass, Christopher K %K Animals %K Cell Line %K Co-Repressor Proteins %K Drosophila %K Gene Expression Regulation %K HEK293 Cells %K Histone-Lysine N-Methyltransferase %K Histones %K Humans %K Inflammation %K Macrophages %K Methylation %K Mice %K Models, Biological %K NF-kappa B %K Promoter Regions, Genetic %K Signal Transduction %K Toll-Like Receptor 4 %XRegulation of genes that initiate and amplify inflammatory programs of gene expression is achieved by signal-dependent exchange of coregulator complexes that function to read, write, and erase specific histone modifications linked to transcriptional activation or repression. Here, we provide evidence for the role of trimethylated histone H4 lysine 20 (H4K20me3) as a repression checkpoint that restricts expression of toll-like receptor 4 (TLR4) target genes in macrophages. H4K20me3 is deposited at the promoters of a subset of these genes by the SMYD5 histone methyltransferase through its association with NCoR corepressor complexes. Signal-dependent erasure of H4K20me3 is required for effective gene activation and is achieved by NF-κB-dependent delivery of the histone demethylase PHF2. Liver X receptors antagonize TLR4-dependent gene activation by maintaining NCoR/SMYD5-mediated repression. These findings reveal a histone H4K20 trimethylation/demethylation strategy that integrates positive and negative signaling inputs that control immunity and homeostasis.
%B Mol Cell %V 48 %P 28-38 %8 2012 Oct 12 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/22921934?dopt=Abstract %R 10.1016/j.molcel.2012.07.020 %0 Journal Article %J Cell %D 2012 %T Drosophila cytokine unpaired 2 regulates physiological homeostasis by remotely controlling insulin secretion. %A Rajan, Akhila %A Perrimon, Norbert %K Animals %K Carbohydrate Metabolism %K Drosophila melanogaster %K Drosophila Proteins %K Energy Metabolism %K Fat Body %K Fats %K Female %K Humans %K Insulin %K Janus Kinases %K Leptin %K Male %K Neuropeptides %XIn Drosophila, the fat body (FB), a functional analog of the vertebrate adipose tissue, is the nutrient sensor that conveys the nutrient status to the insulin-producing cells (IPCs) in the fly brain to release Drosophila insulin-like peptides (Dilps). Dilp secretion in turn regulates energy balance and promotes systemic growth. We identify Unpaired 2 (Upd2), a protein with similarities to type I cytokines, as a secreted factor produced by the FB in the fed state. When upd2 function is perturbed specifically in the FB, it results in a systemic reduction in growth and alters energy metabolism. Upd2 activates JAK/STAT signaling in a population of GABAergic neurons that project onto the IPCs. This activation relieves the inhibitory tone of the GABAergic neurons on the IPCs, resulting in the secretion of Dilps. Strikingly, we find that human Leptin can rescue the upd2 mutant phenotypes, suggesting that Upd2 is the functional homolog of Leptin.
%B Cell %V 151 %P 123-37 %8 2012 Sep 28 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/23021220?dopt=Abstract %R 10.1016/j.cell.2012.08.019 %0 Journal Article %J Nucleic Acids Res %D 2012 %T FlyRNAi.org--the database of the Drosophila RNAi screening center: 2012 update. %A Flockhart, Ian T %A Booker, Matthew %A Hu, Yanhui %A McElvany, Benjamin %A Gilly, Quentin %A Mathey-Prevot, Bernard %A Perrimon, Norbert %A Mohr, Stephanie E %K Animals %K Databases, Genetic %K Drosophila %K Genes, Insect %K Genome, Insect %K Indicators and Reagents %K Internet %K RNA Interference %K Software %XFlyRNAi (http://www.flyrnai.org), the database and website of the Drosophila RNAi Screening Center (DRSC) at Harvard Medical School, serves a dual role, tracking both production of reagents for RNA interference (RNAi) screening in Drosophila cells and RNAi screen results. The database and website is used as a platform for community availability of protocols, tools, and other resources useful to researchers planning, conducting, analyzing or interpreting the results of Drosophila RNAi screens. Based on our own experience and user feedback, we have made several changes. Specifically, we have restructured the database to accommodate new types of reagents; added information about new RNAi libraries and other reagents; updated the user interface and website; and added new tools of use to the Drosophila community and others. Overall, the result is a more useful, flexible and comprehensive website and database.
%B Nucleic Acids Res %V 40 %P D715-9 %8 2012 Jan %G eng %N Database issue %1 http://www.ncbi.nlm.nih.gov/pubmed/22067456?dopt=Abstract %R 10.1093/nar/gkr953 %0 Journal Article %J Cell Metab %D 2012 %T Intramyocellular fatty-acid metabolism plays a critical role in mediating responses to dietary restriction in Drosophila melanogaster. %A Katewa, Subhash D %A Demontis, Fabio %A Kolipinski, Marysia %A Hubbard, Alan %A Gill, Matthew S %A Perrimon, Norbert %A Melov, Simon %A Kapahi, Pankaj %K Acetyl-CoA Carboxylase %K Animals %K Caloric Restriction %K Drosophila melanogaster %K Drosophila Proteins %K Fat Body %K Fatty Acids %K Female %K Gene Expression %K Gene Knockdown Techniques %K Insect Hormones %K Lipogenesis %K Lipolysis %K Longevity %K Male %K Motor Activity %K Muscle Cells %K Muscles %K Oligopeptides %K Pyrrolidonecarboxylic Acid %K RNA Interference %K Triglycerides %XChanges in fat content have been associated with dietary restriction (DR), but whether they play a causal role in mediating various responses to DR remains unknown. We demonstrate that upon DR, Drosophila melanogaster shift their metabolism toward increasing fatty-acid synthesis and breakdown, which is required for various responses to DR. Inhibition of fatty-acid synthesis or oxidation genes specifically in the muscle tissue inhibited life-span extension upon DR. Furthermore, DR enhances spontaneous activity of flies, which was found to be dependent on the enhanced fatty-acid metabolism. This increase in activity was found to be at least partially required for the life-span extension upon DR. Overexpression of adipokinetic hormone (dAKH), the functional ortholog of glucagon, enhances fat metabolism, spontaneous activity, and life span. Together, these results suggest that enhanced fat metabolism in the muscle and physical activity play a key role in the protective effects of DR.
%B Cell Metab %V 16 %P 97-103 %8 2012 Jul 3 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/22768842?dopt=Abstract %R 10.1016/j.cmet.2012.06.005 %0 Journal Article %J Wiley Interdiscip Rev RNA %D 2012 %T RNAi screening: new approaches, understandings, and organisms. %A Mohr, Stephanie E %A Perrimon, Norbert %K Animals %K Gene Knockdown Techniques %K Genetic Testing %K Genomics %K High-Throughput Screening Assays %K Humans %K Phenotype %K RNA Interference %K RNA, Small Interfering %XRNA interference (RNAi) leads to sequence-specific knockdown of gene function. The approach can be used in large-scale screens to interrogate function in various model organisms and an increasing number of other species. Genome-scale RNAi screens are routinely performed in cultured or primary cells or in vivo in organisms such as C. elegans. High-throughput RNAi screening is benefitting from the development of sophisticated new instrumentation and software tools for collecting and analyzing data, including high-content image data. The results of large-scale RNAi screens have already proved useful, leading to new understandings of gene function relevant to topics such as infection, cancer, obesity, and aging. Nevertheless, important caveats apply and should be taken into consideration when developing or interpreting RNAi screens. Some level of false discovery is inherent to high-throughput approaches and specific to RNAi screens, false discovery due to off-target effects (OTEs) of RNAi reagents remains a problem. The need to improve our ability to use RNAi to elucidate gene function at large scale and in additional systems continues to be addressed through improved RNAi library design, development of innovative computational and analysis tools and other approaches.
%B Wiley Interdiscip Rev RNA %V 3 %P 145-58 %8 2012 Mar-Apr %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/21953743?dopt=Abstract %R 10.1002/wrna.110 %0 Journal Article %J Cold Spring Harb Perspect Biol %D 2012 %T Signaling mechanisms controlling cell fate and embryonic patterning. %A Perrimon, Norbert %A Pitsouli, Chrysoula %A Shilo, Ben-Zion %K Animals %K Body Patterning %K Cell Differentiation %K Drosophila %K Gene Expression Regulation, Developmental %K Humans %K Models, Biological %K Paracrine Communication %K Receptors, Notch %K Signal Transduction %XDuring development, signaling pathways specify cell fates by activating transcriptional programs in response to extracellular signals. Extensive studies in the past 30 years have revealed that surprisingly few pathways exist to regulate developmental programs and that dysregulation of these can lead to human diseases, including cancer. Although these pathways use distinct signaling components and signaling strategies, a number of common themes have emerged regarding their organization and regulation in time and space. Examples from Drosophila, such as Notch, Hedgehog, Wingless/WNT, BMP (bone morphogenetic proteins), EGF (epidermal growth factor), and FGF (fibroblast growth factor) signaling, illustrate their abilities to act either at a short range or over a long distance, and in some instances to generate morphogen gradients that pattern fields of cells in a concentration-dependent manner. They also show how feedback loops and transcriptional cascades are part of the logic of developmental regulation.
%B Cold Spring Harb Perspect Biol %V 4 %P a005975 %8 2012 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/22855721?dopt=Abstract %R 10.1101/cshperspect.a005975 %0 Journal Article %J Genetics %D 2012 %T Stringent analysis of gene function and protein-protein interactions using fluorescently tagged genes. %A Neumüller, Ralph A %A Wirtz-Peitz, Frederik %A Lee, Stella %A Kwon, Young %A Buckner, Michael %A Hoskins, Roger A %A Venken, Koen J T %A Bellen, Hugo J %A Mohr, Stephanie E %A Perrimon, Norbert %K Animals %K Cell Line %K Cell Survival %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Female %K Fluorescent Dyes %K Gene Order %K Gene Silencing %K Green Fluorescent Proteins %K Multiprotein Complexes %K Peptide Elongation Factors %K Protein Binding %K Protein Interaction Mapping %K Recombinant Fusion Proteins %K RNA, Small Interfering %K Stem Cells %XIn Drosophila collections of green fluorescent protein (GFP) trap lines have been used to probe the endogenous expression patterns of trapped genes or the subcellular localization of their protein products. Here, we describe a method, based on nonoverlapping, highly specific, shRNA transgenes directed against GFP, that extends the utility of these collections to loss-of-function studies. Furthermore, we used a MiMIC transposon to generate GFP traps in Drosophila cell lines with distinct subcellular localization patterns, which will permit high-throughput screens using fluorescently tagged proteins. Finally, we show that fluorescent traps, paired with recombinant nanobodies and mass spectrometry, allow the study of endogenous protein complexes in Drosophila.
%B Genetics %V 190 %P 931-40 %8 2012 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/22174071?dopt=Abstract %R 10.1534/genetics.111.136465 %0 Journal Article %J J Vis Exp %D 2011 %T Primary cell cultures from Drosophila gastrula embryos. %A Perrimon, Norbert %A Zirin, Jonathan %A Bai, Jianwu %K Animals %K Cell Culture Techniques %K Culture Media %K Drosophila %K RNA Interference %K RNA, Double-Stranded %XHere we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.
%B J Vis Exp %8 2011 %G eng %N 48 %1 http://www.ncbi.nlm.nih.gov/pubmed/21403631?dopt=Abstract %R 10.3791/2215 %0 Journal Article %J J Cell Biol %D 2011 %T Comparative RNAi screening identifies a conserved core metazoan actinome by phenotype. %A Rohn, Jennifer L %A Sims, David %A Liu, Tao %A Fedorova, Marina %A Schöck, Frieder %A Dopie, Joseph %A Vartiainen, Maria K %A Kiger, Amy A %A Perrimon, Norbert %A Baum, Buzz %K Actin Cytoskeleton %K Actins %K Animals %K Carboxy-Lyases %K Carrier Proteins %K Cell Line %K Cell Nucleus %K Cell Shape %K Cluster Analysis %K DNA %K Drosophila melanogaster %K Drosophila Proteins %K HeLa Cells %K Hemocytes %K High-Throughput Screening Assays %K Humans %K Microfilament Proteins %K Phenotype %K rho GTP-Binding Proteins %K RNA Interference %K RNA Splicing %K RNA, Double-Stranded %K RNA, Small Interfering %K SKP Cullin F-Box Protein Ligases %K Tubulin %XAlthough a large number of actin-binding proteins and their regulators have been identified through classical approaches, gaps in our knowledge remain. Here, we used genome-wide RNA interference as a systematic method to define metazoan actin regulators based on visual phenotype. Using comparative screens in cultured Drosophila and human cells, we generated phenotypic profiles for annotated actin regulators together with proteins bearing predicted actin-binding domains. These phenotypic clusters for the known metazoan "actinome" were used to identify putative new core actin regulators, together with a number of genes with conserved but poorly studied roles in the regulation of the actin cytoskeleton, several of which we studied in detail. This work suggests that although our search for new components of the core actin machinery is nearing saturation, regulation at the level of nuclear actin export, RNA splicing, ubiquitination, and other upstream processes remains an important but unexplored frontier of actin biology.
%B J Cell Biol %V 194 %P 789-805 %8 2011 Sep 5 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/21893601?dopt=Abstract %R 10.1083/jcb.201103168 %0 Journal Article %J Cell %D 2011 %T Control of the mitotic cleavage plane by local epithelial topology. %A Gibson, William T %A Veldhuis, James H %A Rubinstein, Boris %A Cartwright, Heather N %A Perrimon, Norbert %A Brodland, G Wayne %A Radhika Nagpal %A Gibson, Matthew C %K Animals %K Cell Division %K Cell Shape %K Cell Size %K Cucumis sativus %K Drosophila melanogaster %K Epithelial Cells %K Spindle Apparatus %K Wings, Animal %XFor nearly 150 years, it has been recognized that cell shape strongly influences the orientation of the mitotic cleavage plane (e.g., Hofmeister, 1863). However, we still understand little about the complex interplay between cell shape and cleavage-plane orientation in epithelia, where polygonal cell geometries emerge from multiple factors, including cell packing, cell growth, and cell division itself. Here, using mechanical simulations, we show that the polygonal shapes of individual cells can systematically bias the long-axis orientations of their adjacent mitotic neighbors. Strikingly, analyses of both animal epithelia and plant epidermis confirm a robust and nearly identical correlation between local cell topology and cleavage-plane orientation in vivo. Using simple mathematics, we show that this effect derives from fundamental packing constraints. Our results suggest that local epithelial topology is a key determinant of cleavage-plane orientation, and that cleavage-plane bias may be a widespread property of polygonal cell sheets in plants and animals.
%B Cell %V 144 %P 427-38 %8 2011 Feb 4 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/21295702?dopt=Abstract %R 10.1016/j.cell.2010.12.035 %0 Journal Article %J Genome Res %D 2011 %T Deep annotation of Drosophila melanogaster microRNAs yields insights into their processing, modification, and emergence. %A Berezikov, Eugene %A Robine, Nicolas %A Samsonova, Anastasia %A Westholm, Jakub O %A Naqvi, Ammar %A Hung, Jui-Hung %A Okamura, Katsutomo %A Dai, Qi %A Bortolamiol-Becet, Diane %A Martin, Raquel %A Zhao, Yongjun %A Zamore, Phillip D %A Hannon, Gregory J %A Marra, Marco A %A Weng, Zhiping %A Perrimon, Norbert %A Lai, Eric C %K Animals %K Base Sequence %K Cell Line %K Computational Biology %K Drosophila melanogaster %K Female %K Gene Expression Regulation %K Male %K MicroRNAs %K Molecular Sequence Annotation %K Ribonuclease III %K RNA Editing %K RNA, Antisense %K RNA, Messenger %K Sequence Alignment %XSince the initial annotation of miRNAs from cloned short RNAs by the Ambros, Tuschl, and Bartel groups in 2001, more than a hundred studies have sought to identify additional miRNAs in various species. We report here a meta-analysis of short RNA data from Drosophila melanogaster, aggregating published libraries with 76 data sets that we generated for the modENCODE project. In total, we began with more than 1 billion raw reads from 187 libraries comprising diverse developmental stages, specific tissue- and cell-types, mutant conditions, and/or Argonaute immunoprecipitations. We elucidated several features of known miRNA loci, including multiple phased byproducts of cropping and dicing, abundant alternative 5' termini of certain miRNAs, frequent 3' untemplated additions, and potential editing events. We also identified 49 novel genomic locations of miRNA production, and 61 additional candidate loci with limited evidence for miRNA biogenesis. Although these loci broaden the Drosophila miRNA catalog, this work supports the notion that a restricted set of cellular transcripts is competent to be specifically processed by the Drosha/Dicer-1 pathway. Unexpectedly, we detected miRNA production from coding and untranslated regions of mRNAs and found the phenomenon of miRNA production from the antisense strand of known loci to be common. Altogether, this study lays a comprehensive foundation for the study of miRNA diversity and evolution in a complex animal model.
%B Genome Res %V 21 %P 203-15 %8 2011 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/21177969?dopt=Abstract %R 10.1101/gr.116657.110 %0 Journal Article %J Nature %D 2011 %T The developmental transcriptome of Drosophila melanogaster. %A Graveley, Brenton R %A Brooks, Angela N %A Carlson, Joseph W %A Duff, Michael O %A Landolin, Jane M %A Yang, Li %A Artieri, Carlo G %A van Baren, Marijke J %A Boley, Nathan %A Booth, Benjamin W %A Brown, James B %A Cherbas, Lucy %A Davis, Carrie A %A Dobin, Alex %A Li, Renhua %A Lin, Wei %A Malone, John H %A Mattiuzzo, Nicolas R %A Miller, David %A Sturgill, David %A Tuch, Brian B %A Zaleski, Chris %A Zhang, Dayu %A Blanchette, Marco %A Dudoit, Sandrine %A Eads, Brian %A Green, Richard E %A Hammonds, Ann %A Jiang, Lichun %A Kapranov, Phil %A Langton, Laura %A Perrimon, Norbert %A Sandler, Jeremy E %A Wan, Kenneth H %A Willingham, Aarron %A Zhang, Yu %A Zou, Yi %A Andrews, Justen %A Bickel, Peter J %A Brenner, Steven E %A Brent, Michael R %A Cherbas, Peter %A Gingeras, Thomas R %A Hoskins, Roger A %A Kaufman, Thomas C %A Oliver, Brian %A Celniker, Susan E %K Alternative Splicing %K Animals %K Base Sequence %K Drosophila melanogaster %K Drosophila Proteins %K Exons %K Female %K Gene Expression Profiling %K Gene Expression Regulation, Developmental %K Genes, Insect %K Genome, Insect %K Male %K MicroRNAs %K Oligonucleotide Array Sequence Analysis %K Protein Isoforms %K RNA Editing %K RNA, Messenger %K RNA, Small Untranslated %K Sequence Analysis %K Sex Characteristics %K Transcription, Genetic %XDrosophila melanogaster is one of the most well studied genetic model organisms; nonetheless, its genome still contains unannotated coding and non-coding genes, transcripts, exons and RNA editing sites. Full discovery and annotation are pre-requisites for understanding how the regulation of transcription, splicing and RNA editing directs the development of this complex organism. Here we used RNA-Seq, tiling microarrays and cDNA sequencing to explore the transcriptome in 30 distinct developmental stages. We identified 111,195 new elements, including thousands of genes, coding and non-coding transcripts, exons, splicing and editing events, and inferred protein isoforms that previously eluded discovery using established experimental, prediction and conservation-based approaches. These data substantially expand the number of known transcribed elements in the Drosophila genome and provide a high-resolution view of transcriptome dynamics throughout development.
%B Nature %V 471 %P 473-9 %8 2011 Mar 24 %G eng %N 7339 %1 http://www.ncbi.nlm.nih.gov/pubmed/21179090?dopt=Abstract %R 10.1038/nature09715 %0 Journal Article %J BMC Genomics %D 2011 %T False negative rates in Drosophila cell-based RNAi screens: a case study. %A Booker, Matthew %A Samsonova, Anastasia A %A Kwon, Young %A Flockhart, Ian %A Mohr, Stephanie E %A Perrimon, Norbert %K Animals %K Cluster Analysis %K Drosophila %K Gene Expression Profiling %K Proteasome Endopeptidase Complex %K Ribosomes %K RNA Interference %K RNA, Double-Stranded %XBACKGROUND: High-throughput screening using RNAi is a powerful gene discovery method but is often complicated by false positive and false negative results. Whereas false positive results associated with RNAi reagents has been a matter of extensive study, the issue of false negatives has received less attention. RESULTS: We performed a meta-analysis of several genome-wide, cell-based Drosophila RNAi screens, together with a more focused RNAi screen, and conclude that the rate of false negative results is at least 8%. Further, we demonstrate how knowledge of the cell transcriptome can be used to resolve ambiguous results and how the number of false negative results can be reduced by using multiple, independently-tested RNAi reagents per gene. CONCLUSIONS: RNAi reagents that target the same gene do not always yield consistent results due to false positives and weak or ineffective reagents. False positive results can be partially minimized by filtering with transcriptome data. RNAi libraries with multiple reagents per gene also reduce false positive and false negative outcomes when inconsistent results are disambiguated carefully.
%B BMC Genomics %V 12 %P 50 %8 2011 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/21251254?dopt=Abstract %R 10.1186/1471-2164-12-50 %0 Journal Article %J Sci Signal %D 2011 %T A genome-wide RNAi screen identifies core components of the G₂-M DNA damage checkpoint. %A Kondo, Shu %A Perrimon, Norbert %K Animals %K Cell Cycle Proteins %K Cell Division %K DNA Damage %K Drosophila %K G1 Phase %K Genome %K RNA, Small Interfering %XThe DNA damage checkpoint, the first pathway known to be activated in response to DNA damage, is a mechanism by which the cell cycle is temporarily arrested to allow DNA repair. The checkpoint pathway transmits signals from the sites of DNA damage to the cell cycle machinery through the evolutionarily conserved ATM (ataxia telangiectasia mutated) and ATR (ATM- and Rad3-related) kinase cascades. We conducted a genome-wide RNAi (RNA interference) screen in Drosophila cells to identify previously unknown genes and pathways required for the G₂-M checkpoint induced by DNA double-strand breaks (DSBs). Our large-scale analysis provided a systems-level view of the G₂-M checkpoint and revealed the coordinated actions of particular classes of proteins, which include those involved in DNA repair, DNA replication, cell cycle control, chromatin regulation, and RNA processing. Further, from the screen and in vivo analysis, we identified previously unrecognized roles of two DNA damage response genes, mus101 and mus312. Our results suggest that the DNA replication preinitiation complex, which includes MUS101, and the MUS312-containing nuclease complexes, which are important for DSB repair, also function in the G₂-M checkpoint. Our results provide insight into the diverse mechanisms that link DNA damage and the checkpoint signaling pathway.
%B Sci Signal %V 4 %P rs1 %8 2011 %G eng %N 154 %1 http://www.ncbi.nlm.nih.gov/pubmed/21205937?dopt=Abstract %R 10.1126/scisignal.2001350 %0 Journal Article %J Genome Res %D 2011 %T The transcriptional diversity of 25 Drosophila cell lines. %A Cherbas, Lucy %A Willingham, Aarron %A Zhang, Dayu %A Yang, Li %A Zou, Yi %A Eads, Brian D %A Carlson, Joseph W %A Landolin, Jane M %A Kapranov, Philipp %A Dumais, Jacqueline %A Samsonova, Anastasia %A Choi, Jeong-Hyeon %A Roberts, Johnny %A Davis, Carrie A %A Tang, Haixu %A van Baren, Marijke J %A Ghosh, Srinka %A Dobin, Alexander %A Bell, Kim %A Lin, Wei %A Langton, Laura %A Duff, Michael O %A Tenney, Aaron E %A Zaleski, Chris %A Brent, Michael R %A Hoskins, Roger A %A Kaufman, Thomas C %A Andrews, Justen %A Graveley, Brenton R %A Perrimon, Norbert %A Celniker, Susan E %A Gingeras, Thomas R %A Cherbas, Peter %K Animals %K Cell Line %K Cluster Analysis %K Drosophila melanogaster %K Exons %K Female %K Gene Expression Profiling %K Genetic Variation %K Male %K Molecular Sequence Data %K Signal Transduction %K Transcription Factors %K Transcription, Genetic %XDrosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are "off" and survival/growth pathways "on." Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common "cell line" gene expression pattern.
%B Genome Res %V 21 %P 301-14 %8 2011 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/21177962?dopt=Abstract %R 10.1101/gr.112961.110 %0 Journal Article %J Genome Res %D 2011 %T Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs. %A Schnall-Levin, Michael %A Rissland, Olivia S %A Johnston, Wendy K %A Perrimon, Norbert %A Bartel, David P %A Berger, Bonnie %K Amino Acid Motifs %K Animals %K Gene Duplication %K Gene Expression Regulation %K Gene Knockdown Techniques %K HEK293 Cells %K HeLa Cells %K Humans %K MicroRNAs %K Open Reading Frames %K Repetitive Sequences, Nucleic Acid %K RNA, Messenger %K Zinc Fingers %XMicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3' untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3' UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C(2)H(2) zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C(2)H(2) zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted.
%B Genome Res %V 21 %P 1395-403 %8 2011 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/21685129?dopt=Abstract %R 10.1101/gr.121210.111 %0 Journal Article %J Developmental Cell %D 2011 %T Deciphering gene expression patterns (Paper Pick) %A Perrimon, Norbert %B Developmental Cell %V 21 %P e1 %G eng %0 Audiovisual Material %D 2011 %T Science Signaling Podcast: 25 October 2011 %A Friedman, Adam A %A Perrimon, Norbert %E VanHook, Annalisa M %XThis Podcast features a conversation with authors of a Research Resource published in the 25 October 2011 issue of Science Signaling. Although the extracellular signal–regulated kinase (ERK) pathway has been extensively studied, our understanding of all the regulatory interactions that modulate signaling is by no means complete. Adam Friedman and Norbert Perrimon discuss their group’s strategy of combining functional and genomics approaches to identify common and specific regulators of ERK signaling in the fruit fly Drosophila melanogaster. The ERK pathway plays an important role in normal developmental and physiological contexts as well as in disease states, such as tumorigenesis.
%B ScienceMag.org %G eng %U http://stke.sciencemag.org/content/4/196/pc22 %0 Journal Article %J Dev Cell %D 2011 %T Drosophila as a model for interorgan communication: lessons from studies on energy homeostasis. %A Rajan, Akhila %A Perrimon, Norbert %K Aging %K Animals %K Drosophila %K Energy Metabolism %K Homeostasis %K Models, Animal %K Stem Cells %XCurrent studies of physiological communication between Drosophila organs are beginning to address the fundamental problem of how nutrients regulate organismal growth, stem cell behavior, immunity, and aging. Advances in the Drosophila genetic tool kit will allow the design of genetic screens to systematically identify factors involved in organ communication.
%B Dev Cell %V 21 %P 29-31 %8 2011 Jul 19 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/21763605?dopt=Abstract %R 10.1016/j.devcel.2011.06.034 %0 Journal Article %J Curr Opin Genet Dev %D 2011 %T The era of systems developmental biology. %A Perrimon, Norbert %A Barkai, Naama %K Animals %K Body Patterning %K Cell Polarity %K Gene Expression Regulation, Developmental %K Genomics %K Models, Biological %K Proteomics %K Systems Biology %B Curr Opin Genet Dev %V 21 %P 681-3 %8 2011 Dec %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/22079435?dopt=Abstract %R 10.1016/j.gde.2011.10.004 %0 Journal Article %J Hum Mol Genet %D 2011 %T Expanded polyglutamine domain possesses nuclear export activity which modulates subcellular localization and toxicity of polyQ disease protein via exportin-1. %A Chan, Wing Man %A Tsoi, Ho %A Wu, Chi Chung %A Wong, Chi Hang %A Cheng, Tat Cheung %A Li, Hoi Yeung %A Lau, Kwok Fai %A Shaw, Pang Chui %A Perrimon, Norbert %A Chan, Ho Yin Edwin %K Active Transport, Cell Nucleus %K Animals %K Cell Line %K Cell Nucleus %K Disease Models, Animal %K Drosophila %K HEK293 Cells %K Humans %K Intracellular Space %K Karyopherins %K Neurodegenerative Diseases %K Peptides %K Protein Binding %K Protein Structure, Tertiary %K Protein Transport %K Proteins %K Receptors, Cytoplasmic and Nuclear %K Trinucleotide Repeat Expansion %XPolyglutamine (polyQ) diseases are a group of late-onset, progressive neurodegenerative disorders caused by CAG trinucleotide repeat expansion in the coding region of disease genes. The cell nucleus is an important site of pathology in polyQ diseases, and transcriptional dysregulation is one of the pathologic hallmarks observed. In this study, we showed that exportin-1 (Xpo1) regulates the nucleocytoplasmic distribution of expanded polyQ protein. We found that expanded polyQ protein, but not its unexpanded form, possesses nuclear export activity and interacts with Xpo1. Genetic manipulation of Xpo1 expression levels in transgenic Drosophila models of polyQ disease confirmed the specific nuclear export role of Xpo1 on expanded polyQ protein. Upon Xpo1 knockdown, the expanded polyQ protein was retained in the nucleus. The nuclear disease protein enhanced polyQ toxicity by binding to heat shock protein (hsp) gene promoter and abolished hsp gene induction. Further, we uncovered a developmental decline of Xpo1 protein levels in vivo that contributes to the accumulation of expanded polyQ protein in the nucleus of symptomatic polyQ transgenic mice. Taken together, we first showed that Xpo1 is a nuclear export receptor for expanded polyQ domain, and our findings establish a direct link between protein nuclear export and the progressive nature of polyQ neurodegeneration.
%B Hum Mol Genet %V 20 %P 1738-50 %8 2011 May 1 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/21300695?dopt=Abstract %R 10.1093/hmg/ddr049 %0 Journal Article %J Nat Methods %D 2011 %T A genome-scale shRNA resource for transgenic RNAi in Drosophila. %A Ni, Jian-Quan %A Zhou, Rui %A Czech, Benjamin %A Liu, Lu-Ping %A Holderbaum, Laura %A Yang-Zhou, Donghui %A Shim, Hye-Seok %A Tao, Rong %A Handler, Dominik %A Karpowicz, Phillip %A Binari, Richard %A Booker, Matthew %A Brennecke, Julius %A Perkins, Lizabeth A %A Hannon, Gregory J %A Perrimon, Norbert %K Animals %K Animals, Genetically Modified %K Base Sequence %K DNA Primers %K Drosophila melanogaster %K Female %K Gene Knockdown Techniques %K Genetic Techniques %K Genetic Vectors %K Genome, Insect %K MicroRNAs %K Oogenesis %K RNA Interference %K RNA, Small Interfering %XExisting transgenic RNAi resources in Drosophila melanogaster based on long double-stranded hairpin RNAs are powerful tools for functional studies, but they are ineffective in gene knockdown during oogenesis, an important model system for the study of many biological questions. We show that shRNAs, modeled on an endogenous microRNA, are extremely effective at silencing gene expression during oogenesis. We also describe our progress toward building a genome-wide shRNA resource.
%B Nat Methods %V 8 %P 405-7 %8 2011 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/21460824?dopt=Abstract %R 10.1038/nmeth.1592 %0 Journal Article %J Gene Expr Patterns %D 2011 %T Identification of adult midgut precursors in Drosophila. %A Micchelli, Craig A %A Sudmeier, Lisa %A Perrimon, Norbert %A Tang, Shan %A Beehler-Evans, Ryan %K Animals %K Cell Lineage %K Digestive System %K Drosophila %K Ecdysone %K Larva %K Stem Cells %XThe adult Drosophila midgut is thought to arise from an endodermal rudiment specified during embryogenesis. Previous studies have reported the presence of individual cells termed adult midgut precursors (AMPs) as well as "midgut islands" or "islets" in embryonic and larval midgut tissue. Yet the precise relationship between progenitor cell populations and the cells of the adult midgut has not been characterized. Using a combination of molecular markers and directed cell lineage tracing, we provide evidence that the adult midgut arises from a molecularly distinct population of single cells present by the embryonic/larval transition. AMPs reside in a distinct basal position in the larval midgut where they remain through all subsequent larval and pupal stages and into adulthood. At least five phases of AMP activity are associated with the stepwise process of midgut formation. Our data shows that during larval stages AMPs give rise to the presumptive adult epithelium; during pupal stages AMPs contribute to the final size, cell number and form. Finally, a genetic screen has led to the identification of the Ecdysone receptor as a regulator of AMP expansion.
%B Gene Expr Patterns %V 11 %P 12-21 %8 2011 Jan-Feb %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/20804858?dopt=Abstract %R 10.1016/j.gep.2010.08.005 %0 Journal Article %J BMC Bioinformatics %D 2011 %T An integrative approach to ortholog prediction for disease-focused and other functional studies. %A Hu, Yanhui %A Flockhart, Ian %A Vinayagam, Arunachalam %A Bergwitz, Clemens %A Berger, Bonnie %A Perrimon, Norbert %A Mohr, Stephanie E %K Animals %K Databases, Genetic %K Disease %K Disease Models, Animal %K Evolution, Molecular %K Genetic Predisposition to Disease %K Genome-Wide Association Study %K Humans %XBACKGROUND: Mapping of orthologous genes among species serves an important role in functional genomics by allowing researchers to develop hypotheses about gene function in one species based on what is known about the functions of orthologs in other species. Several tools for predicting orthologous gene relationships are available. However, these tools can give different results and identification of predicted orthologs is not always straightforward. RESULTS: We report a simple but effective tool, the Drosophila RNAi Screening Center Integrative Ortholog Prediction Tool (DIOPT; http://www.flyrnai.org/diopt), for rapid identification of orthologs. DIOPT integrates existing approaches, facilitating rapid identification of orthologs among human, mouse, zebrafish, C. elegans, Drosophila, and S. cerevisiae. As compared to individual tools, DIOPT shows increased sensitivity with only a modest decrease in specificity. Moreover, the flexibility built into the DIOPT graphical user interface allows researchers with different goals to appropriately 'cast a wide net' or limit results to highest confidence predictions. DIOPT also displays protein and domain alignments, including percent amino acid identity, for predicted ortholog pairs. This helps users identify the most appropriate matches among multiple possible orthologs. To facilitate using model organisms for functional analysis of human disease-associated genes, we used DIOPT to predict high-confidence orthologs of disease genes in Online Mendelian Inheritance in Man (OMIM) and genes in genome-wide association study (GWAS) data sets. The results are accessible through the DIOPT diseases and traits query tool (DIOPT-DIST; http://www.flyrnai.org/diopt-dist). CONCLUSIONS: DIOPT and DIOPT-DIST are useful resources for researchers working with model organisms, especially those who are interested in exploiting model organisms such as Drosophila to study the functions of human disease genes.
%B BMC Bioinformatics %V 12 %P 357 %8 2011 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/21880147?dopt=Abstract %R 10.1186/1471-2105-12-357 %0 Journal Article %J Sci Signal %D 2011 %T Proteomic and functional genomic landscape of receptor tyrosine kinase and ras to extracellular signal-regulated kinase signaling. %A Friedman, Adam A %A Tucker, George %A Singh, Rohit %A Yan, Dong %A Vinayagam, Arunachalam %A Hu, Yanhui %A Binari, Richard %A Hong, Pengyu %A Sun, Xiaoyun %A Porto, Maura %A Pacifico, Svetlana %A Murali, Thilakam %A Finley, Russell L %A Asara, John M %A Berger, Bonnie %A Perrimon, Norbert %K Algorithms %K Animals %K Blotting, Western %K Cell Line %K Drosophila %K Drosophila Proteins %K Extracellular Signal-Regulated MAP Kinases %K Gene Regulatory Networks %K Genomics %K Immunoprecipitation %K MAP Kinase Signaling System %K Models, Genetic %K Protein Binding %K Protein Interaction Mapping %K Proteomics %K ras Proteins %K Receptor Protein-Tyrosine Kinases %K RNA Interference %K Wings, Animal %XCharacterizing the extent and logic of signaling networks is essential to understanding specificity in such physiological and pathophysiological contexts as cell fate decisions and mechanisms of oncogenesis and resistance to chemotherapy. Cell-based RNA interference (RNAi) screens enable the inference of large numbers of genes that regulate signaling pathways, but these screens cannot provide network structure directly. We describe an integrated network around the canonical receptor tyrosine kinase (RTK)-Ras-extracellular signal-regulated kinase (ERK) signaling pathway, generated by combining parallel genome-wide RNAi screens with protein-protein interaction (PPI) mapping by tandem affinity purification-mass spectrometry. We found that only a small fraction of the total number of PPI or RNAi screen hits was isolated under all conditions tested and that most of these represented the known canonical pathway components, suggesting that much of the core canonical ERK pathway is known. Because most of the newly identified regulators are likely cell type- and RTK-specific, our analysis provides a resource for understanding how output through this clinically relevant pathway is regulated in different contexts. We report in vivo roles for several of the previously unknown regulators, including CG10289 and PpV, the Drosophila orthologs of two components of the serine/threonine-protein phosphatase 6 complex; the Drosophila ortholog of TepIV, a glycophosphatidylinositol-linked protein mutated in human cancers; CG6453, a noncatalytic subunit of glucosidase II; and Rtf1, a histone methyltransferase.
%B Sci Signal %V 4 %P rs10 %8 2011 %G eng %N 196 %1 http://www.ncbi.nlm.nih.gov/pubmed/22028469?dopt=Abstract %R 10.1126/scisignal.2002029 %0 Journal Article %J Wiley Interdiscip Rev Syst Biol Med %D 2011 %T Where gene discovery turns into systems biology: genome-scale RNAi screens in Drosophila. %A Neumüller, Ralph A %A Perrimon, Norbert %K Animals %K Cells, Cultured %K Databases, Genetic %K Drosophila %K Genetic Association Studies %K Genome, Insect %K RNA Interference %K Systems Biology %XSystems biology aims to describe the complex interplays between cellular building blocks which, in their concurrence, give rise to the emergent properties observed in cellular behaviors and responses. This approach tries to determine the molecular players and the architectural principles of their interactions within the genetic networks that control certain biological processes. Large-scale loss-of-function screens, applicable in various different model systems, have begun to systematically interrogate entire genomes to identify the genes that contribute to a certain cellular response. In particular, RNA interference (RNAi)-based high-throughput screens have been instrumental in determining the composition of regulatory systems and paired with integrative data analyses have begun to delineate the genetic networks that control cell biological and developmental processes. Through the creation of tools for both, in vitro and in vivo genome-wide RNAi screens, Drosophila melanogaster has emerged as one of the key model organisms in systems biology research and over the last years has massively contributed to and hence shaped this discipline. WIREs Syst Biol Med 2011 3 471-478 DOI: 10.1002/wsbm.127
%B Wiley Interdiscip Rev Syst Biol Med %V 3 %P 471-8 %8 2011 Jul-Aug %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/21197652?dopt=Abstract %R 10.1002/wsbm.127 %0 Journal Article %J Science %D 2010 %T Identification of functional elements and regulatory circuits by Drosophila modENCODE. %A modENCODE, %A Roy, Sushmita %A Ernst, Jason %A Kharchenko, Peter V %A Kheradpour, Pouya %A Negre, Nicolas %A Eaton, Matthew L %A Landolin, Jane M %A Bristow, Christopher A %A Ma, Lijia %A Lin, Michael F %A Washietl, Stefan %A Arshinoff, Bradley I %A Ay, Ferhat %A Meyer, Patrick E %A Robine, Nicolas %A Washington, Nicole L %A Di Stefano, Luisa %A Berezikov, Eugene %A Brown, Christopher D %A Candeias, Rogerio %A Carlson, Joseph W %A Carr, Adrian %A Jungreis, Irwin %A Marbach, Daniel %A Sealfon, Rachel %A Tolstorukov, Michael Y %A Will, Sebastian %A Alekseyenko, Artyom A %A Artieri, Carlo %A Booth, Benjamin W %A Brooks, Angela N %A Dai, Qi %A Davis, Carrie A %A Duff, Michael O %A Feng, Xin %A Gorchakov, Andrey A %A Gu, Tingting %A Henikoff, Jorja G %A Kapranov, Philipp %A Li, Renhua %A MacAlpine, Heather K %A Malone, John %A Minoda, Aki %A Nordman, Jared %A Okamura, Katsutomo %A Perry, Marc %A Powell, Sara K %A Riddle, Nicole C %A Sakai, Akiko %A Samsonova, Anastasia %A Sandler, Jeremy E %A Schwartz, Yuri B %A Sher, Noa %A Spokony, Rebecca %A Sturgill, David %A van Baren, Marijke %A Wan, Kenneth H %A Yang, Li %A Yu, Charles %A Feingold, Elise %A Good, Peter %A Guyer, Mark %A Lowdon, Rebecca %A Ahmad, Kami %A Andrews, Justen %A Berger, Bonnie %A Brenner, Steven E %A Brent, Michael R %A Cherbas, Lucy %A Elgin, Sarah C R %A Gingeras, Thomas R %A Grossman, Robert %A Hoskins, Roger A %A Kaufman, Thomas C %A Kent, William %A Kuroda, Mitzi I %A Orr-Weaver, Terry %A Perrimon, Norbert %A Pirrotta, Vincenzo %A Posakony, James W %A Ren, Bing %A Russell, Steven %A Cherbas, Peter %A Graveley, Brenton R %A Lewis, Suzanna %A Micklem, Gos %A Oliver, Brian %A Park, Peter J %A Celniker, Susan E %A Henikoff, Steven %A Karpen, Gary H %A Lai, Eric C %A MacAlpine, David M %A Stein, Lincoln D %A White, Kevin P %A Kellis, Manolis %K Animals %K Binding Sites %K Chromatin %K Computational Biology %K Drosophila melanogaster %K Drosophila Proteins %K Epigenesis, Genetic %K Gene Expression Regulation %K Gene Regulatory Networks %K Genes, Insect %K Genome, Insect %K Genomics %K Histones %K Molecular Sequence Annotation %K Nucleosomes %K Promoter Regions, Genetic %K RNA, Small Untranslated %K Transcription Factors %K Transcription, Genetic %XTo gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation.
%B Science %V 330 %P 1787-97 %8 2010 Dec 24 %G eng %N 6012 %1 http://www.ncbi.nlm.nih.gov/pubmed/21177974?dopt=Abstract %R 10.1126/science.1198374 %0 Journal Article %J F1000 Biol Rep %D 2010 %T All for one, and one for all: the clonality of the intestinal stem cell niche. %A Karpowicz, Phillip %A Perrimon, Norbert %XIntestinal epithelia are maintained by intestinal stem cells (ISCs) that divide to replace dying absorptive and secretory cells that make up this tissue. Lineage labeling studies, both in vertebrates and Drosophila, have revealed the relationships between ISCs and their progeny. In addition, a number of signaling pathways involved in ISC proliferation and differentiation have been identified. Further studies will clarify the signals originating from the ISC niche and determine the processes that control the number and uniform distribution of niches throughout the epithelium.
%B F1000 Biol Rep %V 2 %P 73 %8 2010 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/21173846?dopt=Abstract %R 10.3410/B2-73 %0 Journal Article %J Cell Metab %D 2010 %T Steroids make you bigger? Fat chance says Myc. %A Rajan, Akhila %A Perrimon, Norbert %XIn flies, ecdysone integrates growth with developmental transitions by antagonizing insulin signaling, which links growth with nutritional status. Work in Developmental Cell (Delanoue et. al, 2010) finds that ecdysone represses the transcription factor Myc in the larval fat body to inhibit systemic growth, revealing a mechanism for such coordination.
%B Cell Metab %V 12 %P 7-9 %8 2010 Jul 7 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/20620990?dopt=Abstract %R 10.1016/j.cmet.2010.06.003 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2010 %T Conserved microRNA targeting in Drosophila is as widespread in coding regions as in 3'UTRs. %A Schnall-Levin, Michael %A Zhao, Yong %A Perrimon, Norbert %A Berger, Bonnie %K 3' Untranslated Regions %K Algorithms %K Animals %K Drosophila %K MicroRNAs %K Open Reading Frames %XMicroRNAs (miRNAs) are a class of short noncoding RNAs that regulate protein-coding genes posttranscriptionally. In animals, most known miRNA targeting occurs within the 3'UTR of mRNAs, but the extent of biologically relevant targeting in the ORF or 5'UTR of mRNAs remains unknown. Here, we develop an algorithm (MinoTar-miRNA ORF Targets) to identify conserved regulatory motifs within protein-coding regions and use it to estimate the number of preferentially conserved miRNA-target sites in ORFs. We show that, in Drosophila, preferentially conserved miRNA targeting in ORFs is as widespread as it is in 3'UTRs and that, while far less abundant, conserved targets in Drosophila 5'UTRs number in the hundreds. Using our algorithm, we predicted a set of high-confidence ORF targets and selected seven miRNA-target pairs from among these for experimental validation. We observed down-regulation by the miRNA in five out of seven cases, indicating our approach can recover functional sites with high confidence. Additionally, we observed additive targeting by multiple sites within a single ORF. Altogether, our results demonstrate that the scale of biologically important miRNA targeting in ORFs is extensive and that computational tools such as ours can aid in the identification of such targets. Further evidence suggests that our results extend to mammals, but that the extent of ORF and 5'UTR targeting relative to 3'UTR targeting may be greater in Drosophila.
%B Proc Natl Acad Sci U S A %V 107 %P 15751-6 %8 2010 Sep 7 %G eng %N 36 %1 http://www.ncbi.nlm.nih.gov/pubmed/20729470?dopt=Abstract %R 10.1073/pnas.1006172107 %0 Journal Article %J Development %D 2010 %T Embryonic multipotent progenitors remodel the Drosophila airways during metamorphosis. %A Pitsouli, Chrysoula %A Perrimon, Norbert %K Aging %K Airway Remodeling %K Animals %K Animals, Genetically Modified %K Drosophila %K Embryo, Nonmammalian %K Embryonic Stem Cells %K Genes, Developmental %K Metamorphosis, Biological %K Mitosis %K Models, Biological %K Multipotent Stem Cells %K Trachea %XAdult structures in holometabolous insects such as Drosophila are generated by groups of imaginal cells dedicated to the formation of different organs. Imaginal cells are specified in the embryo and remain quiescent until the larval stages, when they proliferate and differentiate to form organs. The Drosophila tracheal system is extensively remodeled during metamorphosis by a small number of airway progenitors. Among these, the spiracular branch tracheoblasts are responsible for the generation of the pupal and adult abdominal airways. To understand the coordination of proliferation and differentiation during organogenesis of tubular organs, we analyzed the remodeling of Drosophila airways during metamorphosis. We show that the embryonic spiracular branch tracheoblasts are multipotent cells that express the homeobox transcription factor Cut, which is necessary for their survival and normal development. They give rise to three distinct cell populations at the end of larval development, which generate the adult tracheal tubes, the spiracle and the epidermis surrounding the spiracle. Our study establishes the series of events that lead to the formation of an adult tubular structure in Drosophila.
%B Development %V 137 %P 3615-24 %8 2010 Nov %G eng %N 21 %1 http://www.ncbi.nlm.nih.gov/pubmed/20940225?dopt=Abstract %R 10.1242/dev.056408 %0 Journal Article %J PLoS One %D 2010 %T A genome-wide gene function prediction resource for Drosophila melanogaster. %A Yan, Han %A Venkatesan, Kavitha %A Beaver, John E %A Klitgord, Niels %A Muhammed A. Yildirim %A Hao, Tong %A Hill, David E %A Cusick, Michael E %A Perrimon, Norbert %A Roth, Frederick P %A Vidal, Marc %K Animals %K Databases, Genetic %K Drosophila melanogaster %K Genes, Insect %K Genomics %K Reproducibility of Results %K RNA Interference %XPredicting gene functions by integrating large-scale biological data remains a challenge for systems biology. Here we present a resource for Drosophila melanogaster gene function predictions. We trained function-specific classifiers to optimize the influence of different biological datasets for each functional category. Our model predicted GO terms and KEGG pathway memberships for Drosophila melanogaster genes with high accuracy, as affirmed by cross-validation, supporting literature evidence, and large-scale RNAi screens. The resulting resource of prioritized associations between Drosophila genes and their potential functions offers a guide for experimental investigations.
%B PLoS One %V 5 %P e12139 %8 2010 %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/20711346?dopt=Abstract %R 10.1371/journal.pone.0012139 %0 Journal Article %J Genetics %D 2010 %T A genomewide RNA interference screen for modifiers of aggregates formation by mutant Huntingtin in Drosophila. %A Zhang, Sheng %A Binari, Richard %A Zhou, Rui %A Perrimon, Norbert %K Age Factors %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Genome, Insect %K Genomics %K High-Throughput Screening Assays %K HSP110 Heat-Shock Proteins %K Humans %K Microtubule-Associated Proteins %K Mutant Proteins %K Mutation %K Peptides %K Protein Multimerization %K Protein Structure, Quaternary %K RNA Interference %K Transcription Factors %XProtein aggregates are a common pathological feature of most neurodegenerative diseases (NDs). Understanding their formation and regulation will help clarify their controversial roles in disease pathogenesis. To date, there have been few systematic studies of aggregates formation in Drosophila, a model organism that has been applied extensively in modeling NDs and screening for toxicity modifiers. We generated transgenic fly lines that express enhanced-GFP-tagged mutant Huntingtin (Htt) fragments with different lengths of polyglutamine (polyQ) tract and showed that these Htt mutants develop protein aggregates in a polyQ-length- and age-dependent manner in Drosophila. To identify central regulators of protein aggregation, we further generated stable Drosophila cell lines expressing these Htt mutants and also established a cell-based quantitative assay that allows automated measurement of aggregates within cells. We then performed a genomewide RNA interference screen for regulators of mutant Htt aggregation and isolated 126 genes involved in diverse cellular processes. Interestingly, although our screen focused only on mutant Htt aggregation, several of the identified candidates were known previously as toxicity modifiers of NDs. Moreover, modulating the in vivo activity of hsp110 (CG6603) or tra1, two hits from the screen, affects neurodegeneration in a dose-dependent manner in a Drosophila model of Huntington's disease. Thus, other aggregates regulators isolated in our screen may identify additional genes involved in the protein-folding pathway and neurotoxicity.
%B Genetics %V 184 %P 1165-79 %8 2010 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/20100940?dopt=Abstract %R 10.1534/genetics.109.112516 %0 Journal Article %J Development %D 2010 %T The Hippo tumor suppressor pathway regulates intestinal stem cell regeneration. %A Karpowicz, Phillip %A Perez, Jessica %A Perrimon, Norbert %K Animals %K Cadherins %K Cell Differentiation %K Cell Proliferation %K Drosophila %K Drosophila Proteins %K Enterocytes %K Intestines %K Intracellular Signaling Peptides and Proteins %K Microscopy, Fluorescence %K Nuclear Proteins %K Protein-Serine-Threonine Kinases %K Reverse Transcriptase Polymerase Chain Reaction %K Signal Transduction %K STAT Transcription Factors %K Stem Cells %K Temperature %K Trans-Activators %XIdentification of the signaling pathways that control the proliferation of stem cells (SCs), and whether they act in a cell or non-cell autonomous manner, is key to our understanding of tissue homeostasis and cancer. In the adult Drosophila midgut, the Jun N-Terminal Kinase (JNK) pathway is activated in damaged enterocyte cells (ECs) following injury. This leads to the production of Upd cytokines from ECs, which in turn activate the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway in Intestinal SCs (ISCs), stimulating their proliferation. In addition, the Hippo pathway has been recently implicated in the regulation of Upd production from the ECs. Here, we show that the Hippo pathway target, Yorkie (Yki), also plays a crucial and cell-autonomous role in ISCs. Activation of Yki in ISCs is sufficient to increase ISC proliferation, a process involving Yki target genes that promote division, survival and the Upd cytokines. We further show that prior to injury, Yki activity is constitutively repressed by the upstream Hippo pathway members Fat and Dachsous (Ds). These findings demonstrate a cell-autonomous role for the Hippo pathway in SCs, and have implications for understanding the role of this pathway in tumorigenesis and cancer stem cells.
%B Development %V 137 %P 4135-45 %8 2010 Dec %G eng %N 24 %1 http://www.ncbi.nlm.nih.gov/pubmed/21098564?dopt=Abstract %R 10.1242/dev.060483 %0 Journal Article %J Genome Res %D 2010 %T Inference of RhoGAP/GTPase regulation using single-cell morphological data from a combinatorial RNAi screen. %A Nir, Oaz %A Bakal, Chris %A Perrimon, Norbert %A Berger, Bonnie %K Animals %K Drosophila %K GTP Phosphohydrolases %K GTPase-Activating Proteins %K Protein Processing, Post-Translational %K RNA Interference %K Signal Transduction %XBiological networks are highly complex systems, consisting largely of enzymes that act as molecular switches to activate/inhibit downstream targets via post-translational modification. Computational techniques have been developed to perform signaling network inference using some high-throughput data sources, such as those generated from transcriptional and proteomic studies, but comparable methods have not been developed to use high-content morphological data, which are emerging principally from large-scale RNAi screens, to these ends. Here, we describe a systematic computational framework based on a classification model for identifying genetic interactions using high-dimensional single-cell morphological data from genetic screens, apply it to RhoGAP/GTPase regulation in Drosophila, and evaluate its efficacy. Augmented by knowledge of the basic structure of RhoGAP/GTPase signaling, namely, that GAPs act directly upstream of GTPases, we apply our framework for identifying genetic interactions to predict signaling relationships between these proteins. We find that our method makes mediocre predictions using only RhoGAP single-knockdown morphological data, yet achieves vastly improved accuracy by including original data from a double-knockdown RhoGAP genetic screen, which likely reflects the redundant network structure of RhoGAP/GTPase signaling. We consider other possible methods for inference and show that our primary model outperforms the alternatives. This work demonstrates the fundamental fact that high-throughput morphological data can be used in a systematic, successful fashion to identify genetic interactions and, using additional elementary knowledge of network structure, to infer signaling relations.
%B Genome Res %V 20 %P 372-80 %8 2010 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/20144944?dopt=Abstract %R 10.1101/gr.100248.109 %0 Journal Article %J Semin Immunopathol %D 2010 %T Drosophila as a model system to study autophagy. %A Zirin, Jonathan %A Perrimon, Norbert %K Animals %K Autophagy %K Drosophila melanogaster %K Homeostasis %K Humans %K Models, Immunological %XOriginally identified as a response to starvation in yeast, autophagy is now understood to fulfill a variety of roles in higher eukaryotes, from the maintenance of cellular homeostasis to the cellular response to stress, starvation, and infection. Although genetics and biochemical studies in yeast have identified many components involved in autophagy, the findings that some of the essential components of the yeast pathway are missing in higher organisms underscore the need to study autophagy in more complex systems. This review focuses on the use of the fruitfly, Drosophila melanogaster as a model system for analysis of autophagy. Drosophila is an organism well-suited for genetic analysis and represents an intermediate between yeast and mammals with respect to conservation of the autophagy machinery. Furthermore, the complex biology and physiology of Drosophila presents an opportunity to model human diseases in a tissue specific and analogous context.
%B Semin Immunopathol %V 32 %P 363-72 %8 2010 Dec %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/20798940?dopt=Abstract %R 10.1007/s00281-010-0223-y %0 Journal Article %J PLoS Genet %D 2010 %T Dynamic switch of negative feedback regulation in Drosophila Akt-TOR signaling. %A Kockel, Lutz %A Kerr, Kimberly S %A Melnick, Michael %A Brückner, Katja %A Hebrok, Matthias %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Enzyme Activation %K Epistasis, Genetic %K Genome-Wide Association Study %K Phosphorylation %K Protein Kinases %K Proto-Oncogene Proteins c-akt %K Ribosomal Protein S6 Kinases, 70-kDa %K Signal Transduction %K TOR Serine-Threonine Kinases %XAkt represents a nodal point between the Insulin receptor and TOR signaling, and its activation by phosphorylation controls cell proliferation, cell size, and metabolism. The activity of Akt must be carefully balanced, as increased Akt signaling is frequently associated with cancer and as insufficient Akt signaling is linked to metabolic disease and diabetes mellitus. Using a genome-wide RNAi screen in Drosophila cells in culture, and in vivo analyses in the third instar wing imaginal disc, we studied the regulatory circuitries that define dAkt activation. We provide evidence that negative feedback regulation of dAkt occurs during normal Drosophila development in vivo. Whereas in cell culture dAkt is regulated by S6 Kinase (S6K)-dependent negative feedback, this feedback inhibition only plays a minor role in vivo. In contrast, dAkt activation under wild-type conditions is defined by feedback inhibition that depends on TOR Complex 1 (TORC1), but is S6K-independent. This feedback inhibition is switched from TORC1 to S6K only in the context of enhanced TORC1 activity, as triggered by mutations in tsc2. These results illustrate how the Akt-TOR pathway dynamically adapts the routing of negative feedback in response to the activity load of its signaling circuit in vivo.
%B PLoS Genet %V 6 %P e1000990 %8 2010 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/20585550?dopt=Abstract %R 10.1371/journal.pgen.1000990 %0 Journal Article %J Cell %D 2010 %T FOXO/4E-BP signaling in Drosophila muscles regulates organism-wide proteostasis during aging. %A Demontis, Fabio %A Perrimon, Norbert %K Aging %K Animals %K Autophagy %K Drosophila melanogaster %K Drosophila Proteins %K Forkhead Transcription Factors %K Humans %K Intracellular Signaling Peptides and Proteins %K Lysosomes %K Models, Animal %K Muscle Proteins %K Muscles %K Peptide Initiation Factors %K Signal Transduction %XThe progressive loss of muscle strength during aging is a common degenerative event of unclear pathogenesis. Although muscle functional decline precedes age-related changes in other tissues, its contribution to systemic aging is unknown. Here, we show that muscle aging is characterized in Drosophila by the progressive accumulation of protein aggregates that associate with impaired muscle function. The transcription factor FOXO and its target 4E-BP remove damaged proteins at least in part via the autophagy/lysosome system, whereas foxo mutants have dysfunctional proteostasis. Both FOXO and 4E-BP delay muscle functional decay and extend life span. Moreover, FOXO/4E-BP signaling in muscles decreases feeding behavior and the release of insulin from producing cells, which in turn delays the age-related accumulation of protein aggregates in other tissues. These findings reveal an organism-wide regulation of proteostasis in response to muscle aging and a key role of FOXO/4E-BP signaling in the coordination of organismal and tissue aging.
%B Cell %V 143 %P 813-25 %8 2010 Nov 24 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/21111239?dopt=Abstract %R 10.1016/j.cell.2010.10.007 %0 Journal Article %J Annu Rev Biochem %D 2010 %T Genomic screening with RNAi: results and challenges. %A Mohr, Stephanie %A Bakal, Chris %A Perrimon, Norbert %K Animals %K Genes %K Genetic Techniques %K Genome %K Humans %K RNA Interference %XRNA interference (RNAi) is an effective tool for genome-scale, high-throughput analysis of gene function. In the past five years, a number of genome-scale RNAi high-throughput screens (HTSs) have been done in both Drosophila and mammalian cultured cells to study diverse biological processes, including signal transduction, cancer biology, and host cell responses to infection. Results from these screens have led to the identification of new components of these processes and, importantly, have also provided insights into the complexity of biological systems, forcing new and innovative approaches to understanding functional networks in cells. Here, we review the main findings that have emerged from RNAi HTS and discuss technical issues that remain to be improved, in particular the verification of RNAi results and validation of their biological relevance. Furthermore, we discuss the importance of multiplexed and integrated experimental data analysis pipelines to RNAi HTS.
%B Annu Rev Biochem %V 79 %P 37-64 %8 2010 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/20367032?dopt=Abstract %R 10.1146/annurev-biochem-060408-092949 %0 Book Section %B Cold Spring Harb Perspect Biol %D 2010 %T In vivo RNAi: today and tomorrow. %A Perrimon, Norbert %A Ni, Jian-Quan %A Perkins, Lizabeth %K Animals %K Caenorhabditis elegans %K Drosophila %K Evolution, Molecular %K Genetic Techniques %K Humans %K Mice %K Models, Animal %K Models, Genetic %K Molecular Biology %K Plants, Genetically Modified %K RNA Interference %XRNA interference (RNAi) provides a powerful reverse genetics approach to analyze gene functions both in tissue culture and in vivo. Because of its widespread applicability and effectiveness it has become an essential part of the tool box kits of model organisms such as Caenorhabditis elegans, Drosophila, and the mouse. In addition, the use of RNAi in animals in which genetic tools are either poorly developed or nonexistent enables a myriad of fundamental questions to be asked. Here, we review the methods and applications of in vivo RNAi to characterize gene functions in model organisms and discuss their impact to the study of developmental as well as evolutionary questions. Further, we discuss the applications of RNAi technologies to crop improvement, pest control and RNAi therapeutics, thus providing an appreciation of the potential for phenomenal applications of RNAi to agriculture and medicine.
%B Cold Spring Harb Perspect Biol %V 2 %P 1-11 %8 2010 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/20534712?dopt=Abstract %R 10.1101/cshperspect.a003640 %0 Journal Article %J Dev Cell %D 2010 %T Realizing the promise of RNAi high throughput screening. %A Bakal, Chris %A Perrimon, Norbert %XRecently reporting in Nature, Collinet et al. describes the application of quantitative multiparametric methods to a genome-wide RNAi screen for regulators of endocytosis. The study illustrates the power of this approach beyond the identification of new endocytic components to providing insights into the design principles of the endocytic system.
%B Dev Cell %V 18 %P 506-7 %8 2010 Apr 20 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/20412765?dopt=Abstract %R 10.1016/j.devcel.2010.04.005 %0 Journal Article %J PLoS Genet %D 2010 %T Sarcomere formation occurs by the assembly of multiple latent protein complexes. %A Rui, Yanning %A Bai, Jianwu %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Integrins %K Models, Biological %K Multiprotein Complexes %K Muscle Proteins %K Myosin Heavy Chains %K Sarcomeres %K Tropomyosin %K Troponin %XThe stereotyped striation of myofibrils is a conserved feature of muscle organization that is critical to its function. Although most components that constitute the basic myofibrils are well-characterized biochemically and are conserved across the animal kingdom, the mechanisms leading to the precise assembly of sarcomeres, the basic units of myofibrils, are poorly understood. To gain insights into this process, we investigated the functional relationships of sarcomeric protein complexes. Specifically, we systematically analyzed, using either RNAi in primary muscle cells or available genetic mutations, the organization of myofibrils in Drosophila muscles that lack one or more sarcomeric proteins. Our study reveals that the thin and thick filaments are mutually dependent on each other for striation. Further, the tension sensor complex comprised of zipper/Zasp/α-actinin is involved in stabilizing the sarcomere but not in its initial formation. Finally, integrins appear essential for the interdigitation of thin and thick filaments that occurs prior to striation. Thus, sarcomere formation occurs by the coordinated assembly of multiple latent protein complexes, as opposed to sequential assembly.
%B PLoS Genet %V 6 %P e1001208 %8 2010 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/21124995?dopt=Abstract %R 10.1371/journal.pgen.1001208 %0 Journal Article %J J Vis Exp %D 2009 %T Mesoscopic fluorescence tomography for in-vivo imaging of developing Drosophila. %A Vinegoni, Claudio %A Razansky, Daniel %A Pitsouli, Chrysoula %A Perrimon, Norbert %A Ntziachristos, Vasilis %A Weissleder, Ralph %K Animals %K Drosophila melanogaster %K Fluorescence %K Imaging, Three-Dimensional %K Tomography %XVisualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (>1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery. We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism. The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size. We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.
%B J Vis Exp %8 2009 %G eng %N 30 %1 http://www.ncbi.nlm.nih.gov/pubmed/19696720?dopt=Abstract %R 10.3791/1510 %0 Journal Article %J Genetics %D 2009 %T Cross-species RNAi rescue platform in Drosophila melanogaster. %A Kondo, Shu %A Booker, Matthew %A Perrimon, Norbert %K Animals %K Animals, Genetically Modified %K Aurora Kinases %K Base Sequence %K Caspases %K Drosophila %K Drosophila melanogaster %K Drosophila Proteins %K Genetic Vectors %K Molecular Sequence Data %K Mutation %K Phenotype %K Phylogeny %K Protein-Serine-Threonine Kinases %K RNA Interference %K RNA, Double-Stranded %K Sequence Homology, Nucleic Acid %K Species Specificity %K Transfection %XRNAi-mediated gene knockdown in Drosophila melanogaster is a powerful method to analyze loss-of-function phenotypes both in cell culture and in vivo. However, it has also become clear that false positives caused by off-target effects are prevalent, requiring careful validation of RNAi-induced phenotypes. The most rigorous proof that an RNAi-induced phenotype is due to loss of its intended target is to rescue the phenotype by a transgene impervious to RNAi. For large-scale validations in the mouse and Caenorhabditis elegans, this has been accomplished by using bacterial artificial chromosomes (BACs) of related species. However, in Drosophila, this approach is not feasible because transformation of large BACs is inefficient. We have therefore developed a general RNAi rescue approach for Drosophila that employs Cre/loxP-mediated recombination to rapidly retrofit existing fosmid clones into rescue constructs. Retrofitted fosmid clones carry a selection marker and a phiC31 attB site, which facilitates the production of transgenic animals. Here, we describe our approach and demonstrate proof-of-principle experiments showing that D. pseudoobscura fosmids can successfully rescue RNAi-induced phenotypes in D. melanogaster, both in cell culture and in vivo. Altogether, the tools and method that we have developed provide a gold standard for validation of Drosophila RNAi experiments.
%B Genetics %V 183 %P 1165-73 %8 2009 Nov %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/19720858?dopt=Abstract %R 10.1534/genetics.109.106567 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2009 %T Droplet microfluidic technology for single-cell high-throughput screening. %A Brouzes, Eric %A Medkova, Martina %A Savenelli, Neal %A Marran, Dave %A Twardowski, Mariusz %A Hutchison, J. Brian %A Rothberg, Jonathan M %A Link, Darren R. %A Perrimon, Norbert %A Samuels, Michael L. %K Cell Survival %K Drug Evaluation, Preclinical %K Emulsions %K Fluorescent Dyes %K Humans %K Microfluidic Analytical Techniques %K Microfluidics %K Mitomycin %K Reproducibility of Results %K Time Factors %K U937 Cells %XWe present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.
%B Proc Natl Acad Sci U S A %V 106 %P 14195-200 %8 2009 Aug 25 %G eng %N 34 %1 http://www.ncbi.nlm.nih.gov/pubmed/19617544?dopt=Abstract %R 10.1073/pnas.0903542106 %0 Journal Article %J Mol Cell %D 2009 %T Hierarchical rules for Argonaute loading in Drosophila. %A Czech, Benjamin %A Zhou, Rui %A Erlich, Yaniv %A Brennecke, Julius %A Binari, Richard %A Villalta, Christians %A Gordon, Assaf %A Perrimon, Norbert %A Hannon, Gregory J %K 3' Untranslated Regions %K Animals %K Arabidopsis Proteins %K Argonaute Proteins %K Base Pairing %K Blotting, Northern %K Cell Line %K Drosophila melanogaster %K Drosophila Proteins %K Immunoprecipitation %K MicroRNAs %K Models, Biological %K Nucleic Acid Conformation %K Protein Binding %K RNA Interference %K RNA Precursors %K RNA, Double-Stranded %K RNA, Messenger %K RNA, Small Interfering %K RNA-Induced Silencing Complex %K Thermodynamics %XDrosophila Argonaute-1 and Argonaute-2 differ in function and small RNA content. AGO2 binds to siRNAs, whereas AGO1 is almost exclusively occupied by microRNAs. MicroRNA duplexes are intrinsically asymmetric, with one strand, the miR strand, preferentially entering AGO1 to recognize and regulate the expression of target mRNAs. The other strand, miR*, has been viewed as a byproduct of microRNA biogenesis. Here, we show that miR*s are often loaded as functional species into AGO2. This indicates that each microRNA precursor can potentially produce two mature small RNA strands that are differentially sorted within the RNAi pathway. miR* biogenesis depends upon the canonical microRNA pathway, but loading into AGO2 is mediated by factors traditionally dedicated to siRNAs. By inferring and validating hierarchical rules that predict differential AGO loading, we find that intrinsic determinants, including structural and thermodynamic properties of the processed duplex, regulate the fate of each RNA strand within the RNAi pathway.
%B Mol Cell %V 36 %P 445-56 %8 2009 Nov 13 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/19917252?dopt=Abstract %R 10.1016/j.molcel.2009.09.028 %0 Journal Article %J J Biomed Inform %D 2009 %T An image score inference system for RNAi genome-wide screening based on fuzzy mixture regression modeling. %A Wang, Jun %A Zhou, Xiaobo %A Li, Fuhai %A Bradley, Pamela L %A Chang, Shih-Fu %A Perrimon, Norbert %A Wong, Stephen T C %K Algorithms %K Animals %K Cells, Cultured %K Databases, Genetic %K Drosophila %K Fuzzy Logic %K Gene Knockdown Techniques %K Genome %K Genomics %K Image Processing, Computer-Assisted %K Information Storage and Retrieval %K Microscopy, Fluorescence %K Models, Genetic %K Pattern Recognition, Automated %K Phenotype %K Regression Analysis %K Reproducibility of Results %K RNA Interference %XWith recent advances in fluorescence microscopy imaging techniques and methods of gene knock down by RNA interference (RNAi), genome-scale high-content screening (HCS) has emerged as a powerful approach to systematically identify all parts of complex biological processes. However, a critical barrier preventing fulfillment of the success is the lack of efficient and robust methods for automating RNAi image analysis and quantitative evaluation of the gene knock down effects on huge volume of HCS data. Facing such opportunities and challenges, we have started investigation of automatic methods towards the development of a fully automatic RNAi-HCS system. Particularly important are reliable approaches to cellular phenotype classification and image-based gene function estimation. We have developed a HCS analysis platform that consists of two main components: fluorescence image analysis and image scoring. For image analysis, we used a two-step enhanced watershed method to extract cellular boundaries from HCS images. Segmented cells were classified into several predefined phenotypes based on morphological and appearance features. Using statistical characteristics of the identified phenotypes as a quantitative description of the image, a score is generated that reflects gene function. Our scoring model integrates fuzzy gene class estimation and single regression models. The final functional score of an image was derived using the weighted combination of the inference from several support vector-based regression models. We validated our phenotype classification method and scoring system on our cellular phenotype and gene database with expert ground truth labeling. We built a database of high-content, 3-channel, fluorescence microscopy images of Drosophila Kc(167) cultured cells that were treated with RNAi to perturb gene function. The proposed informatics system for microscopy image analysis is tested on this database. Both of the two main components, automated phenotype classification and image scoring system, were evaluated. The robustness and efficiency of our system were validated in quantitatively predicting the biological relevance of genes.
%B J Biomed Inform %V 42 %P 32-40 %8 2009 Feb %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/18547870?dopt=Abstract %R 10.1016/j.jbi.2008.04.007 %0 Journal Article %J Dis Model Mech %D 2009 %T Inactivation of Drosophila Huntingtin affects long-term adult functioning and the pathogenesis of a Huntington's disease model. %A Zhang, Sheng %A Feany, Mel B %A Saraswati, Sudipta %A Littleton, J Troy %A Perrimon, Norbert %K Aging %K Animals %K Axons %K Biological Transport %K Brain %K Cytoplasm %K Disease Models, Animal %K Drosophila melanogaster %K Drosophila Proteins %K Gene Deletion %K Gene Silencing %K Huntington Disease %K Microtubule-Associated Proteins %K Phenotype %K Repetitive Sequences, Amino Acid %K Synapses %K Synaptic Transmission %K Time Factors %XA polyglutamine expansion in the huntingtin (HTT) gene causes neurodegeneration in Huntington's disease (HD), but the in vivo function of the native protein (Htt) is largely unknown. Numerous biochemical and in vitro studies have suggested a role for Htt in neuronal development, synaptic function and axonal trafficking. To test these models, we generated a null mutant in the putative Drosophila HTT homolog (htt, hereafter referred to asdhtt) and, surprisingly, found that dhtt mutant animals are viable with no obvious developmental defects. Instead, dhtt is required for maintaining the mobility and long-term survival of adult animals, and for modulating axonal terminal complexity in the adult brain. Furthermore, removing endogenous dhtt significantly accelerates the neurodegenerative phenotype associated with a Drosophila model of polyglutamine Htt toxicity (HD-Q93), providing in vivo evidence that disrupting the normal function of Htt might contribute to HD pathogenesis.
%B Dis Model Mech %V 2 %P 247-66 %8 2009 May-Jun %G eng %N 5-6 %1 http://www.ncbi.nlm.nih.gov/pubmed/19380309?dopt=Abstract %R 10.1242/dmm.000653 %0 Journal Article %J RNA %D 2009 %T Processing of Drosophila endo-siRNAs depends on a specific Loquacious isoform. %A Zhou, Rui %A Czech, Benjamin %A Brennecke, Julius %A Sachidanandam, Ravi %A Wohlschlegel, James A %A Perrimon, Norbert %A Hannon, Gregory J %K Animals %K DNA Transposable Elements %K Drosophila melanogaster %K Gene Silencing %K Germ Cells %K RNA, Small Interfering %XDrosophila melanogaster expresses three classes of small RNAs, which are classified according to their mechanisms of biogenesis. MicroRNAs are approximately 22-23 nucleotides (nt), ubiquitously expressed small RNAs that are sequentially processed from hairpin-like precursors by Drosha/Pasha and Dcr-1/Loquacious complexes. MicroRNAs usually associate with AGO1 and regulate the expression of protein-coding genes. Piwi-interacting RNAs (piRNAs) of approximately 24-28 nt associate with Piwi-family proteins and can arise from single-stranded precursors. piRNAs function in transposon silencing and are mainly restricted to gonadal tissues. Endo-siRNAs are found in both germline and somatic tissues. These approximately 21-nt RNAs are produced by a distinct Dicer, Dcr-2, and do not depend on Drosha/Pasha complexes. They predominantly bind to AGO2 and target both mobile elements and protein-coding genes. Surprisingly, a subset of endo-siRNAs strongly depend for their production on the dsRNA-binding protein Loquacious (Loqs), thought generally to be a partner for Dcr-1 and a cofactor for miRNA biogenesis. Endo-siRNA production depends on a specific Loqs isoform, Loqs-PD, which is distinct from the one, Loqs-PB, required for the production of microRNAs. Paralleling their roles in the biogenesis of distinct small RNA classes, Loqs-PD and Loqs-PB bind to different Dicer proteins, with Dcr-1/Loqs-PB complexes and Dcr-2/Loqs-PD complexes driving microRNA and endo-siRNA biogenesis, respectively.
%B RNA %V 15 %P 1886-95 %8 2009 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/19635780?dopt=Abstract %R 10.1261/rna.1611309 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2009 %T Synergy between bacterial infection and genetic predisposition in intestinal dysplasia. %A Apidianakis, Yiorgos %A Pitsouli, Chrysoula %A Perrimon, Norbert %A Rahme, Laurence %K Animals %K Apoptosis %K Cell Count %K Cell Differentiation %K Cell Division %K Cell Proliferation %K Drosophila melanogaster %K Drosophila Proteins %K Enterocytes %K Enzyme Activation %K Gastrointestinal Tract %K Genes, ras %K Genetic Predisposition to Disease %K Homeostasis %K Hyperplasia %K Intestinal Neoplasms %K Intestines %K JNK Mitogen-Activated Protein Kinases %K Pseudomonas aeruginosa %K Pseudomonas Infections %K Stem Cells %XAccumulating evidence suggests that hyperproliferating intestinal stem cells (SCs) and progenitors drive cancer initiation, maintenance, and metastasis. In addition, chronic inflammation and infection have been increasingly recognized for their roles in cancer. Nevertheless, the mechanisms by which bacterial infections can initiate SC-mediated tumorigenesis remain elusive. Using a Drosophila model of gut pathogenesis, we show that intestinal infection with Pseudomonas aeruginosa, a human opportunistic bacterial pathogen, activates the c-Jun N-terminal kinase (JNK) pathway, a hallmark of the host stress response. This, in turn, causes apoptosis of enterocytes, the largest class of differentiated intestinal cells, and promotes a dramatic proliferation of SCs and progenitors that serves as a homeostatic compensatory mechanism to replenish the apoptotic enterocytes. However, we find that this homeostatic mechanism can lead to massive over-proliferation of intestinal cells when infection occurs in animals with a latent oncogenic form of the Ras1 oncogene. The affected intestines develop excess layers of cells with altered apicobasal polarity reminiscent of dysplasia, suggesting that infection can directly synergize with the genetic background in predisposed individuals to initiate SC-mediated tumorigenesis. Our results provide a framework for the study of intestinal bacterial infections and their effects on undifferentiated and mature enteric epithelial cells in the initial stages of intestinal cancer. Assessment of progenitor cell responses to pathogenic intestinal bacteria could provide a measure of predisposition for apoptotic enterocyte-assisted intestinal dysplasias in humans.
%B Proc Natl Acad Sci U S A %V 106 %P 20883-8 %8 2009 Dec 8 %G eng %N 49 %1 http://www.ncbi.nlm.nih.gov/pubmed/19934041?dopt=Abstract %R 10.1073/pnas.0911797106 %0 Journal Article %J Nature %D 2009 %T Unlocking the secrets of the genome. %A Celniker, Susan E %A Dillon, Laura A L %A Gerstein, Mark B %A Gunsalus, Kristin C %A Henikoff, Steven %A Karpen, Gary H %A Kellis, Manolis %A Lai, Eric C %A Lieb, Jason D %A MacAlpine, David M %A Micklem, Gos %A Piano, Fabio %A Snyder, Michael %A Stein, Lincoln %A White, Kevin P %A Waterston, Robert H %A modENCODE Consortium %K Animals %K Caenorhabditis elegans %K Drosophila melanogaster %K Genome %K Genomics %K Human Genome Project %K Humans %B Nature %V 459 %P 927-30 %8 2009 Jun 18 %G eng %N 7249 %1 http://www.ncbi.nlm.nih.gov/pubmed/19536255?dopt=Abstract %R 10.1038/459927a %0 Journal Article %J Nature Reviews Molecular and Cell Biology %D 2009 %T Building on the Shoulders of Giants (Journal Club) %A Perrimon, Norbert %B Nature Reviews Molecular and Cell Biology %V 10 %P 144 %G eng %0 Journal Article %J Nat Protoc %D 2009 %T Culture of Drosophila primary cells dissociated from gastrula embryos and their use in RNAi screening. %A Bai, Jianwu %A Sepp, Katharine J %A Perrimon, Norbert %K Animals %K Cell Culture Techniques %K Cells, Cultured %K Culture Media %K Drosophila %K Gastrula %K Genomics %K RNA Interference %K RNA, Double-Stranded %XWe provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes approximately 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.
%B Nat Protoc %V 4 %P 1502-12 %8 2009 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/19798083?dopt=Abstract %R 10.1038/nprot.2009.147 %0 Journal Article %J Genetics %D 2009 %T A Drosophila resource of transgenic RNAi lines for neurogenetics. %A Ni, Jian-Quan %A Liu, Lu-Ping %A Binari, Richard %A Hardy, Robert %A Shim, Hye-Seok %A Cavallaro, Amanda %A Booker, Matthew %A Pfeiffer, Barret D %A Markstein, Michele %A Wang, Hui %A Villalta, Christians %A Laverty, Todd R %A Perkins, Lizabeth A %A Perrimon, Norbert %K Animals %K Carrier Proteins %K Drosophila %K Gene Knockdown Techniques %K Ion Channels %K Methods %K Nervous System %K RNA Interference %K RNA, Small Interfering %K Transcription Factors %XConditional expression of hairpin constructs in Drosophila is a powerful method to disrupt the activity of single genes with a spatial and temporal resolution that is impossible, or exceedingly difficult, using classical genetic methods. We previously described a method (Ni et al. 2008) whereby RNAi constructs are targeted into the genome by the phiC31-mediated integration approach using Vermilion-AttB-Loxp-Intron-UAS-MCS (VALIUM), a vector that contains vermilion as a selectable marker, an attB sequence to allow for phiC31-targeted integration at genomic attP landing sites, two pentamers of UAS, the hsp70 core promoter, a multiple cloning site, and two introns. As the level of gene activity knockdown associated with transgenic RNAi depends on the level of expression of the hairpin constructs, we generated a number of derivatives of our initial vector, called the "VALIUM" series, to improve the efficiency of the method. Here, we report the results from the systematic analysis of these derivatives and characterize VALIUM10 as the most optimal vector of this series. A critical feature of VALIUM10 is the presence of gypsy insulator sequences that boost dramatically the level of knockdown. We document the efficacy of VALIUM as a vector to analyze the phenotype of genes expressed in the nervous system and have generated a library of 2282 constructs targeting 2043 genes that will be particularly useful for studies of the nervous system as they target, in particular, transcription factors, ion channels, and transporters.
%B Genetics %V 182 %P 1089-100 %8 2009 Aug %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/19487563?dopt=Abstract %R 10.1534/genetics.109.103630 %0 Journal Article %J Cell Host Microbe %D 2009 %T Homeostasis in infected epithelia: stem cells take the lead. %A Pitsouli, Chrysoula %A Apidianakis, Yiorgos %A Perrimon, Norbert %K Animals %K Drosophila %K Epithelium %K Homeostasis %K Mammals %K Regeneration %K Stem Cells %XTo maintain tissue homeostasis and avoid disease, epithelial cells damaged by pathogens need to be readily replenished, and this is mainly achieved by the activation of stem cells. In this Short Review, we discuss recent developments in the exciting field of host epithelia-pathogen interaction in Drosophila as well as in mammals.
%B Cell Host Microbe %V 6 %P 301-7 %8 2009 Oct 22 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/19837370?dopt=Abstract %R 10.1016/j.chom.2009.10.001 %0 Journal Article %J Development %D 2009 %T Integration of Insulin receptor/Foxo signaling and dMyc activity during muscle growth regulates body size in Drosophila. %A Demontis, Fabio %A Perrimon, Norbert %K Animals %K Cell Nucleus %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Feeding Behavior %K Forkhead Transcription Factors %K Gene Expression Regulation, Developmental %K Muscles %K Receptor, Insulin %K Signal Transduction %K Transcription Factors %K Transcription, Genetic %XDrosophila larval skeletal muscles are single, multinucleated cells of different sizes that undergo tremendous growth within a few days. The mechanisms underlying this growth in concert with overall body growth are unknown. We find that the size of individual muscles correlates with the number of nuclei per muscle cell and with increasing nuclear ploidy during development. Inhibition of Insulin receptor (InR; Insulin-like receptor) signaling in muscles autonomously reduces muscle size and systemically affects the size of other tissues, organs and indeed the entire body, most likely by regulating feeding behavior. In muscles, InR/Tor signaling, Foxo and dMyc (Diminutive) are key regulators of endoreplication, which is necessary but not sufficient to induce growth. Mechanistically, InR/Foxo signaling controls cell cycle progression by modulating dmyc expression and dMyc transcriptional activity. Thus, maximal dMyc transcriptional activity depends on InR to control muscle mass, which in turn induces a systemic behavioral response to allocate body size and proportions.
%B Development %V 136 %P 983-93 %8 2009 Mar %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/19211682?dopt=Abstract %R 10.1242/dev.027466 %0 Journal Article %J Nat Methods %D 2009 %T RNAiCut: automated detection of significant genes from functional genomic screens. %A Kaplow, Irene M %A Singh, Rohit %A Friedman, Adam %A Bakal, Chris %A Perrimon, Norbert %A Berger, Bonnie %K Animals %K Databases, Genetic %K Genome, Insect %K Genomics %K Insulin %K MAP Kinase Signaling System %K Protein Interaction Mapping %K RNA Interference %K Software %B Nat Methods %V 6 %P 476-7 %8 2009 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/19564846?dopt=Abstract %R 10.1038/nmeth0709-476 %0 Journal Article %J Nat Methods %D 2009 %T The twin spot generator for differential Drosophila lineage analysis. %A Griffin, Ruth %A Sustar, Anne %A Bonvin, Marianne %A Binari, Richard %A del Valle Rodriguez, Alberto %A Hohl, Amber M %A Bateman, Jack R %A Villalta, Christians %A Heffern, Elleard %A Grunwald, Didier %A Bakal, Chris %A Desplan, Claude %A Schubiger, Gerold %A Wu, C-ting %A Perrimon, Norbert %K Animals %K Cell Lineage %K Clone Cells %K Drosophila melanogaster %K Fluorometry %K Genomics %K Mitosis %K Mutation %K Recombination, Genetic %XIn Drosophila melanogaster, widely used mitotic recombination-based strategies generate mosaic flies with positive readout for only one daughter cell after division. To differentially label both daughter cells, we developed the twin spot generator (TSG) technique, which through mitotic recombination generates green and red twin spots that are detectable after the first cell division as single cells. We propose wide applications of TSG to lineage and genetic mosaic studies.
%B Nat Methods %V 6 %P 600-2 %8 2009 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/19633664?dopt=Abstract %R 10.1038/nmeth.1349 %0 Journal Article %J J Biomol Tech %D 2009 %T Use of a label-free quantitative platform based on MS/MS average TIC to calculate dynamics of protein complexes in insulin signaling. %A Yang, Xuemei %A Friedman, Adam %A Nagpal, Shailender %A Perrimon, Norbert %A Asara, John M %K Animals %K Biochemistry %K Computational Biology %K Drosophila %K Fourier Analysis %K Insulin %K Ions %K MAP Kinase Signaling System %K Models, Biological %K Protein Interaction Mapping %K Proteome %K Proteomics %K Signal Transduction %K Software %K Tandem Mass Spectrometry %XA label-free quantification strategy including the development of in-house software (NakedQuant) to calculate the average TIC across all spectral counts in tandem affinity purification (TAP)-tagging liquid chromatography-mass spectrometry MS/MS (LC/MS/MS) experiments was applied to a large-scale study of protein complexes in the MAPK portion of the insulin signaling pathway from Drosophila cells. Dynamics were calculated under basal and stimulating conditions as fold changes. These experiments were performed in the context of a core service model with the user performing the TAP immunoprecipitation and the MS core performing the MS and informatics stops. The MS strategy showed excellent coverage of known components in addition to potentially novel interactions.
%B J Biomol Tech %V 20 %P 272-7 %8 2009 Dec %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/19949701?dopt=Abstract %0 Journal Article %J J Biomol Screen %D 2008 %T Cellular phenotype recognition for high-content RNA interference genome-wide screening. %A Wang, Jun %A Zhou, Xiaobo %A Bradley, Pamela L %A Chang, Shih-Fu %A Perrimon, Norbert %A Wong, Stephen T C %K Algorithms %K Animals %K Cell Line %K Cell Shape %K Cytoskeleton %K Drosophila %K Drosophila Proteins %K Genomics %K Microscopy, Fluorescence %K Phenotype %K rac GTP-Binding Proteins %K RNA Interference %K Signal Transduction %XGenome-wide, cell-based screens using high-content screening (HCS) techniques and automated fluorescence microscopy generate thousands of high-content images that contain an enormous wealth of cell biological information. Such screens are key to the analysis of basic cell biological principles, such as control of cell cycle and cell morphology. However, these screens will ultimately only shed light on human disease mechanisms and potential cures if the analysis can keep up with the generation of data. A fundamental step toward automated analysis of high-content screening is to construct a robust platform for automatic cellular phenotype identification. The authors present a framework, consisting of microscopic image segmentation and analysis components, for automatic recognition of cellular phenotypes in the context of the Rho family of small GTPases. To implicate genes involved in Rac signaling, RNA interference (RNAi) was used to perturb gene functions, and the corresponding cellular phenotypes were analyzed for changes. The data used in the experiments are high-content, 3-channel, fluorescence microscopy images of Drosophila Kc167 cultured cells stained with markers that allow visualization of DNA, polymerized actin filaments, and the constitutively activated Rho protein Rac(V12). The performance of this approach was tested using a cellular database that contained more than 1000 samples of 3 predefined cellular phenotypes, and the generalization error was estimated using a cross-validation technique. Moreover, the authors applied this approach to analyze the whole high-content fluorescence images of Drosophila cells for further HCS-based gene function analysis.
%B J Biomol Screen %V 13 %P 29-39 %8 2008 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/18227224?dopt=Abstract %R 10.1177/1087057107311223 %0 Journal Article %J Mol Cell %D 2008 %T Comparative analysis of argonaute-dependent small RNA pathways in Drosophila. %A Zhou, Rui %A Hotta, Ikuko %A Denli, Ahmet M %A Hong, Pengyu %A Perrimon, Norbert %A Hannon, Gregory J %K Animals %K Argonaute Proteins %K Cell Line %K Drosophila %K Drosophila melanogaster %K Drosophila Proteins %K Eukaryotic Initiation Factors %K Gene Silencing %K Genes, Insect %K MicroRNAs %K Models, Biological %K RNA Interference %K RNA, Small Interfering %K RNA, Untranslated %K RNA-Induced Silencing Complex %XThe specificity of RNAi pathways is determined by several classes of small RNAs, which include siRNAs, piRNAs, endo-siRNAs, and microRNAs (miRNAs). These small RNAs are invariably incorporated into large Argonaute (Ago)-containing effector complexes known as RNA-induced silencing complexes (RISCs), which they guide to silencing targets. Both genetic and biochemical strategies have yielded conserved molecular components of small RNA biogenesis and effector machineries. However, given the complexity of these pathways, there are likely to be additional components and regulators that remain to be uncovered. We have undertaken a comparative and comprehensive RNAi screen to identify genes that impact three major Ago-dependent small RNA pathways that operate in Drosophila S2 cells. We identify subsets of candidates that act positively or negatively in siRNA, endo-siRNA, and miRNA pathways. Our studies indicate that many components are shared among all three Argonaute-dependent silencing pathways, though each is also impacted by discrete sets of genes.
%B Mol Cell %V 32 %P 592-9 %8 2008 Nov 21 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/19026789?dopt=Abstract %R 10.1016/j.molcel.2008.10.018 %0 Journal Article %J Nature %D 2008 %T An endogenous small interfering RNA pathway in Drosophila. %A Czech, Benjamin %A Malone, Colin D %A Zhou, Rui %A Stark, Alexander %A Schlingeheyde, Catherine %A Dus, Monica %A Perrimon, Norbert %A Kellis, Manolis %A Wohlschlegel, James A %A Sachidanandam, Ravi %A Hannon, Gregory J %A Brennecke, Julius %K Animals %K Argonaute Proteins %K Cell Line %K Drosophila melanogaster %K Drosophila Proteins %K Protein Binding %K Retroelements %K Ribonuclease III %K RNA Helicases %K RNA Interference %K RNA, Small Interfering %K RNA-Binding Proteins %K RNA-Induced Silencing Complex %XDrosophila endogenous small RNAs are categorized according to their mechanisms of biogenesis and the Argonaute protein to which they bind. MicroRNAs are a class of ubiquitously expressed RNAs of approximately 22 nucleotides in length, which arise from structured precursors through the action of Drosha-Pasha and Dicer-1-Loquacious complexes. These join Argonaute-1 to regulate gene expression. A second endogenous small RNA class, the Piwi-interacting RNAs, bind Piwi proteins and suppress transposons. Piwi-interacting RNAs are restricted to the gonad, and at least a subset of these arises by Piwi-catalysed cleavage of single-stranded RNAs. Here we show that Drosophila generates a third small RNA class, endogenous small interfering RNAs, in both gonadal and somatic tissues. Production of these RNAs requires Dicer-2, but a subset depends preferentially on Loquacious rather than the canonical Dicer-2 partner, R2D2 (ref. 14). Endogenous small interfering RNAs arise both from convergent transcription units and from structured genomic loci in a tissue-specific fashion. They predominantly join Argonaute-2 and have the capacity, as a class, to target both protein-coding genes and mobile elements. These observations expand the repertoire of small RNAs in Drosophila, adding a class that blurs distinctions based on known biogenesis mechanisms and functional roles.
%B Nature %V 453 %P 798-802 %8 2008 Jun 5 %G eng %N 7196 %1 http://www.ncbi.nlm.nih.gov/pubmed/18463631?dopt=Abstract %R 10.1038/nature07007 %0 Journal Article %J Nat Genet %D 2008 %T Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. %A Markstein, Michele %A Pitsouli, Chrysoula %A Villalta, Christians %A Celniker, Susan E %A Perrimon, Norbert %K Animals %K Animals, Genetically Modified %K Attachment Sites, Microbiological %K Chromatin %K Cytoskeletal Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Gene Expression Regulation %K Genome, Insect %K HSP70 Heat-Shock Proteins %K Insulator Elements %K Larva %K Molecular Sequence Data %K Myogenic Regulatory Factors %K Phenotype %K Plasmids %K Receptors, Notch %K Recombination, Genetic %K Retroviridae %K Saccharomyces cerevisiae Proteins %K Tissue Distribution %K Transgenes %K Wings, Animal %XA major obstacle to creating precisely expressed transgenes lies in the epigenetic effects of the host chromatin that surrounds them. Here we present a strategy to overcome this problem, employing a Gal4-inducible luciferase assay to systematically quantify position effects of host chromatin and the ability of insulators to counteract these effects at phiC31 integration loci randomly distributed throughout the Drosophila genome. We identify loci that can be exploited to deliver precise doses of transgene expression to specific tissues. Moreover, we uncover a previously unrecognized property of the gypsy retrovirus insulator to boost gene expression to levels severalfold greater than at most or possibly all un-insulated loci, in every tissue tested. These findings provide the first opportunity to create a battery of transgenes that can be reliably expressed at high levels in virtually any tissue by integration at a single locus, and conversely, to engineer a controlled phenotypic allelic series by exploiting several loci. The generality of our approach makes it adaptable to other model systems to identify and modify loci for optimal transgene expression.
%B Nat Genet %V 40 %P 476-83 %8 2008 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/18311141?dopt=Abstract %R 10.1038/ng.101 %0 Journal Article %J Development %D 2008 %T Hedgehog and Wingless stabilize but do not induce cell fate during Drosophila dorsal embryonic epidermal patterning. %A Vincent, Stephane %A Perrimon, Norbert %A Axelrod, Jeffrey D %K Animals %K Body Patterning %K Cell Differentiation %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Epidermis %K Hedgehog Proteins %K Wnt1 Protein %XA fundamental concept in development is that secreted molecules such as Wingless (Wg) and Hedgehog (Hh) generate pattern by inducing cell fate. By following markers of cellular identity posterior to the Wg- and Hh-expressing cells in the Drosophila dorsal embryonic epidermis, we provide evidence that neither Wg nor Hh specifies the identity of the cell types they pattern. Rather, they maintain pre-existing cellular identities that are otherwise unstable and progress stepwise towards a default fate. Wg and Hh therefore generate pattern by inhibiting specific switches in cell identity, showing that the specification and the patterning of a given cell are uncoupled. Sequential binary decisions without induction of cell identity give rise to both the groove cells and their posterior neighbors. The combination of independent progression of cell identity and arrest of progression by signals facilitates accurate patterning of an extremely plastic developing epidermis.
%B Development %V 135 %P 2767-75 %8 2008 Aug %G eng %N 16 %1 http://www.ncbi.nlm.nih.gov/pubmed/18614578?dopt=Abstract %R 10.1242/dev.017814 %0 Journal Article %J PLoS Genet %D 2008 %T Identification of neural outgrowth genes using genome-wide RNAi. %A Sepp, Katharine J %A Hong, Pengyu %A Lizarraga, Sofia B %A Liu, Judy S %A Mejia, Luis A %A Walsh, Christopher A %A Perrimon, Norbert %K Animals %K Cells, Cultured %K Drosophila %K Genome %K Genomics %K Mice %K Nervous System %K Neurons %K Phenotype %K ran GTP-Binding Protein %K RNA Interference %K RNA, Small Interfering %XWhile genetic screens have identified many genes essential for neurite outgrowth, they have been limited in their ability to identify neural genes that also have earlier critical roles in the gastrula, or neural genes for which maternally contributed RNA compensates for gene mutations in the zygote. To address this, we developed methods to screen the Drosophila genome using RNA-interference (RNAi) on primary neural cells and present the results of the first full-genome RNAi screen in neurons. We used live-cell imaging and quantitative image analysis to characterize the morphological phenotypes of fluorescently labelled primary neurons and glia in response to RNAi-mediated gene knockdown. From the full genome screen, we focused our analysis on 104 evolutionarily conserved genes that when downregulated by RNAi, have morphological defects such as reduced axon extension, excessive branching, loss of fasciculation, and blebbing. To assist in the phenotypic analysis of the large data sets, we generated image analysis algorithms that could assess the statistical significance of the mutant phenotypes. The algorithms were essential for the analysis of the thousands of images generated by the screening process and will become a valuable tool for future genome-wide screens in primary neurons. Our analysis revealed unexpected, essential roles in neurite outgrowth for genes representing a wide range of functional categories including signalling molecules, enzymes, channels, receptors, and cytoskeletal proteins. We also found that genes known to be involved in protein and vesicle trafficking showed similar RNAi phenotypes. We confirmed phenotypes of the protein trafficking genes Sec61alpha and Ran GTPase using Drosophila embryo and mouse embryonic cerebral cortical neurons, respectively. Collectively, our results showed that RNAi phenotypes in primary neural culture can parallel in vivo phenotypes, and the screening technique can be used to identify many new genes that have important functions in the nervous system.
%B PLoS Genet %V 4 %P e1000111 %8 2008 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/18604272?dopt=Abstract %R 10.1371/journal.pgen.1000111 %0 Journal Article %J Nat Methods %D 2008 %T In vivo imaging of Drosophila melanogaster pupae with mesoscopic fluorescence tomography. %A Vinegoni, Claudio %A Pitsouli, Chrysoula %A Razansky, Daniel %A Perrimon, Norbert %A Ntziachristos, Vasilis %K Animals %K Drosophila melanogaster %K Image Enhancement %K Imaging, Three-Dimensional %K Lighting %K Microscopy, Fluorescence %K Pupa %K Tomography, Optical %XWe report a technique for fluorescence tomography that operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The method uses multi-projection illumination and photon transport description in opaque tissues. We demonstrate whole-body three-dimensional visualization of the morphogenesis of GFP-expressing salivary glands and wing imaginal discs in living Drosophila melanogaster pupae in vivo and over time.
%B Nat Methods %V 5 %P 45-7 %8 2008 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/18066071?dopt=Abstract %R 10.1038/nmeth1149 %0 Journal Article %J Science %D 2008 %T Phosphorylation networks regulating JNK activity in diverse genetic backgrounds. %A Bakal, Chris %A Linding, Rune %A Llense, Flora %A Heffern, Elleard %A Martin-Blanco, Enrique %A Pawson, Tony %A Perrimon, Norbert %K Algorithms %K Animals %K Cell Line %K Computational Biology %K Drosophila %K Drosophila Proteins %K Fluorescence Resonance Energy Transfer %K Genes, Insect %K JNK Mitogen-Activated Protein Kinases %K MAP Kinase Signaling System %K Metabolic Networks and Pathways %K Phosphorylation %K Proteomics %K RNA Interference %K Signal Transduction %XCellular signaling networks have evolved to enable swift and accurate responses, even in the face of genetic or environmental perturbation. Thus, genetic screens may not identify all the genes that regulate different biological processes. Moreover, although classical screening approaches have succeeded in providing parts lists of the essential components of signaling networks, they typically do not provide much insight into the hierarchical and functional relations that exist among these components. We describe a high-throughput screen in which we used RNA interference to systematically inhibit two genes simultaneously in 17,724 combinations to identify regulators of Drosophila JUN NH(2)-terminal kinase (JNK). Using both genetic and phosphoproteomics data, we then implemented an integrative network algorithm to construct a JNK phosphorylation network, which provides structural and mechanistic insights into the systems architecture of JNK signaling.
%B Science %V 322 %P 453-6 %8 2008 Oct 17 %G eng %N 5900 %1 http://www.ncbi.nlm.nih.gov/pubmed/18927396?dopt=Abstract %R 10.1126/science.1158739 %0 Journal Article %J Development %D 2008 %T RNA interference screening in Drosophila primary cells for genes involved in muscle assembly and maintenance. %A Bai, Jianwu %A Binari, Richard %A Ni, Jian-Quan %A Vijayakanthan, Marina %A Li, Hong-Sheng %A Perrimon, Norbert %K Animals %K Animals, Genetically Modified %K Base Sequence %K Cells, Cultured %K DNA Primers %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Genes, Insect %K Humans %K Muscle Development %K Muscular Diseases %K Phenotype %K Plasma Membrane Calcium-Transporting ATPases %K RNA Interference %XTo facilitate the genetic analysis of muscle assembly and maintenance, we have developed a method for efficient RNA interference (RNAi) in Drosophila primary cells using double-stranded RNAs (dsRNAs). First, using molecular markers, we confirm and extend the observation that myogenesis in primary cultures derived from Drosophila embryonic cells follows the same developmental course as that seen in vivo. Second, we apply this approach to analyze 28 Drosophila homologs of human muscle disease genes and find that 19 of them, when disrupted, lead to abnormal muscle phenotypes in primary culture. Third, from an RNAi screen of 1140 genes chosen at random, we identify 49 involved in late muscle differentiation. We validate our approach with the in vivo analyses of three genes. We find that Fermitin 1 and Fermitin 2, which are involved in integrin-containing adhesion structures, act in a partially redundant manner to maintain muscle integrity. In addition, we characterize CG2165, which encodes a plasma membrane Ca2+-ATPase, and show that it plays an important role in maintaining muscle integrity. Finally, we discuss how Drosophila primary cells can be manipulated to develop cell-based assays to model human diseases for RNAi and small-molecule screens.
%B Development %V 135 %P 1439-49 %8 2008 Apr %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/18359903?dopt=Abstract %R 10.1242/dev.012849 %0 Journal Article %J BMC Bioinformatics %D 2008 %T Using iterative cluster merging with improved gap statistics to perform online phenotype discovery in the context of high-throughput RNAi screens. %A Yin, Zheng %A Zhou, Xiaobo %A Bakal, Chris %A Li, Fuhai %A Sun, Youxian %A Perrimon, Norbert %A Wong, Stephen T C %K Animals %K Cluster Analysis %K Computer Simulation %K Cytological Techniques %K Drosophila %K HeLa Cells %K Humans %K Internet %K Pattern Recognition, Automated %K Phenotype %K RNA Interference %K RNA, Small Interfering %XBACKGROUND: The recent emergence of high-throughput automated image acquisition technologies has forever changed how cell biologists collect and analyze data. Historically, the interpretation of cellular phenotypes in different experimental conditions has been dependent upon the expert opinions of well-trained biologists. Such qualitative analysis is particularly effective in detecting subtle, but important, deviations in phenotypes. However, while the rapid and continuing development of automated microscope-based technologies now facilitates the acquisition of trillions of cells in thousands of diverse experimental conditions, such as in the context of RNA interference (RNAi) or small-molecule screens, the massive size of these datasets precludes human analysis. Thus, the development of automated methods which aim to identify novel and biological relevant phenotypes online is one of the major challenges in high-throughput image-based screening. Ideally, phenotype discovery methods should be designed to utilize prior/existing information and tackle three challenging tasks, i.e. restoring pre-defined biological meaningful phenotypes, differentiating novel phenotypes from known ones and clarifying novel phenotypes from each other. Arbitrarily extracted information causes biased analysis, while combining the complete existing datasets with each new image is intractable in high-throughput screens. RESULTS: Here we present the design and implementation of a novel and robust online phenotype discovery method with broad applicability that can be used in diverse experimental contexts, especially high-throughput RNAi screens. This method features phenotype modelling and iterative cluster merging using improved gap statistics. A Gaussian Mixture Model (GMM) is employed to estimate the distribution of each existing phenotype, and then used as reference distribution in gap statistics. This method is broadly applicable to a number of different types of image-based datasets derived from a wide spectrum of experimental conditions and is suitable to adaptively process new images which are continuously added to existing datasets. Validations were carried out on different dataset, including published RNAi screening using Drosophila embryos [Additional files 1, 2], dataset for cell cycle phase identification using HeLa cells [Additional files 1, 3, 4] and synthetic dataset using polygons, our methods tackled three aforementioned tasks effectively with an accuracy range of 85%-90%. When our method is implemented in the context of a Drosophila genome-scale RNAi image-based screening of cultured cells aimed to identifying the contribution of individual genes towards the regulation of cell-shape, it efficiently discovers meaningful new phenotypes and provides novel biological insight. We also propose a two-step procedure to modify the novelty detection method based on one-class SVM, so that it can be used to online phenotype discovery. In different conditions, we compared the SVM based method with our method using various datasets and our methods consistently outperformed SVM based method in at least two of three tasks by 2% to 5%. These results demonstrate that our methods can be used to better identify novel phenotypes in image-based datasets from a wide range of conditions and organisms. CONCLUSION: We demonstrate that our method can detect various novel phenotypes effectively in complex datasets. Experiment results also validate that our method performs consistently under different order of image input, variation of starting conditions including the number and composition of existing phenotypes, and dataset from different screens. In our findings, the proposed method is suitable for online phenotype discovery in diverse high-throughput image-based genetic and chemical screens.
%B BMC Bioinformatics %V 9 %P 264 %8 2008 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/18534020?dopt=Abstract %R 10.1186/1471-2105-9-264 %0 Journal Article %J Nature %D 2008 %T Journal Club %A Perrimon, Norbert %B Nature %V 452 %P 669 %G eng %0 Journal Article %J Nature %D 2008 %T Developmental biology: Our fly cousins' gut. %A Pitsouli, Chrysoula %A Perrimon, Norbert %K Animals %K Cell Differentiation %K Cell Proliferation %K Drosophila melanogaster %K Epithelial Cells %K Humans %K Intestinal Mucosa %K Signal Transduction %B Nature %V 454 %P 592-3 %8 2008 Jul 31 %G eng %N 7204 %1 http://www.ncbi.nlm.nih.gov/pubmed/18668098?dopt=Abstract %R 10.1038/454592a %0 Journal Article %J Proc Natl Acad Sci U S A %D 2008 %T ESCRT factors restrict mycobacterial growth. %A Philips, Jennifer A %A Porto, Maura C %A Wang, Hui %A Rubin, Eric J %A Perrimon, Norbert %K Animals %K Cell Line %K DNA-Binding Proteins %K Drosophila %K Endosomal Sorting Complexes Required for Transport %K Endosomes %K Microscopy, Fluorescence %K Multiprotein Complexes %K Mycobacterium %K Mycobacterium Infections %K rab GTP-Binding Proteins %K RNA Interference %K Species Specificity %K Transcription Factors %K Vesicular Transport Proteins %K Virulence %XNearly 1.7 billion people are infected with Mycobacterium tuberculosis. Its ability to survive intracellularly is thought to be central to its success as a pathogen, but how it does this is poorly understood. Using a Drosophila model of infection, we identify three host cell activities, Rab7, CG8743, and the ESCRT machinery, that modulate the mycobacterial phagosome. In the absence of these factors the cell no longer restricts growth of the non-pathogen Mycobacterium smegmatis. Hence, we identify factors that represent unique vulnerabilities of the host cell, because manipulation of any one of them alone is sufficient to allow a nonpathogenic mycobacterial species to proliferate. Furthermore, we demonstrate that, in mammalian cells, the ESCRT machinery plays a conserved role in restricting bacterial growth.
%B Proc Natl Acad Sci U S A %V 105 %P 3070-5 %8 2008 Feb 26 %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/18287038?dopt=Abstract %R 10.1073/pnas.0707206105 %0 Journal Article %J Nat Methods %D 2008 %T Vector and parameters for targeted transgenic RNA interference in Drosophila melanogaster. %A Ni, Jian-Quan %A Markstein, Michele %A Binari, Richard %A Pfeiffer, Barret %A Liu, Lu-Ping %A Villalta, Christians %A Booker, Matthew %A Perkins, Lizabeth %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Gene Targeting %K Genetic Vectors %K RNA Interference %XThe conditional expression of hairpin constructs in Drosophila melanogaster has emerged in recent years as a method of choice in functional genomic studies. To date, upstream activating site-driven RNA interference constructs have been inserted into the genome randomly using P-element-mediated transformation, which can result in false negatives due to variable expression. To avoid this problem, we have developed a transgenic RNA interference vector based on the phiC31 site-specific integration method.
%B Nat Methods %V 5 %P 49-51 %8 2008 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/18084299?dopt=Abstract %R 10.1038/nmeth1146 %0 Journal Article %J Drug Discov Today %D 2007 %T Drug-target identification in Drosophila cells: combining high-throughout RNAi and small-molecule screens. %A Perrimon, Norbert %A Friedman, Adam %A Mathey-Prevot, Bernard %A Eggert, Ulrike S %K Animals %K Drosophila %K Drug Delivery Systems %K Drug Design %K Drug Evaluation, Preclinical %K RNA Interference %K RNA, Small Interfering %XRNA interference (RNAi) and small-molecule approaches are synergistic on multiple levels, from technology and high-throughput screen development to target identification and functional studies. Here, we describe the RNAi screening platform that we have established and made available to the community through the Drosophila RNAi Screening Center at Harvard Medical School. We then illustrate how the combination of RNAi and small-molecule HTS can lead to effective identification of targets in drug discovery.
%B Drug Discov Today %V 12 %P 28-33 %8 2007 Jan %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/17198970?dopt=Abstract %R 10.1016/j.drudis.2006.10.006 %0 Journal Article %J Fly (Austin) %D 2007 %T Matter arising: off-targets and genome-scale RNAi screens in Drosophila. %A Perrimon, Norbert %A Mathey-Prevot, Bernard %K Animals %K Drosophila %K False Positive Reactions %K RNA Interference %K RNA, Double-Stranded %XRecently, the issue of off-target effects (OTEs) associated with long double stranded RNAs (dsRNAs) used in RNAi screens, such as those performed at the Drosophila RNAi Screening Center and other laboratories, has become a focus of great interest and some concern. Although OTEs have been recognized as an important source of false positives in mammalian studies (where short siRNAs are used as triggers), they were generally thought to be inconsequential in Drosophila RNAi experiments because of the use of long dsRNAs. Two recent papers have disputed this contention and show that significant off-target effects can take place with the use of some long dsRNAs in Drosophila cells. Together, these studies provide evidence that OTEs mediated by short homology stretches of 19nt or greater within long dsRNAs can contribute to false positives in Drosophila RNAi screens. Here, we address how widespread the occurrence of OTE is in Drosophila screens, focusing on the DRSC dsRNA collections, and we discuss the implication for the interpretation of results reported in RNAi screens to-date. Lastly, we summarize steps taken by the DRSC to redress that situation and include a set of recommendations to observe in future RNAi screens.
%B Fly (Austin) %V 1 %P 1-5 %8 2007 Jan-Feb %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/18705022?dopt=Abstract %0 Journal Article %J Nat Protoc %D 2007 %T Design and implementation of high-throughput RNAi screens in cultured Drosophila cells. %A Ramadan, Nadire %A Flockhart, Ian %A Booker, Matthew %A Perrimon, Norbert %A Mathey-Prevot, Bernard %K Animals %K Artifacts %K Cells, Cultured %K Drosophila %K Gene Library %K Genomics %K Polymerase Chain Reaction %K RNA Interference %K RNA, Double-Stranded %XThis protocol describes the various steps and considerations involved in planning and carrying out RNA interference (RNAi) genome-wide screens in cultured Drosophila cells. We focus largely on the procedures that have been modified as a result of our experience over the past 3 years and of our better understanding of the underlying technology. Specifically, our protocol offers a set of suggestions and considerations for screen optimization and a step-by-step description of the procedures successfully used at the Drosophila RNAi Screening Center for screen implementation, data collection and analysis to identify potential hits. In addition, this protocol briefly covers postscreen analysis approaches that are often needed to finalize the hit list. Depending on the scope of the screen and subsequent analysis and validation involved, the full protocol can take anywhere from 3 months to 2 years to complete.
%B Nat Protoc %V 2 %P 2245-64 %8 2007 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/17853882?dopt=Abstract %R 10.1038/nprot.2007.250 %0 Journal Article %J Nat Methods %D 2007 %T Do-it-yourself RNAi made easy? %A Mathey-Prevot, Bernard %A Perrimon, Norbert %K Animals %K Humans %K Models, Genetic %K RNA Interference %K RNA, Small Interfering %B Nat Methods %V 4 %P 308-9 %8 2007 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/17396126?dopt=Abstract %R 10.1038/nmeth0407-308 %0 Journal Article %J Nature %D 2007 %T Drosophila and the genetics of the internal milieu. %A Leopold, Pierre %A Perrimon, Norbert %K Animals %K Drosophila %K Genome, Insect %K Homeostasis %K Humans %X'Homeostasis', from the Greek words for 'same' and 'steady', refers to ways in which the body acts to maintain a stable internal environment despite perturbations. Recent studies in Drosophila exemplify the conservation of regulatory mechanisms involved in metabolic homeostasis. These new findings underscore the use of Drosophila as a model for the study of various human disorders.
%B Nature %V 450 %P 186-8 %8 2007 Nov 8 %G eng %N 7167 %1 http://www.ncbi.nlm.nih.gov/pubmed/17994083?dopt=Abstract %R 10.1038/nature06286 %0 Journal Article %J Cell %D 2007 %T Genetic screening for signal transduction in the era of network biology. %A Friedman, Adam %A Perrimon, Norbert %K Animals %K Computational Biology %K Gene Expression Regulation %K Gene Regulatory Networks %K Genetic Diseases, Inborn %K Genetic Testing %K Humans %K Molecular Biology %K Neoplasms %K Signal Transduction %XIn contrast to animal-based mutant phenotype assays, recent biochemical and quantitative genetic studies have identified hundreds of potential regulators of known signaling pathways. We discuss the discrepancy between previous models and new data, put forward a different signaling conceptual framework incorporating time-dependent quantitative contributions, and suggest how this new framework can impact our study of human disease.
%B Cell %V 128 %P 225-31 %8 2007 Jan 26 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/17254958?dopt=Abstract %R 10.1016/j.cell.2007.01.007 %0 Journal Article %J Gene Expr Patterns %D 2007 %T GFP reporters detect the activation of the Drosophila JAK/STAT pathway in vivo. %A Bach, Erika A %A Ekas, Laura A %A Ayala-Camargo, Aidee %A Flaherty, Maria Sol %A Lee, Haeryun %A Perrimon, Norbert %A Baeg, Gyeong-Hun %K Animals %K Animals, Genetically Modified %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Gene Expression Regulation, Developmental %K Genes, Reporter %K Green Fluorescent Proteins %K Janus Kinases %K Signal Transduction %K STAT Transcription Factors %K Suppressor of Cytokine Signaling Proteins %K Transcription Factors %XJAK/STAT signaling is essential for a wide range of developmental processes in Drosophila melanogaster. The mechanism by which the JAK/STAT pathway contributes to these processes has been the subject of recent investigation. However, a reporter that reflects activity of the JAK/STAT pathway in all Drosophila tissues has not yet been developed. By placing a fragment of the Stat92E target gene Socs36E, which contains at least two putative Stat92E binding sites, upstream of GFP, we generated three constructs that can be used to monitor JAK/STAT pathway activity in vivo. These constructs differ by the number of Stat92E binding sites and the stability of GFP. The 2XSTAT92E-GFP and 10XSTAT92E-GFP constructs contain 2 and 10 Stat92E binding sites, respectively, driving expression of enhanced GFP, while 10XSTAT92E-DGFP drives expression of destabilized GFP. We show that these reporters are expressed in the embryo in an overlapping pattern with Stat92E protein and in tissues where JAK/STAT signaling is required. In addition, these reporters accurately reflect JAK/STAT pathway activity at larval stages, as their expression pattern overlaps that of the activating ligand unpaired in imaginal discs. Moreover, the STAT92E-GFP reporters are activated by ectopic JAK/STAT signaling. STAT92E-GFP fluorescence is increased in response to ectopic upd in the larval eye disc and mis-expression of the JAK kinase hopscotch in the adult fat body. Lastly, these reporters are specifically activated by Stat92E, as STAT92E-GFP reporter expression is lost cell-autonomously in stat92E homozygous mutant tissue. In sum, we have generated in vivo GFP reporters that accurately reflect JAK/STAT pathway activation in a variety of tissues. These reporters are valuable tools to further investigate and understand the role of JAK/STAT signaling in Drosophila.
%B Gene Expr Patterns %V 7 %P 323-31 %8 2007 Jan %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/17008134?dopt=Abstract %R 10.1016/j.modgep.2006.08.003 %0 Journal Article %J Science %D 2007 %T Quantitative morphological signatures define local signaling networks regulating cell morphology. %A Bakal, Chris %A Aach, John %A Church, George %A Perrimon, Norbert %K Animals %K Cell Line %K Cell Movement %K Cell Shape %K Drosophila %K Green Fluorescent Proteins %K Metabolic Networks and Pathways %K Phenotype %K RNA Interference %K Signal Transduction %XAlthough classical genetic and biochemical approaches have identified hundreds of proteins that function in the dynamic remodeling of cell shape in response to upstream signals, there is currently little systems-level understanding of the organization and composition of signaling networks that regulate cell morphology. We have developed quantitative morphological profiling methods to systematically investigate the role of individual genes in the regulation of cell morphology in a fast, robust, and cost-efficient manner. We analyzed a compendium of quantitative morphological signatures and described the existence of local signaling networks that act to regulate cell protrusion, adhesion, and tension.
%B Science %V 316 %P 1753-6 %8 2007 Jun 22 %G eng %N 5832 %1 http://www.ncbi.nlm.nih.gov/pubmed/17588932?dopt=Abstract %R 10.1126/science.1140324 %0 Journal Article %J Dev Cell %D 2007 %T SALS, a WH2-domain-containing protein, promotes sarcomeric actin filament elongation from pointed ends during Drosophila muscle growth. %A Bai, Jianwu %A Hartwig, John H %A Perrimon, Norbert %K Actin Cytoskeleton %K Amino Acid Sequence %K Animals %K Animals, Genetically Modified %K Cells, Cultured %K Drosophila %K Drosophila Proteins %K Fluorescent Antibody Technique %K Microfilament Proteins %K Molecular Sequence Data %K Muscle, Striated %K Myofibrils %K Phenotype %K RNA, Small Interfering %K Sarcomeres %K Sequence Homology, Amino Acid %XOrganization of actin filaments into a well-organized sarcomere structure is critical for muscle development and function. However, it is not completely understood how sarcomeric actin/thin filaments attain their stereotyped lengths. In an RNAi screen in Drosophila primary muscle cells, we identified a gene, sarcomere length short (sals), which encodes an actin-binding, WH2 domain-containing protein, required for proper sarcomere size. When sals is knocked down by RNAi, primary muscles display thin myofibrils with shortened sarcomeres and increased sarcomere number. Both loss- and gain-of-function analyses indicate that SALS may influence sarcomere lengths by promoting thin-filament lengthening from pointed ends. Furthermore, the complex localization of SALS and other sarcomeric proteins in myofibrils reveals that the full length of thin filaments is achieved in a two-step process, and that SALS is required for the second elongation phase, most likely because it antagonizes the pointed-end capping protein Tropomodulin.
%B Dev Cell %V 13 %P 828-42 %8 2007 Dec %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/18061565?dopt=Abstract %R 10.1016/j.devcel.2007.10.003 %0 Journal Article %J Genetics %D 2007 %T Applications of high-throughput RNA interference screens to problems in cell and developmental biology. %A Perrimon, Norbert %A Mathey-Prevot, Bernard %K Animals %K Developmental Biology %K Genome %K Humans %K Phenotype %K RNA Interference %XRNA interference (RNAi) in tissue culture cells has emerged as an excellent methodology for identifying gene functions systematically and in an unbiased manner. Here, we describe how RNAi high-throughput screening (HTS) in Drosophila cells are currently being performed and emphasize the strengths and weaknesses of the approach. Further, to demonstrate the versatility of the technology, we provide examples of the various applications of the method to problems in signal transduction and cell and developmental biology. Finally, we discuss emerging technological advances that will extend RNAi-based screening methods.
%B Genetics %V 175 %P 7-16 %8 2007 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/17244760?dopt=Abstract %R 10.1534/genetics.106.069963 %0 Journal Article %J Genome Biol %D 2007 %T A case study of the reproducibility of transcriptional reporter cell-based RNAi screens in Drosophila. %A Dasgupta, Ramanuj %A Nybakken, Kent %A Booker, Matthew %A Mathey-Prevot, Bernard %A Gonsalves, Foster %A Changkakoty, Binita %A Perrimon, Norbert %K Animals %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation %K Gene Library %K Genes, Insect %K Genetic Techniques %K Genome %K Models, Genetic %K RNA %K RNA Interference %K RNA, Double-Stranded %K Sensitivity and Specificity %K Transcription, Genetic %XOff-target effects have been demonstrated to be a major source of false-positives in RNA interference (RNAi) high-throughput screens. In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog signaling pathways using second generation double-stranded RNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization, which determine the efficacy of RNAi-knockdown of target genes.
%B Genome Biol %V 8 %P R203 %8 2007 %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/17903264?dopt=Abstract %R 10.1186/gb-2007-8-9-r203 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2007 %T Functional screening identifies miR-315 as a potent activator of Wingless signaling. %A Silver, Serena J %A Hagen, Joshua W %A Okamura, Katsutomo %A Perrimon, Norbert %A Lai, Eric C %K 3' Untranslated Regions %K Animals %K Animals, Genetically Modified %K Base Sequence %K DNA Primers %K Drosophila %K Drosophila Proteins %K MicroRNAs %K Proto-Oncogene Proteins %K Signal Transduction %K Wnt1 Protein %XThe existence of vast regulatory networks mediated by microRNAs (miRNAs) suggests broad potential for miRNA dysfunction to contribute to disease. However, relatively few miRNA-target interactions are likely to make detectable contributions to phenotype, and effective strategies to identify these few interactions are currently wanting. We hypothesized that signaling cascades represent critical points of susceptibility to miRNA dysfunction, and we developed a strategy to test this theory by using quantitative cell-based screens. Here we report a screen for miRNAs that affect the Wingless (Wg) pathway, a conserved pathway that regulates growth and tissue specification. This process identified ectopic miR-315 as a potent and specific activator of Wg signaling, an activity that we corroborated in transgenic animals. This miR-315 activity was mediated by direct inhibition of Axin and Notum, which encode essential, negatively acting components of the Wg pathway. Genetic interaction tests substantiated both of these genes as key functional targets of miR-315. The ability of ectopic miR-315 to activate Wg signaling was not a trivial consequence of predicted miRNA-target relationships because other miRNAs with conserved sites in the Axin 3' UTR neither activated Wg outputs nor inhibited an Axin sensor. In summary, activity-based screening can selectively identify miRNAs whose deregulation can lead to interpretable phenotypic consequences.
%B Proc Natl Acad Sci U S A %V 104 %P 18151-6 %8 2007 Nov 13 %G eng %N 46 %1 http://www.ncbi.nlm.nih.gov/pubmed/17989227?dopt=Abstract %R 10.1073/pnas.0706673104 %0 Journal Article %J Proc Natl Acad Sci U S A %D 2007 %T A genome-wide RNA interference screen identifies putative chromatin regulators essential for E2F repression. %A Lu, Jianrong %A Ruhf, Marie-Laure %A Perrimon, Norbert %A Leder, Philip %K Animals %K Cells, Cultured %K Chromatin %K Cyclin H %K Cyclins %K Down-Regulation %K Drosophila melanogaster %K Drosophila Proteins %K E2F Transcription Factors %K Genetic Testing %K Genome, Insect %K Mutation %K Phenotype %K Promoter Regions, Genetic %K Protein Binding %K Protein Kinases %K RNA Interference %K Transcription Factors %XRegulation of chromatin structure is critical in many fundamental cellular processes. Previous studies have suggested that the Rb tumor suppressor may recruit multiple chromatin regulatory proteins to repress E2F, a key regulator of cell proliferation and differentiation. Taking advantage of the evolutionary conservation of the E2F pathway, we have conducted a genome-wide RNAi screen in cultured Drosophila cells for genes required for repression of E2F activity. Among the genes identified are components of the putative Domino chromatin remodeling complex, as well as the Polycomb Group (PcG) protein-like fly tumor suppressor, L3mbt, and the related CG16975/dSfmbt. These factors are recruited to E2F-responsive promoters through physical association with E2F and are required for repression of endogenous E2F target genes. Surprisingly, their inhibitory activities on E2F appear to be independent of Rb. In Drosophila, domino mutation enhances cell proliferation induced by E2F overexpression and suppresses a loss-of-function cyclin E mutation. These findings suggest that potential chromatin regulation mediated by Domino and PcG-like factors plays an important role in controlling E2F activity and cell growth.
%B Proc Natl Acad Sci U S A %V 104 %P 9381-6 %8 2007 May 29 %G eng %N 22 %1 http://www.ncbi.nlm.nih.gov/pubmed/17517653?dopt=Abstract %R 10.1073/pnas.0610279104 %0 Journal Article %J Methods %D 2006 %T High-throughput approaches to dissecting MAPK signaling pathways. %A Friedman, Adam %A Perrimon, Norbert %K Animals %K Antibody Specificity %K Cells, Cultured %K Combinatorial Chemistry Techniques %K Drosophila %K Extracellular Signal-Regulated MAP Kinases %K MAP Kinase Signaling System %K Mitogen-Activated Protein Kinases %K Phosphorylation %K Receptor Protein-Tyrosine Kinases %K Reproducibility of Results %K RNA Interference %K RNA, Double-Stranded %XWith the development of genome-wide RNAi libraries, it is now possible to screen for novel components of mitogen-activated protein kinase (MAPK) pathways in cell culture. Although genetic dissection in model organisms and biochemical approaches in mammalian cells have been successful in identifying the core signaling cassettes of these pathways, high-throughput assays can yield unbiased, functional genomic insight into pathway regulation. We describe general high-throughput approaches to assaying MAPK signaling and the receptor tyrosine kinase (RTK)/extracellular signal-regulated kinase (ERK) pathway in particular using a phospho-specific antibody-based readout of pathway activity. We also provide examples of secondary validation screens and methods for managing large datasets for future in vivo functional characterization.
%B Methods %V 40 %P 262-71 %8 2006 Nov %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/16844385?dopt=Abstract %R 10.1016/j.ymeth.2006.05.002 %0 Journal Article %J Nat Rev Genet %D 2006 %T High-throughput RNAi screening in cultured cells: a user's guide. %A Echeverri, Christophe J %A Perrimon, Norbert %K Animals %K Automation %K Cells, Cultured %K Drosophila melanogaster %K Gene Expression Profiling %K Gene Transfer Techniques %K Humans %K Models, Biological %K Phenotype %K Quality Control %K RNA Interference %K RNA, Small Interfering %XRNA interference has re-energized the field of functional genomics by enabling genome-scale loss-of-function screens in cultured cells. Looking back on the lessons that have been learned from the first wave of technology developments and applications in this exciting field, we provide both a user's guide for newcomers to the field and a detailed examination of some more complex issues, particularly concerning optimization and quality control, for more advanced users. From a discussion of cell lines, screening paradigms, reagent types and read-out methodologies, we explore in particular the complexities of designing optimal controls and normalization strategies for these challenging but extremely powerful studies.
%B Nat Rev Genet %V 7 %P 373-84 %8 2006 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/16607398?dopt=Abstract %R 10.1038/nrg1836 %0 Journal Article %J Nat Methods %D 2006 %T Minimizing the risk of reporting false positives in large-scale RNAi screens. %A Echeverri, Christophe J %A Beachy, Philip A %A Baum, Buzz %A Boutros, Michael %A Buchholz, Frank %A Chanda, Sumit K %A Downward, Julian %A Ellenberg, Jan %A Fraser, Andrew G %A Hacohen, Nir %A Hahn, William C %A Jackson, Aimee L %A Kiger, Amy %A Linsley, Peter S %A Lum, Lawrence %A Ma, Yong %A Mathey-Prévôt, Bernard %A Root, David E %A Sabatini, David M %A Taipale, Jussi %A Perrimon, Norbert %A Bernards, René %K Databases, Genetic %K False Positive Reactions %K Genetic Testing %K Phenotype %K Risk %K RNA Interference %K Sensitivity and Specificity %XLarge-scale RNA interference (RNAi)-based analyses, very much as other 'omic' approaches, have inherent rates of false positives and negatives. The variability in the standards of care applied to validate results from these studies, if left unchecked, could eventually begin to undermine the credibility of RNAi as a powerful functional approach. This Commentary is an invitation to an open discussion started among various users of RNAi to set forth accepted standards that would insure the quality and accuracy of information in the large datasets coming out of genome-scale screens.
%B Nat Methods %V 3 %P 777-9 %8 2006 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/16990807?dopt=Abstract %R 10.1038/nmeth1006-777 %0 Journal Article %J Nature %D 2006 %T The emergence of geometric order in proliferating metazoan epithelia. %A Gibson, Matthew C %A Patel, Ankit B %A Radhika Nagpal %A Perrimon, Norbert %K Animals %K Cell Proliferation %K Drosophila %K Epithelial Cells %K Epithelium %K Markov Chains %K Mitosis %K Morphogenesis %K Wings, Animal %XThe predominantly hexagonal cell pattern of simple epithelia was noted in the earliest microscopic analyses of animal tissues, a topology commonly thought to reflect cell sorting into optimally packed honeycomb arrays. Here we use a discrete Markov model validated by time-lapse microscopy and clonal analysis to demonstrate that the distribution of polygonal cell types in epithelia is not a result of cell packing, but rather a direct mathematical consequence of cell proliferation. On the basis of in vivo analysis of mitotic cell junction dynamics in Drosophila imaginal discs, we mathematically predict the convergence of epithelial topology to a fixed equilibrium distribution of cellular polygons. This distribution is empirically confirmed in tissue samples from vertebrate, arthropod and cnidarian organisms, suggesting that a similar proliferation-dependent cell pattern underlies pattern formation and morphogenesis throughout the metazoa.
%B Nature %V 442 %P 1038-41 %8 2006 Aug 31 %G eng %N 7106 %1 http://www.ncbi.nlm.nih.gov/pubmed/16900102?dopt=Abstract %R 10.1038/nature05014 %0 Journal Article %J Nat Methods %D 2006 %T Evidence of off-target effects associated with long dsRNAs in Drosophila melanogaster cell-based assays. %A Kulkarni, Meghana M %A Booker, Matthew %A Silver, Serena J %A Friedman, Adam %A Hong, Pengyu %A Perrimon, Norbert %A Mathey-Prevot, Bernard %K Animals %K Drosophila melanogaster %K False Positive Reactions %K Gene Library %K Genetic Testing %K RNA Interference %K RNA, Double-Stranded %K Sensitivity and Specificity %XTo evaluate the specificity of long dsRNAs used in high-throughput RNA interference (RNAi) screens performed at the Drosophila RNAi Screening Center (DRSC), we performed a global analysis of their activity in 30 genome-wide screens completed at our facility. Notably, our analysis predicts that dsRNAs containing > or = 19-nucleotide perfect matches identified in silico to unintended targets may contribute to a significant false positive error rate arising from off-target effects. We confirmed experimentally that such sequences in dsRNAs lead to false positives and to efficient knockdown of a cross-hybridizing transcript, raising a cautionary note about interpreting results based on the use of a single dsRNA per gene. Although a full appreciation of all causes of false positive errors remains to be determined, we suggest simple guidelines to help ensure high-quality information from RNAi high-throughput screens.
%B Nat Methods %V 3 %P 833-8 %8 2006 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/16964256?dopt=Abstract %R 10.1038/nmeth935 %0 Journal Article %J Nature %D 2006 %T Evidence that stem cells reside in the adult Drosophila midgut epithelium. %A Micchelli, Craig A %A Perrimon, Norbert %K Aging %K Animals %K Cell Differentiation %K Cell Lineage %K Cell Proliferation %K Drosophila melanogaster %K Drosophila Proteins %K Epithelial Cells %K Epithelium %K Female %K Gastrointestinal Tract %K Male %K Receptors, Notch %K Signal Transduction %K Stem Cells %K Temperature %XAdult stem cells maintain organ systems throughout the course of life and facilitate repair after injury or disease. A fundamental property of stem and progenitor cell division is the capacity to retain a proliferative state or generate differentiated daughter cells; however, little is currently known about signals that regulate the balance between these processes. Here, we characterize a proliferating cellular compartment in the adult Drosophila midgut. Using genetic mosaic analysis we demonstrate that differentiated cells in the epithelium arise from a common lineage. Furthermore, we show that reduction of Notch signalling leads to an increase in the number of midgut progenitor cells, whereas activation of the Notch pathway leads to a decrease in proliferation. Thus, the midgut progenitor's default state is proliferation, which is inhibited through the Notch signalling pathway. The ability to identify, manipulate and genetically trace cell lineages in the midgut should lead to the discovery of additional genes that regulate stem and progenitor cell biology in the gastrointestinal tract.
%B Nature %V 439 %P 475-9 %8 2006 Jan 26 %G eng %N 7075 %1 http://www.ncbi.nlm.nih.gov/pubmed/16340959?dopt=Abstract %R 10.1038/nature04371 %0 Journal Article %J Nature %D 2006 %T Functional genomics reveals genes involved in protein secretion and Golgi organization. %A Bard, Frederic %A Casano, Laetitia %A Mallabiabarrena, Arrate %A Wallace, Erin %A Saito, Kota %A Kitayama, Hitoshi %A Guizzunti, Gianni %A Hu, Yue %A Wendler, Franz %A Dasgupta, Ramanuj %A Perrimon, Norbert %A Malhotra, Vivek %K Animals %K Cell Line %K Drosophila %K Drosophila Proteins %K Endoplasmic Reticulum %K Genes, Insect %K Genes, Reporter %K Genomics %K Golgi Apparatus %K Horseradish Peroxidase %K Intracellular Membranes %K Protein Sorting Signals %K RNA Interference %XYeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.
%B Nature %V 439 %P 604-7 %8 2006 Feb 2 %G eng %N 7076 %1 http://www.ncbi.nlm.nih.gov/pubmed/16452979?dopt=Abstract %R 10.1038/nature04377 %0 Journal Article %J Nature %D 2006 %T A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling. %A Friedman, Adam %A Perrimon, Norbert %K Animals %K Drosophila %K Drug Evaluation, Preclinical %K Enzyme Activation %K Extracellular Signal-Regulated MAP Kinases %K Genome, Insect %K Genomics %K Ligands %K MAP Kinase Signaling System %K Receptor Protein-Tyrosine Kinases %K RNA Interference %XReceptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs or downstream pathway components such as Ras and Raf. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components-in multiple cell types with different RTK ligands and oncogenic stimuli-validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.
%B Nature %V 444 %P 230-4 %8 2006 Nov 9 %G eng %N 7116 %1 http://www.ncbi.nlm.nih.gov/pubmed/17086199?dopt=Abstract %R 10.1038/nature05280 %0 Journal Article %J Cell Signal %D 2006 %T Mutational analysis reveals separable DNA binding and trans-activation of Drosophila STAT92E. %A Karsten, Peter %A Plischke, Iris %A Perrimon, Norbert %A Zeidler, Martin P %K Amino Acid Sequence %K Amino Acid Substitution %K Animals %K Cells, Cultured %K DNA Mutational Analysis %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Molecular Sequence Data %K Mutagenesis, Site-Directed %K Mutation, Missense %K Sequence Alignment %K src Homology Domains %K STAT Transcription Factors %K Transcriptional Activation %XIn the canonical model of JAK/STAT signalling STAT transcription factors are activated by JAK mediated tyrosine phosphorylation following pathway stimulation by external cytokines. Activated STAT molecules then homo- or heterodimerise before translocating to the nucleus where they bind to DNA sequences within the promoters of pathway target genes. DNA-bound STAT dimers then activate transcription of their targets via interaction with components of the basal transcription machinery. Here we describe a missense mutation in the SH2 domain of the single Drosophila STAT92E homologue which results in an amino-acid substitution conserved in both the canonical SH2 domain and STAT-like molecules previously identified in C. elegans and the mosquito Anopheles gambiae. This mutation leads to nuclear accumulation and constitutive DNA binding of Drosophila STAT92E even in the absence of JAK stimulation. Strikingly, this mutant shows only limited transcriptional activity in tissue culture based assays and functions as a dominant-negative at both the phenotypic and molecular levels in vivo. These features represent aspects of both dominant gain-of-function and dominant-negative activities and imply that the functions of DNA binding can be functionally separated from the role of STAT92E as a transcriptional activator. It is thus possible that an alternative post-translational modification, in addition to tyrosine phosphorylation, may be required to allow STAT to act as a transcriptional activator and suggests the existence of an alternative mechanism by which STAT transcriptional activity may be regulated in vivo.
%B Cell Signal %V 18 %P 819-29 %8 2006 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/16129580?dopt=Abstract %R 10.1016/j.cellsig.2005.07.006 %0 Journal Article %J Curr Biol %D 2006 %T Weckle is a zinc finger adaptor of the toll pathway in dorsoventral patterning of the Drosophila embryo. %A Chen, Li-Ying %A Wang, Juinn-Chin %A Hyvert, Yann %A Lin, Hui-Ping %A Perrimon, Norbert %A Imler, Jean-Luc %A Hsu, Jui-Chou %K Adaptor Proteins, Signal Transducing %K Animals %K Antigens, Differentiation %K Body Patterning %K Cell Membrane %K Dimerization %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Epistasis, Genetic %K Immunity, Innate %K Models, Biological %K Mutation %K Phenotype %K Phosphoproteins %K Receptors, Immunologic %K Toll-Like Receptors %K Transcription Factors %K Zinc Fingers %XBACKGROUND: The Drosophila Toll pathway takes part in both establishment of the embryonic dorsoventral axis and induction of the innate immune response in adults. Upon activation by the cytokine Spätzle, Toll interacts with the adaptor proteins DmMyD88 and Tube and the kinase Pelle and triggers degradation of the inhibitor Cactus, thus allowing the nuclear translocation of the transcription factor Dorsal/Dif. weckle (wek) was previously identified as a new dorsal group gene that encodes a putative zinc finger transcription factor. However, its role in the Toll pathway was unknown. RESULTS: Here, we isolated new wek alleles and demonstrated that cactus is epistatic to wek, which in turn is epistatic to Toll. Consistent with this, Wek localizes to the plasma membrane of embryos, independently of Toll signaling. Wek homodimerizes and associates with Toll. Moreover, Wek binds to and localizes DmMyD88 to the plasma membrane. Thus, Wek acts as an adaptor to assemble/stabilize a Toll/Wek/DmMyD88/Tube complex. Remarkably, unlike the DmMyD88/tube/pelle/cactus gene cassette of the Toll pathway, wek plays a minimal role, if any, in the immune defense against Gram-positive bacteria and fungi. CONCLUSIONS: We conclude that Wek is an adaptor to link Toll and DmMyD88 and is required for efficient recruitment of DmMyD88 to Toll. Unexpectedly, wek is dispensable for innate immune response, thus revealing differences in the Toll-mediated activation of Dorsal in the embryo and Dif in the fat body of adult flies.
%B Curr Biol %V 16 %P 1183-93 %8 2006 Jun 20 %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/16782008?dopt=Abstract %R 10.1016/j.cub.2006.05.050 %0 Journal Article %J Cold Spring Harb Symp Quant Biol %D 2006 %T Drosophila genome-wide RNAi screens: are they delivering the promise? %A Mathey-Prevot, Bermard %A Perrimon, Norbert %K Animals %K Antibodies %K Drosophila %K Genes, Reporter %K Genetic Techniques %K Genome, Insect %K Genomics %K Oligonucleotide Array Sequence Analysis %K RNA Interference %K Systems Biology %K Transcription, Genetic %XThe emergence of RNA interference (RNAi) on the heels of the successful completion of the Drosophila genome project was seen by many as the ace in functional genomics: Its application would quickly assign a function to all genes in this organism and help delineate the complex web of interactions or networks linking them at the systemic level. A few years wiser and a number of genome-wide Drosophila RNAi screens later, we reflect on the state of high-throughput RNAi screens in Drosophila and ask whether the initial promise was fulfilled. We review the impact that this approach has had in the field of Drosophila research and chart out strategies to extract maximal benefit from the application of RNAi to gene discovery and pursuit of systems biology.
%B Cold Spring Harb Symp Quant Biol %V 71 %P 141-8 %8 2006 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/17381290?dopt=Abstract %R 10.1101/sqb.2006.71.027 %0 Journal Article %J PLoS Pathog %D 2006 %T COPI activity coupled with fatty acid biosynthesis is required for viral replication. %A Cherry, Sara %A Kunte, Amit %A Wang, Hui %A Coyne, Carolyn %A Rawson, Robert B %A Perrimon, Norbert %K Animals %K Cell Line %K Coat Protein Complex I %K Drosophila %K Fatty Acids %K Golgi Apparatus %K Humans %K Nodaviridae %K Poliovirus %K RNA Interference %K RNA, Viral %K Virus Replication %XDuring infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.
%B PLoS Pathog %V 2 %P e102 %8 2006 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/17040126?dopt=Abstract %R 10.1371/journal.ppat.0020102 %0 Journal Article %J Nucleic Acids Res %D 2006 %T FlyRNAi: the Drosophila RNAi screening center database. %A Flockhart, Ian %A Booker, Matthew %A Kiger, Amy %A Boutros, Michael %A Armknecht, Susan %A Ramadan, Nadire %A Richardson, Kris %A Xu, Andrew %A Perrimon, Norbert %A Mathey-Prevot, Bernard %K Animals %K Computational Biology %K Databases, Genetic %K Drosophila %K Gene Library %K Genome, Insect %K Internet %K RNA Interference %K RNA, Double-Stranded %K Software %K User-Computer Interface %XRNA interference (RNAi) has become a powerful tool for genetic screening in Drosophila. At the Drosophila RNAi Screening Center (DRSC), we are using a library of over 21,000 double-stranded RNAs targeting known and predicted genes in Drosophila. This library is available for the use of visiting scientists wishing to perform full-genome RNAi screens. The data generated from these screens are collected in the DRSC database (http://flyRNAi.org/cgi-bin/RNAi_screens.pl) in a flexible format for the convenience of the scientist and for archiving data. The long-term goal of this database is to provide annotations for as many of the uncharacterized genes in Drosophila as possible. Data from published screens are available to the public through a highly configurable interface that allows detailed examination of the data and provides access to a number of other databases and bioinformatics tools.
%B Nucleic Acids Res %V 34 %P D489-94 %8 2006 Jan 1 %G eng %N Database issue %1 http://www.ncbi.nlm.nih.gov/pubmed/16381918?dopt=Abstract %R 10.1093/nar/gkj114 %0 Book Section %B Med Image Comput Comput Assist Interv %D 2005 %T Towards automated cellular image segmentation for RNAi genome-wide screening. %A Zhou, Xiaobo %A Liu, K Y %A Bradley, P %A Perrimon, N %A Wong, Stephen T C %K Algorithms %K Artificial Intelligence %K Cluster Analysis %K Gene Expression Profiling %K Genomics %K Image Enhancement %K Image Interpretation, Computer-Assisted %K Microscopy, Fluorescence %K Pattern Recognition, Automated %K Proteome %K Reproducibility of Results %K RNA Interference %K Sensitivity and Specificity %XThe Rho family of small GTPases is essential for morphological changes during normal cell development and migration, as well as during disease states such as cancer. Our goal is to identify novel effectors of Rho proteins using a cell-based assay for Rho activity to perform genome-wide functional screens using double stranded RNA (dsRNAs) interference. We aim to discover genes could cause the cell phenotype changed dramatically. Biologists currently attempt to perform the genome-wide RNAi screening to identify various image phenotypes. RNAi genome-wide screening, however, could easily generate more than a million of images per study, manual analysis is thus prohibitive. Image analysis becomes a bottleneck in realizing high content imaging screens. We propose a two-step segmentation approach to solve this problem. First, we determine the center of a cell using the information in the DNA-channel by segmenting the DNA nuclei and the dissimilarity function is employed to attenuate the over-segmentation problem, then we estimate a rough boundary for each cell using a polygon. Second, we apply fuzzy c-means based multi-threshold segmentation and sharpening technology; for isolation of touching spots, marker-controlled watershed is employed to remove touching cells. Furthermore, Voronoi diagrams are employed to correct the segmentation errors caused by overlapping cells. Image features are extracted for each cell. K-nearest neighbor classifier (KNN) is employed to perform cell phenotype classification. Experimental results indicate that the proposed approach can be used to identify cell phenotypes of RNAi genome-wide screens.
%B Med Image Comput Comput Assist Interv %V 8 %P 885-92 %8 2005 %G eng %N Pt 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/16685930?dopt=Abstract %0 Journal Article %J Sci STKE %D 2005 %T Drosophila Wnt/Fz pathways. %A Dasgupta, Ramanuj %A Boutros, Michael %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Epidermis %K Epistasis, Genetic %K Frizzled Receptors %K GTP-Binding Proteins %K Larva %K Membrane Proteins %K Models, Biological %K Morphogenesis %K Phenotype %K Proto-Oncogene Proteins %K Receptors, G-Protein-Coupled %K Signal Transduction %K Species Specificity %K Wnt Proteins %K Wnt1 Protein %XWnts [also known as Wingless (Wg)] are a family of conserved signaling molecules involved in a plethora of fundamental developmental and cell biological processes, such as cell proliferation, differentiation, and cell polarity. Dysregulation of the pathway can be detrimental, because several components are tumorigenic when mutated and are associated with hepatic, colorectal, breast, and skin cancers. First identified in the fruit fly Drosophila melanogaster as a gene family responsible for patterning the embryonic epidermis, the Wnt gene family, including Wg, encode secreted glycoproteins that activate receptor-mediated signaling pathways leading to numerous transcriptional and cellular responses. The main function of the canonical Wg pathway is to stabilize the cytoplasmic pool of a key mediator, beta-catenin [beta-catenin, known as Armadillo (Arm) in fruit flies], which is otherwise degraded by the proteasome pathway. Initially identified as a key player in stabilizing cell-cell adherens junctions, Arm is now known to also act as a transcription factor by forming a complex with the lymphoid enhancer factor (LEF)/T cell-specific transcription factor (TCF) family of high mobility group (HMG)-box transcription factors. Upon Wnt/Wg stimulation, stabilized Arm translocates to the nucleus, where, together with LEF/TCF transcription factors, it activates downstream target genes that regulate numerous cell biological processes.
%B Sci STKE %V 2005 %P cm5 %8 2005 May 10 %G eng %N 283 %1 http://www.ncbi.nlm.nih.gov/pubmed/15886387?dopt=Abstract %R 10.1126/stke.2832005cm5 %0 Journal Article %J Nat Rev Mol Cell Biol %D 2005 %T Heparan sulphate proteoglycans: the sweet side of development. %A Häcker, Udo %A Nybakken, Kent %A Perrimon, Norbert %K Animals %K Gene Expression Regulation %K Glycosaminoglycans %K Heparan Sulfate Proteoglycans %K Humans %K Mutation %K Signal Transduction %XPattern formation during development is controlled to a great extent by a small number of conserved signal transduction pathways that are activated by extracellular ligands such as Hedgehog, Wingless or Decapentaplegic. Genetic experiments have identified heparan sulphate proteoglycans (HSPGs) as important regulators of the tissue distribution of these extracellular signalling molecules. Several recent reports provide important new insights into the mechanisms by which HSPGs function during development.
%B Nat Rev Mol Cell Biol %V 6 %P 530-41 %8 2005 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/16072037?dopt=Abstract %R 10.1038/nrm1681 %0 Journal Article %J Methods Enzymol %D 2005 %T High-throughput RNA interference screens in Drosophila tissue culture cells. %A Armknecht, Susan %A Boutros, Michael %A Kiger, Amy %A Nybakken, Kent %A Mathey-Prevot, Bernard %A Perrimon, Norbert %K Animals %K Cell Line %K Drosophila %K Genes, Reporter %K RNA Interference %K Tissue Culture Techniques %K Transfection %XThis chapter describes the method used to conduct high-throughput screening (HTs) by RNA interference in Drosophila tissue culture cells. It covers four main topics: (1) a brief description of the existing platforms to conduct RNAi-screens in cell-based assays; (2) a table of the Drosophila cell lines available for these screens and a brief mention of the need to establish other cell lines as well as cultures of primary cells; (3) a discussion of the considerations and protocols involved in establishing assays suitable for HTS in a 384-well format; and (A) a summary of the various ways of handling raw data from an ongoing screen, with special emphasis on how to apply normalization for experimental variation and statistical filters to sort out noise from signals.
%B Methods Enzymol %V 392 %P 55-73 %8 2005 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/15644175?dopt=Abstract %R 10.1016/S0076-6879(04)92004-6 %0 Journal Article %J Cell Metab %D 2005 %T Super-size flies. %A Kulkarni, Meghana M %A Perrimon, Norbert %K Animals %K Caenorhabditis elegans %K Drosophila melanogaster %K Energy Metabolism %K Fats %K Humans %K Lipase %K Obesity %K Recombinant Proteins %XThe increasing prevalence of obesity and other nutrition-related chronic diseases has prompted considerable efforts to understand their pathogenesis and treatment. One experimental approach is to overexpress, inactivate, or manipulate specific genes that regulate energy metabolism and fat storage. Many such techniques are fully established, routine tools in Drosophila and C. elegans, which provide elegant models for dissecting endocrine problems and metabolic pathways.
%B Cell Metab %V 1 %P 288-90 %8 2005 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/16054074?dopt=Abstract %R 10.1016/j.cmet.2005.04.008 %0 Journal Article %J Dev Cell %D 2005 %T BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary. %A Kirilly, Daniel %A Spana, Eric P %A Perrimon, Norbert %A Padgett, Richard W %A Xie, Ting %K Animals %K Bone Morphogenetic Proteins %K Cell Differentiation %K Cell Division %K Cell Proliferation %K Drosophila %K Drosophila Proteins %K Female %K Gene Expression Regulation, Developmental %K Longevity %K Models, Biological %K Ovary %K Proto-Oncogene Proteins %K Signal Transduction %K Stem Cells %K Wnt1 Protein %XBMP signaling is essential for promoting self-renewal of mouse embryonic stem cells and Drosophila germline stem cells and for repressing stem cell proliferation in the mouse intestine and skin. However, it remains unknown whether BMP signaling can promote self-renewal of adult somatic stem cells. In this study, we show that BMP signaling is necessary and sufficient for promoting self-renewal and proliferation of somatic stem cells (SSCs) in the Drosophila ovary. BMP signaling is required in SSCs to directly control their maintenance and division, but is dispensable for proliferation of their differentiated progeny. Furthermore, BMP signaling is required to control SSC self-renewal, but not survival. Moreover, constitutive BMP signaling prolongs the SSC lifespan. Therefore, our study clearly demonstrates that BMP signaling directly promotes SSC self-renewal and proliferation in the Drosophila ovary. Our work further suggests that BMP signaling could promote self-renewal of adult stem cells in other systems.
%B Dev Cell %V 9 %P 651-62 %8 2005 Nov %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/16256740?dopt=Abstract %R 10.1016/j.devcel.2005.09.013 %0 Journal Article %J Science %D 2005 %T Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection. %A Philips, Jennifer A %A Rubin, Eric J %A Perrimon, Norbert %K Animals %K Antigens, CD36 %K Cell Line %K Cytoskeleton %K Drosophila melanogaster %K Escherichia coli %K Humans %K Immunity, Innate %K Lysosome-Associated Membrane Glycoproteins %K Macrophages %K Membrane Proteins %K Mice %K Mycobacterium fortuitum %K Phagocytosis %K Receptors, Immunologic %K Receptors, Scavenger %K RNA Interference %K RNA, Double-Stranded %K Scavenger Receptors, Class B %K Sialoglycoproteins %K Staphylococcus aureus %K Transfection %K Transport Vesicles %XCertain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.
%B Science %V 309 %P 1251-3 %8 2005 Aug 19 %G eng %N 5738 %1 http://www.ncbi.nlm.nih.gov/pubmed/16020694?dopt=Abstract %R 10.1126/science.1116006 %0 Journal Article %J Science %D 2005 %T Extrusion and death of DPP/BMP-compromised epithelial cells in the developing Drosophila wing. %A Gibson, Matthew C %A Perrimon, Norbert %K Actins %K Alleles %K Animals %K Apoptosis %K Body Patterning %K Cell Death %K Cell Shape %K Clone Cells %K Drosophila melanogaster %K Drosophila Proteins %K Epithelial Cells %K Ethyl Methanesulfonate %K JNK Mitogen-Activated Protein Kinases %K Microtubules %K Morphogenesis %K Mutagens %K Mutation %K Phenotype %K Protein-Serine-Threonine Kinases %K Receptors, Cell Surface %K Signal Transduction %K Transforming Growth Factor beta %K Wings, Animal %XDuring animal development, epithelial cell fates are specified according to spatial position by extracellular signaling pathways. Among these, the transforming growth factor beta/bone morphogenetic protein (TGF-beta/BMP) pathways are evolutionarily conserved and play crucial roles in the development and homeostasis of a wide range of multicellular tissues. Here we show that in the developing Drosophila wing imaginal epithelium, cell clones deprived of the BMP-like ligand Decapentaplegic (DPP) do not die as previously thought but rather extrude from the cell layer as viable cysts exhibiting marked abnormalities in cell shape and cytoskeletal organization. We propose that in addition to assigning cell fates, a crucial developmental function of DPP/BMP signaling is the position-specific control of epithelial architecture.
%B Science %V 307 %P 1785-9 %8 2005 Mar 18 %G eng %N 5716 %1 http://www.ncbi.nlm.nih.gov/pubmed/15774762?dopt=Abstract %R 10.1126/science.1104751 %0 Journal Article %J Development %D 2005 %T Function of the ETS transcription factor Yan in border cell migration. %A Schober, Markus %A Rebay, Ilaria %A Perrimon, Norbert %K Animals %K Cell Movement %K Drosophila %K Drosophila Proteins %K Eye Proteins %K Female %K Oogenesis %K Ovary %K Receptor, Epidermal Growth Factor %K Repressor Proteins %K Signal Transduction %K Transcription Factors %XInvasive cell migration in both normal development and metastatic cancer is regulated by various signaling pathways, transcription factors and cell-adhesion molecules. The coordination between these activities in the context of cell migration is poorly understood. During Drosophila oogenesis, a small group of cells called border cells exit the follicular epithelium to perform a stereotypic, invasive migration. We find that the ETS transcription factor Yan is required for border cell migration and that Yan expression is spatiotemporally regulated as border cells migrate from the anterior pole of the egg chamber towards the nurse cell-oocyte boundary. Yan expression is dependent on inputs from the JAK/STAT, Notch and Receptor Tyrosine Kinase pathways in border cells. Mechanistically, Yan functions to modulate the turnover of DE-Cadherin-dependent adhesive complexes to facilitate border cell migration. Our results suggest that Yan acts as a pivotal link between signal transduction, cell adhesion and invasive cell migration in Drosophila border cells.
%B Development %V 132 %P 3493-504 %8 2005 Aug %G eng %N 15 %1 http://www.ncbi.nlm.nih.gov/pubmed/16014514?dopt=Abstract %R 10.1242/dev.01911 %0 Journal Article %J Science %D 2005 %T Functional genomic analysis of the Wnt-wingless signaling pathway. %A Dasgupta, Ramanuj %A Kaykas, Ajamete %A Moon, Randall T %A Perrimon, Norbert %K Animals %K beta Catenin %K Binding Sites %K Cell Line %K Cloning, Molecular %K Computational Biology %K Cytoskeletal Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Embryonic Development %K Epistasis, Genetic %K Gene Expression Regulation %K Genes, Insect %K Genes, Reporter %K Genomics %K Mutation %K Phenotype %K Phosphorylation %K Protein Kinases %K Proteins %K Proto-Oncogene Proteins %K rab5 GTP-Binding Proteins %K RNA Interference %K Signal Transduction %K Trans-Activators %K Transcription Factors %K Transfection %K Wnt Proteins %K Wnt1 Protein %K Wnt3 Protein %K Zebrafish %K Zebrafish Proteins %XThe Wnt-Wingless (Wg) pathway is one of a core set of evolutionarily conserved signaling pathways that regulates many aspects of metazoan development. Aberrant Wnt signaling has been linked to human disease. In the present study, we used a genomewide RNA interference (RNAi) screen in Drosophila cells to screen for regulators of the Wnt pathway. We identified 238 potential regulators, which include known pathway components, genes with functions not previously linked to this pathway, and genes with no previously assigned functions. Reciprocal-Best-Blast analyses reveal that 50% of the genes identified in the screen have human orthologs, of which approximately 18% are associated with human disease. Functional assays of selected genes from the cell-based screen in Drosophila, mammalian cells, and zebrafish embryos demonstrated that these genes have evolutionarily conserved functions in Wnt signaling. High-throughput RNAi screens in cultured cells, followed by functional analyses in model organisms, prove to be a rapid means of identifying regulators of signaling pathways implicated in development and disease.
%B Science %V 308 %P 826-33 %8 2005 May 6 %G eng %N 5723 %1 http://www.ncbi.nlm.nih.gov/pubmed/15817814?dopt=Abstract %R 10.1126/science.1109374 %0 Journal Article %J Nat Genet %D 2005 %T A genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. %A Nybakken, Kent %A Vokes, Steven A %A Lin, Ting-Yi %A McMahon, Andrew P %A Perrimon, Norbert %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Gene Expression Regulation %K Genes, Insect %K Genome, Insect %K Genomics %K Hedgehog Proteins %K Insect Proteins %K Phosphoprotein Phosphatases %K Phosphotransferases %K Protein Phosphatase 2 %K RNA Interference %K RNA Splicing %K RNA, Messenger %K Signal Transduction %XMembers of the Hedgehog (Hh) family of signaling proteins are powerful regulators of developmental processes in many organisms and have been implicated in many human disease states. Here we report the results of a genome-wide RNA interference screen in Drosophila melanogaster cells for new components of the Hh signaling pathway. The screen identified hundreds of potential new regulators of Hh signaling, including many large protein complexes with pleiotropic effects, such as the coat protein complex I (COPI) complex, the ribosome and the proteasome. We identified the multimeric protein phosphatase 2A (PP2A) and two new kinases, the D. melanogaster orthologs of the vertebrate PITSLRE and cyclin-dependent kinase-9 (CDK9) kinases, as Hh regulators. We also identified a large group of constitutive and alternative splicing factors, two nucleoporins involved in mRNA export and several RNA-regulatory proteins as potent regulators of Hh signal transduction, indicating that splicing regulation and mRNA transport have a previously unrecognized role in Hh signaling. Finally, we showed that several of these genes have conserved roles in mammalian Hh signaling.
%B Nat Genet %V 37 %P 1323-32 %8 2005 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/16311596?dopt=Abstract %R 10.1038/ng1682 %0 Journal Article %J Science %D 2005 %T Genome-wide RNAi screen for host factors required for intracellular bacterial infection. %A Agaisse, Hervé %A Burrack, Laura S %A Philips, Jennifer A %A Rubin, Eric J %A Perrimon, Norbert %A Higgins, Darren E %K Animals %K Cell Cycle %K Cell Line %K Cytoskeleton %K Cytosol %K Drosophila melanogaster %K Drosophila Proteins %K Genes, Insect %K Genome %K Green Fluorescent Proteins %K Listeria monocytogenes %K Macrophages %K Mycobacterium fortuitum %K Phenotype %K RNA Interference %K RNA Processing, Post-Transcriptional %K RNA, Double-Stranded %K Signal Transduction %K Vacuoles %K Vesicular Transport Proteins %XMost studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.
%B Science %V 309 %P 1248-51 %8 2005 Aug 19 %G eng %N 5738 %1 http://www.ncbi.nlm.nih.gov/pubmed/16020693?dopt=Abstract %R 10.1126/science.1116008 %0 Magazine Article %D 2005 %T Q & A - Norbert Perrimon %A Perrimon, Norbert %B Current Biology %V 15 %P R481-R482 %G eng %N 13 %0 Journal Article %J Genes Dev %D 2005 %T Genome-wide RNAi analysis of JAK/STAT signaling components in Drosophila. %A Baeg, Gyeong-Hun %A Zhou, Rui %A Perrimon, Norbert %K Active Transport, Cell Nucleus %K Animals %K Cell Line %K Cell Nucleus %K DNA-Binding Proteins %K Drosophila Proteins %K Janus Kinase 1 %K Phosphorylation %K Protein Tyrosine Phosphatases %K Protein Tyrosine Phosphatases, Non-Receptor %K Protein-Tyrosine Kinases %K Repressor Proteins %K RNA Interference %K Signal Transduction %K STAT Transcription Factors %K Suppressor of Cytokine Signaling Proteins %K Trans-Activators %K Tyrosine %XThe cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.
%B Genes Dev %V 19 %P 1861-70 %8 2005 Aug 15 %G eng %N 16 %1 http://www.ncbi.nlm.nih.gov/pubmed/16055650?dopt=Abstract %R 10.1101/gad.1320705 %0 Journal Article %J Genes Dev %D 2005 %T Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition. %A Cherry, Sara %A Doukas, Tammy %A Armknecht, Susan %A Whelan, Sean %A Wang, Hui %A Sarnow, Peter %A Perrimon, Norbert %K Animals %K Base Sequence %K DNA Primers %K Drosophila %K Genome, Viral %K HeLa Cells %K Humans %K Protein Biosynthesis %K RNA Interference %K RNA Viruses %K Virus Replication %XThe widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.
%B Genes Dev %V 19 %P 445-52 %8 2005 Feb 15 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/15713840?dopt=Abstract %R 10.1101/gad.1267905 %0 Journal Article %J Development %D 2005 %T Notch modulates Wnt signalling by associating with Armadillo/beta-catenin and regulating its transcriptional activity. %A Hayward, Penny %A Brennan, Keith %A Sanders, Phil %A Balayo, Tina %A Dasgupta, Ramanuj %A Perrimon, Norbert %A Martinez Arias, Alfonso %K Animals %K Armadillo Domain Proteins %K beta Catenin %K Cytoskeletal Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Immunohistochemistry %K Immunoprecipitation %K Intercellular Signaling Peptides and Proteins %K Luciferases %K Membrane Proteins %K Receptors, Notch %K RNA Interference %K Signal Transduction %K Trans-Activators %K Transcription Factors %K Wnt Proteins %XThe establishment and stability of cell fates during development depend on the integration of multiple signals, which ultimately modulate specific patterns of gene expression. While there is ample evidence for this integration at the level of gene regulatory sequences, little is known about its operation at other levels of cellular activity. Wnt and Notch signalling are important elements of the circuitry that regulates gene expression in development and disease. Genetic analysis has suggested that in addition to convergence on the transcription of specific genes, there are modulatory cross-regulatory interactions between these signalling pathways. We report that the nodal point of these interactions is an activity of Notch that regulates the activity and the amount of the active/oncogenic form of Armadillo/beta-catenin. This activity of Notch is independent of that induced upon cleavage of its intracellular domain and which mediates transcription through Su(H)/CBF1. The modulatory function of Notch described here, contributes to the establishment of a robust threshold for Wnt signalling which is likely to play important roles in both normal and pathological situations.
%B Development %V 132 %P 1819-30 %8 2005 Apr %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/15772135?dopt=Abstract %R 10.1242/dev.01724 %0 Journal Article %J Commentaire %D 2004 %T [Le modèle américain] (French) %A Perrimon, Norbert %B Commentaire %V 106 %P 335-338 %G eng %0 Journal Article %J Curr Opin Genet Dev %D 2004 %T Genome-wide high-throughput screens in functional genomics. %A Friedman, Adam %A Perrimon, Norbert %K Animals %K Genome %K Genomics %K Humans %K RNA Interference %XThe availability of complete genome sequences from many organisms has yielded the ability to perform high-throughput, genome-wide screens of gene function. Within the past year, rapid advances have been made towards this goal in many major model systems, including yeast, worms, flies, and mammals. Yeast genome-wide screens have taken advantage of libraries of deletion strains, but RNA-interference has been used in other organisms to knockdown gene function. Examples of recent large-scale functional genetic screens include drug-target identification in yeast, regulators of fat accumulation in worms, growth and viability in flies, and proteasome-mediated degradation in mammalian cells. Within the next five years, such screens are likely to lead to annotation of function of most genes across multiple organisms. Integration of such data with other genomic approaches will extend our understanding of cellular networks.
%B Curr Opin Genet Dev %V 14 %P 470-6 %8 2004 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/15380236?dopt=Abstract %R 10.1016/j.gde.2004.07.010 %0 Journal Article %J Immunol Rev %D 2004 %T The roles of JAK/STAT signaling in Drosophila immune responses. %A Agaisse, Hervé %A Perrimon, Norbert %K Animals %K Antibody Formation %K Cytokines %K DNA-Binding Proteins %K Drosophila %K Hemocytes %K Immunity, Cellular %K Immunity, Innate %K Models, Immunological %K Protein-Tyrosine Kinases %K Signal Transduction %XInnate immune responses are mediated by the activation of various signaling processes. Here, we describe our current knowledge on Janus kinase (JAK)/signal transducers and activators of transcription (STAT) signaling in the Drosophila immune response. First, we briefly introduce the main effectors involved in the humoral and cellular responses, such as anti-bacterial peptides and hemocytes. Second, we describe the canonical JAK/STAT-signaling pathway, as established from extensive studies in mammalian systems, and we introduce the Drosophila components of the JAK/STAT pathway, as discovered from studies on embryonic development. Third, we describe the various roles of JAK/STAT signaling in both humoral and cellular responses. We present the JAK/STAT-dependent humoral factors, such as the thioester-containing proteins and the Tot peptides, produced by the fat body in response to septic injury. We also discuss the possible involvement of the JAK/STAT pathway in cellular responses, including hemocyte proliferation and differentiation. Finally, we present how cytokines, such as Upd3, might contribute to the integration of the immune responses at the organism level by orchestrating the response of various immune cells and organs, such as fat body, hemocytes, and lymph glands.
%B Immunol Rev %V 198 %P 72-82 %8 2004 Apr %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/15199955?dopt=Abstract %0 Journal Article %J Oncogene %D 2004 %T Using RNAi to catch Drosophila genes in a web of interactions: insights into cancer research. %A Dasgupta, Ramanuj %A Perrimon, Norbert %K Animals %K Databases, Protein %K Drosophila melanogaster %K Genomics %K Neoplasms %K Protein Interaction Mapping %K RNA Interference %XThe completion of whole-genome sequencing of various model organisms and the recent explosion of new technologies in the field of Functional Genomics and Proteomics is poised to revolutionize the way scientists identify and characterize gene function. One of the most significant advances in recent years has been the application of RNA interference (RNAi) as a means of assaying gene function. In the post-genomic era, advances in the field of cancer biology will rely upon the rapid identification and characterization of genes that regulate cell growth, proliferation, and apoptosis. Significant efforts are being directed towards cancer therapy and devising efficient means of selectively delivering drugs to cancerous cells. In this review, we discuss the promise of integrating genome-wide RNAi screens with proteomic approaches and small-molecule chemical genetic screens, towards improving our ability to understand and treat cancer.
%B Oncogene %V 23 %P 8359-65 %8 2004 Nov 1 %G eng %N 51 %1 http://www.ncbi.nlm.nih.gov/pubmed/15517017?dopt=Abstract %R 10.1038/sj.onc.1208028 %0 Journal Article %J Development %D 2004 %T Wingless, hedgehog and heparan sulfate proteoglycans. %A Perrimon, Norbert %A Häcker, Udo %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Hedgehog Proteins %K Heparan Sulfate Proteoglycans %K Membrane Glycoproteins %K Mutation %K Proteoglycans %K Proto-Oncogene Proteins %K Signal Transduction %K Wnt1 Protein %B Development %V 131 %P 2509-11; author reply 2511-3 %8 2004 Jun %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/15148297?dopt=Abstract %R 10.1242/dev.01225 %0 Journal Article %J Nat Immunol %D 2004 %T Entry is a rate-limiting step for viral infection in a Drosophila melanogaster model of pathogenesis. %A Cherry, Sara %A Perrimon, Norbert %K Animals %K Blotting, Western %K Cell Line %K Clathrin %K Cytopathogenic Effect, Viral %K Disease Models, Animal %K Drosophila melanogaster %K Dynamins %K Endocytosis %K Insect Viruses %K Microscopy, Fluorescence %K Picornaviridae %K Picornaviridae Infections %XThe identification of host factors that control susceptibility to infection has been hampered by a lack of amenable genetic systems. We established an in vivo model to determine the host factors that control pathogenesis and identified viral entry as a rate-limiting step for infection. We infected Drosophila melanogaster cells and adults with drosophila C virus and found that the clathrin-mediated endocytotic pathway is essential for both infection and pathogenesis. Heterozygosity for mutations in genes involved in endocytosis is sufficient to protect flies from pathogenicity, indicating the exquisite sensitivity and dependency of the virus on this pathway. Thus, this virus model provides a sensitive and efficient approach for identifying components required for pathogenesis.
%B Nat Immunol %V 5 %P 81-7 %8 2004 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/14691479?dopt=Abstract %R 10.1038/ni1019 %0 Journal Article %J Science %D 2004 %T Genome-wide RNAi analysis of growth and viability in Drosophila cells. %A Boutros, Michael %A Kiger, Amy A %A Armknecht, Susan %A Kerr, Kim %A Hild, Marc %A Koch, Britta %A Haas, Stefan A %A Paro, Renato %A Perrimon, Norbert %A Heidelberg Fly Array Consortium %K Animals %K Apoptosis %K Cell Cycle %K Cell Survival %K Cells, Cultured %K Computational Biology %K Core Binding Factor Alpha 2 Subunit %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Genes, Essential %K Genes, Insect %K Genome %K Humans %K Inhibitor of Apoptosis Proteins %K Phenotype %K Proteome %K Proto-Oncogene Proteins %K Reproducibility of Results %K RNA Interference %K RNA, Double-Stranded %K Sequence Homology %K Transcription Factors %XA crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.
%B Science %V 303 %P 832-5 %8 2004 Feb 6 %G eng %N 5659 %1 http://www.ncbi.nlm.nih.gov/pubmed/14764878?dopt=Abstract %R 10.1126/science.1091266 %0 Journal Article %J Dev Cell %D 2004 %T The PDGF/VEGF receptor controls blood cell survival in Drosophila. %A Brückner, Katja %A Kockel, Lutz %A Duchek, Peter %A Luque, Carlos M %A Rørth, Pernille %A Perrimon, Norbert %K Animals %K Apoptosis %K Blood Cells %K Cell Aggregation %K Cell Survival %K Cells, Cultured %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Gene Expression Regulation, Developmental %K Hematopoiesis %K Macrophages %K Models, Animal %K Mutation %K Phagocytosis %K Proteins %K ras Proteins %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %K Viral Proteins %XThe Drosophila PDGF/VEGF receptor (PVR) has known functions in the guidance of cell migration. We now demonstrate that during embryonic hematopoiesis, PVR has a role in the control of antiapoptotic cell survival. In Pvr mutants, a large fraction of the embryonic hemocyte population undergoes apoptosis, and the remaining blood cells cannibalistically phagocytose their dying peers. Consequently, total hemocyte numbers drop dramatically during embryogenesis, and large aggregates of engorged macrophages carrying multiple apoptotic corpses form. Hemocyte-specific expression of the pan-caspase inhibitor p35 in Pvr mutants eliminates hemocyte aggregates and restores blood cell counts and morphology. Additional rescue experiments suggest involvement of the Ras pathway in PVR-mediated blood cell survival. In cell culture, we demonstrate that PVR directly controls survival of a hemocyte cell line. This function of PVR shows striking conservation with mammalian hematopoiesis and establishes Drosophila as a model to study hematopoietic cell survival in development and disease.
%B Dev Cell %V 7 %P 73-84 %8 2004 Jul %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/15239955?dopt=Abstract %R 10.1016/j.devcel.2004.06.007 %0 Journal Article %J Dev Cell %D 2004 %T Small wing PLCgamma is required for ER retention of cleaved Spitz during eye development in Drosophila. %A Schlesinger, Ayelet %A Kiger, Amy %A Perrimon, Norbert %A Shilo, Ben-Zion %K Alleles %K Animals %K Cells, Cultured %K Drosophila %K Drosophila Proteins %K Endoplasmic Reticulum %K Epidermal Growth Factor %K Eye %K Gene Deletion %K Gene Expression Regulation, Developmental %K Genes, Insect %K Genetic Variation %K Immunohistochemistry %K Insect Proteins %K Membrane Proteins %K Phospholipase C gamma %K Protein Structure, Tertiary %K Recombinant Fusion Proteins %K RNA Interference %K Serine Endopeptidases %K Type C Phospholipases %XThe Drosophila EGF receptor ligand Spitz is cleaved by Rhomboid to generate an active secreted molecule. Surprisingly, when a cleaved variant of Spitz (cSpi) was expressed, it accumulated in the ER, both in embryos and in cell culture. A cell-based RNAi screen for loss-of-function phenotypes that alleviate ER accumulation of cSpi identified several genes, including the small wing (sl) gene encoding a PLCgamma. sl mutants compromised ER accumulation of cSpi in embryos, yet they exhibit EGFR hyperactivation phenotypes predominantly in the eye. Spi processing in the eye is carried out primarily by Rhomboid-3/Roughoid, which cleaves Spi in the ER, en route to the Golgi. The sl mutant phenotype is consistent with decreased cSpi retention in the R8 cells. Retention of cSpi in the ER provides a novel mechanism for restricting active ligand levels and hence the range of EGFR activation in the developing eye.
%B Dev Cell %V 7 %P 535-45 %8 2004 Oct %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/15469842?dopt=Abstract %R 10.1016/j.devcel.2004.09.001 %0 Journal Article %J Nat Biotechnol %D 2004 %T Temperature-sensitive control of protein activity by conditionally splicing inteins. %A Zeidler, Martin P %A Tan, Change %A Bellaiche, Yohanns %A Cherry, Sara %A Häder, Sabine %A Gayko, Urte %A Perrimon, Norbert %K Animals %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Inteins %K Membrane Proteins %K Protein Splicing %K Receptors, Notch %K Repressor Proteins %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %K Temperature %K Transcription Factors %K Vacuolar Proton-Translocating ATPases %XConditional or temperature-sensitive (TS) alleles represent useful tools with which to investigate gene function. Indeed, much of our understanding of yeast has relied on temperature-sensitive mutations which, when available, also provide important insights into other model systems. However, the rarity of temperature-sensitive alleles and difficulty in identifying them has limited their use. Here we describe a system to generate temperature-sensitive alleles based on conditionally active inteins. We have identified temperature-sensitive splicing variants of the yeast Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) intein inserted within Gal4 and transferred these into Gal80. We show that Gal80-intein(TS) is able to efficiently provide temporal regulation of the Gal4/upstream activation sequence (UAS) system in a temperature-dependent manner in Drosophila melanogaster. Given the minimal host requirements necessary for temperature-sensitive intein splicing, this technique has the potential to allow the generation and use of conditionally active inteins in multiple host proteins and model systems, thereby widening the use of temperature-sensitive alleles for functional protein analysis.
%B Nat Biotechnol %V 22 %P 871-6 %8 2004 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/15184905?dopt=Abstract %R 10.1038/nbt979 %0 Journal Article %J Dev Biol %D 2004 %T The Wingless morphogen gradient is established by the cooperative action of Frizzled and Heparan Sulfate Proteoglycan receptors. %A Baeg, Gyeong-Hun %A Selva, Erica M %A Goodman, Robyn M %A Dasgupta, Ramanuj %A Perrimon, Norbert %K Animals %K Clone Cells %K Drosophila %K Drosophila Proteins %K Frizzled Receptors %K Gene Expression Regulation, Developmental %K Genes, Insect %K Heparan Sulfate Proteoglycans %K Insect Proteins %K Membrane Proteins %K Models, Biological %K Mutation %K Proteoglycans %K Proto-Oncogene Proteins %K Receptors, G-Protein-Coupled %K RNA Interference %K Signal Transduction %K Tissue Distribution %K Wings, Animal %K Wnt1 Protein %XWe have examined the respective contribution of Heparan Sulfate Proteoglycans (HSPGs) and Frizzled (Fz) proteins in the establishment of the Wingless (Wg) morphogen gradient. From the analysis of mutant clones of sulfateless/N-deacetylase-sulphotransferase in the wing imaginal disc, we find that lack of Heparan Sulfate (HS) causes a dramatic reduction of both extracellular and intracellular Wg in receiving cells. Our studies, together with others [Kirkpatrick, C.A., Dimitroff, B.D., Rawson, J.M., Selleck, S.B., 2004. Spatial regulation of Wingless morphogen distribution and signalling by Dally-like protein. Dev. Cell (in press)], reveals that the Glypican molecule Dally-like Protein (Dlp) is associated with both negative and positive roles in Wg short- and long-range signaling, respectively. In addition, analyses of the two Fz proteins indicate that the Fz and DFz2 receptors, in addition to transducing the signal, modulate the slope of the Wg gradient by regulating the amount of extracellular Wg. Taken together, our analysis illustrates how the coordinated activities of HSPGs and Fz/DFz2 shape the Wg morphogen gradient.
%B Dev Biol %V 276 %P 89-100 %8 2004 Dec 1 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/15531366?dopt=Abstract %R 10.1016/j.ydbio.2004.08.023 %0 Journal Article %J Dev Biol %D 2004 %T Yantar, a conserved arginine-rich protein is involved in Drosophila hemocyte development. %A Sinenko, Sergey A %A Kim, Eun Kyung %A Wynn, Rhoda %A Manfruelli, Pascal %A Ando, Istvan %A Wharton, Kristi A %A Perrimon, Norbert %A Mathey-Prevot, Bernard %K 3T3 Cells %K Amino Acid Sequence %K Animals %K Blotting, Northern %K Cercopithecus aethiops %K Chromosome Mapping %K COS Cells %K DNA Primers %K Drosophila %K Drosophila Proteins %K Hematopoiesis %K Hemocytes %K Immunohistochemistry %K In Situ Hybridization %K Larva %K Mice %K Molecular Sequence Data %K Mutagenesis %K Mutation %K Nuclear Proteins %K Phenotype %K Polymerase Chain Reaction %K RNA, Messenger %K Sequence Alignment %K Sequence Analysis, DNA %XTo identify novel factors involved in Drosophila hematopoiesis, we screened a collection of lethal recessive mutations that also affected normal hemocyte composition in larvae. We present the characterization of the gene yantar (ytr) for which we isolated null and hypomorphic mutations that were associated with severe defects in hemocyte differentiation and proliferation; ytr is predominantly expressed in the hematopoietic tissue during larval development and encodes an evolutionary conserved protein which is predominantly localized in the nucleus. The hematopoietic phenotype in ytr mutants is consistent with a defect or block in differentiation of precursor hemocytes: mutant larvae have enlarged lymph glands (LGs) and have an excess of circulating hemocytes. In addition, many cells exhibit both lamellocyte and crystal cell markers. Ytr function has been preserved in evolution as hematopoietic specific expression of the Drosophila or mouse Ytr proteins rescue the differentiation defects in mutant hemocytes.
%B Dev Biol %V 273 %P 48-62 %8 2004 Sep 1 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/15302597?dopt=Abstract %R 10.1016/j.ydbio.2004.05.022 %0 Book Section %D 2004 %T RNA interference: Mechanisms and Applications %A Perrimon, Norbert %A Hymen, Tony %I Cahier Les Treilles %G eng %0 Book Section %B Key Experiments in Practical Developmental Biology %D 2004 %T The UAS/GAL4 system for tissue-specific analysis of EGFR gene function in Drosophila melanogaster %A Duffy, JB %A Perrimon, Norbert %E Marí-Beffa, Manuel %E Knight, Jennifer %B Key Experiments in Practical Developmental Biology %I Cambridge University Press %P 269-281 %G eng %0 Journal Article %J PLoS Biol %D 2004 %T Parallel chemical genetic and genome-wide RNAi screens identify cytokinesis inhibitors and targets. %A Eggert, Ulrike S %A Kiger, Amy A %A Richter, Constance %A Perlman, Zachary E %A Perrimon, Norbert %A Mitchison, Timothy J. %A Field, Christine M %K Animals %K Aurora Kinases %K Caenorhabditis elegans %K Cell Line %K Cytokinesis %K Drosophila %K Formamides %K Genetic Techniques %K Genome %K Genomics %K Microscopy, Fluorescence %K Phenotype %K Protein-Serine-Threonine Kinases %K Pyrazoles %K RNA Interference %K Saccharomyces cerevisiae %XCytokinesis involves temporally and spatially coordinated action of the cell cycle and cytoskeletal and membrane systems to achieve separation of daughter cells. To dissect cytokinesis mechanisms it would be useful to have a complete catalog of the proteins involved, and small molecule tools for specifically inhibiting them with tight temporal control. Finding active small molecules by cell-based screening entails the difficult step of identifying their targets. We performed parallel chemical genetic and genome-wide RNA interference screens in Drosophila cells, identifying 50 small molecule inhibitors of cytokinesis and 214 genes important for cytokinesis, including a new protein in the Aurora B pathway (Borr). By comparing small molecule and RNAi phenotypes, we identified a small molecule that inhibits the Aurora B kinase pathway. Our protein list provides a starting point for systematic dissection of cytokinesis, a direction that will be greatly facilitated by also having diverse small molecule inhibitors, which we have identified. Dissection of the Aurora B pathway, where we found a new gene and a specific small molecule inhibitor, should benefit particularly. Our study shows that parallel RNA interference and small molecule screening is a generally useful approach to identifying active small molecules and their target pathways.
%B PLoS Biol %V 2 %P e379 %8 2004 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/15547975?dopt=Abstract %R 10.1371/journal.pbio.0020379 %0 Journal Article %J Development %D 2003 %T Liz and Norbert at the movies %A Perkins, Lizabeth %A Perrimon, Norbert %B Development %V 130 %P 5556-5557 %G eng %N 23 %0 Journal Article %J Curr Opin Cell Biol %D 2003 %T Apicobasal polarization: epithelial form and function. %A Gibson, Matthew C %A Perrimon, Norbert %K Animals %K Cell Membrane %K Drosophila %K Epithelium %K Humans %K Intercellular Junctions %K Ligands %K Models, Biological %K Neuregulin-1 %K Receptor, ErbB-2 %K Signal Transduction %K Tight Junctions %XThe structure and function of epithelial sheets generally depend on apicobasal polarization, which is achieved and maintained by linking asymmetrically distributed intercellular junctions to the cytoskeleton of individual cells. Recent studies in both Drosophila and vertebrate epithelia have yielded new insights into the conserved mechanisms by which apicobasal polarity is established and maintained during development. In mature polarized epithelia, apicobasal polarity is important for the establishment of adhesive junctions and the formation of a paracellular diffusion barrier that prevents the movement of solutes across the epithelium. Recent findings show that segregation of ligand and receptor with one on each side of this barrier can be a crucial regulator of cell-cell signaling events.
%B Curr Opin Cell Biol %V 15 %P 747-52 %8 2003 Dec %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/14644201?dopt=Abstract %0 Journal Article %J Nat Genet %D 2003 %T Tailoring the genome: the power of genetic approaches. %A Nagy, Andras %A Perrimon, Norbert %A Sandmeyer, Suzanne %A Plasterk, Ronald %K Animals %K Caenorhabditis elegans %K Drosophila melanogaster %K Genetic Techniques %K Genetic Variation %K Genome %K Mice %K Models, Genetic %K Saccharomyces cerevisiae %XIn the last century, genetics has developed into one of the most powerful tools for addressing basic questions concerning inheritance, development, individual and social operations and death. Here we summarize the current approaches to these questions in four of the most advanced models organisms: Saccharomyces cerevisiae (yeast), Caenorhabditis elegans (worm), Drosophila melanogaster (fly) and Mus musculus (mouse). The genomes of each of these four models have been sequenced, and all have well developed methods of efficient genetic manipulations.
%B Nat Genet %V 33 Suppl %P 276-84 %8 2003 Mar %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/12610537?dopt=Abstract %R 10.1038/ng1115 %0 Journal Article %J Dev Biol %D 2003 %T Coordinate regulation of small temporal RNAs at the onset of Drosophila metamorphosis. %A Bashirullah, Arash %A Pasquinelli, Amy E %A Kiger, Amy A %A Perrimon, Norbert %A Ruvkun, Gary %A Thummel, Carl S %K Animals %K Base Sequence %K Drosophila %K Drosophila Proteins %K Ecdysone %K Gene Expression Regulation, Developmental %K Metamorphosis, Biological %K MicroRNAs %K Molecular Sequence Data %K Organ Culture Techniques %XThe lin-4 and let-7 small temporal RNAs play a central role in controlling the timing of Caenorhabditis elegans cell fate decisions. let-7 has been conserved through evolution, and its expression correlates with adult development in bilateral animals, including Drosophila [Nature 408 (2000), 86]. The best match for lin-4 in Drosophila, miR-125, is also expressed during pupal and adult stages of Drosophila development [Curr. Biol. 12 (2002), 735]. Here, we ask whether the steroid hormone ecdysone induces let-7 or miR-125 expression at the onset of metamorphosis, attempting to link a known temporal regulator in Drosophila with the heterochronic pathway defined in C. elegans. We find that let-7 and miR-125 are coordinately expressed in late larvae and prepupae, in synchrony with the high titer ecdysone pulses that initiate metamorphosis. Unexpectedly, however, their expression is neither dependent on the EcR ecdysone receptor nor inducible by ecdysone in cultured larval organs. Although let-7 and miR-125 can be induced by ecdysone in Kc tissue culture cells, their expression is significantly delayed relative to that seen in the animal. let-7 and miR-125 are encoded adjacent to one another in the genome, and their induction correlates with the transient appearance of an approximately 500-nt RNA transcribed from this region, providing a mechanism to explain their precise coordinate regulation. We conclude that a common precursor RNA containing both let-7 and miR-125 is induced independently of ecdysone in Drosophila, raising the possibility of a temporal signal that is distinct from the well-characterized ecdysone-EcR pathway.
%B Dev Biol %V 259 %P 1-8 %8 2003 Jul 1 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/12812783?dopt=Abstract %0 Journal Article %J FASEB J %D 2003 %T Gamma-secretase/presenilin inhibitors for Alzheimer's disease phenocopy Notch mutations in Drosophila. %A Micchelli, Craig A %A Esler, William P %A Kimberly, W Taylor %A Jack, Christine %A Berezovska, Oksana %A Kornilova, Anna %A Hyman, Bradley T %A Perrimon, Norbert %A Wolfe, Michael S %K Active Transport, Cell Nucleus %K Alzheimer Disease %K Amyloid Precursor Protein Secretases %K Animals %K Dipeptides %K Drosophila %K Drosophila Proteins %K Endopeptidases %K Kinetics %K Membrane Proteins %K Mutation %K Phenotype %K Presenilins %K Protease Inhibitors %K Receptors, Notch %K Signal Transduction %K Wings, Animal %XSignaling from the Notch (N) receptor is essential for proper cell-fate determinations and tissue patterning in all metazoans. N signaling requires a presenilin (PS)-dependent transmembrane-cleaving activity that is closely related or identical to the gamma-secretase proteolysis of the amyloid-beta precursor protein (APP) involved in Alzheimer's disease pathogenesis. Here, we show that N-[N-(3,5-difluorophenacetyl)-L-alanyl]-(S)-phenylglycine t-butyl ester, a potent gamma-secretase inhibitor reported to reduce amyloid-beta levels in transgenic mice, prevents N processing, translocation, and signaling in cell culture. This compound also induces developmental defects in Drosophila remarkably similar to those caused by genetic reduction of N. The appearance of this phenocopy depends on the timing and dose of compound exposure, and effects on N-dependent signaling molecules established its biochemical mechanism of action in vivo. Other gamma-secretase inhibitors caused similar effects. Thus, the three-dimensional structure of the drug-binding site(s) in Drosophila gamma-secretase is remarkably conserved vis-à-vis the same site(s) in the mammalian enzyme. These results show that genetics and developmental biology can help elucidate the in vivo site of action of pharmacological agents and suggest that organisms such as Drosophila may be used as simple models for in vivo prescreening of drug candidates.
%B FASEB J %V 17 %P 79-81 %8 2003 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/12424225?dopt=Abstract %R 10.1096/fj.02-0394fje %0 Journal Article %J Nat Cell Biol %D 2003 %T Integrated activity of PDZ protein complexes regulates epithelial polarity. %A Bilder, David %A Schober, Markus %A Perrimon, Norbert %K Alleles %K Animals %K Binding Sites %K Carrier Proteins %K Cell Polarity %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Epithelial Cells %K Guanylate Kinase %K Intracellular Signaling Peptides and Proteins %K Membrane Proteins %K Membrane Transport Proteins %K Nucleoside-Phosphate Kinase %XPolarized cells contain numerous membrane domains, but it is unclear how the formation of these domains is coordinated to create a single integrated cell architecture. Genetic screens of Drosophila melanogaster embryos have identified three complexes, each containing one of the PDZ domain proteins--Stardust (Sdt), Bazooka (Baz) and Scribble (Scrib)--that control epithelial polarity and formation of zonula adherens. We find that these complexes can be ordered into a single regulatory hierarchy that is initiated by cell adhesion-dependent recruitment of the Baz complex to the zonula adherens. The Scrib complex represses apical identity along basolateral surfaces by antagonizing Baz-initiated apical polarity. The Sdt-containing Crb complex is recruited apically by the Baz complex to counter antagonistic Scrib activity. Thus, a finely tuned balance between Scrib and Crb complex activity sets the limits of the apical and basolateral membrane domains and positions cell junctions. Our data suggest a model in which the maturation of epithelial cell polarity is driven by integration of the sequential activities of PDZ-based protein complexes.
%B Nat Cell Biol %V 5 %P 53-8 %8 2003 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/12510194?dopt=Abstract %R 10.1038/ncb897 %0 Journal Article %J Development %D 2003 %T Mechanism of inhibition of the Drosophila and mammalian EGF receptors by the transmembrane protein Kekkon 1. %A Ghiglione, Christian %A Amundadottir, Laufey %A Andresdottir, Margret %A Bilder, David %A Diamonti, John A %A Noselli, Stéphane %A Perrimon, Norbert %A Carraway III, Kermit L %K Animals %K Animals, Genetically Modified %K Cell Line %K Drosophila melanogaster %K Drosophila Proteins %K Embryonic Structures %K Epidermal Growth Factor %K Feedback, Physiological %K Female %K Humans %K Membrane Proteins %K Mice %K Photoreceptor Cells, Invertebrate %K Protein Binding %K Protein Structure, Tertiary %K Protein Tyrosine Phosphatases %K Receptor, Epidermal Growth Factor %K Signal Transduction %K Wings, Animal %XThe transmembrane protein Kekkon 1 (Kek1) has previously been shown to act in a negative feedback loop to downregulate the Drosophila Epidermal Growth Factor Receptor (DER) during oogenesis. We show that this protein plays a similar role in other DER-mediated developmental processes. Structure-function analysis reveals that the extracellular Leucine-Rich Repeat (LRR) domains of Kek1 are critical for its function through direct association with DER, whereas its cytoplasmic domain is required for apical subcellular localization. In addition, the use of chimeric proteins between Kek1 extracellular and transmembrane domains fused to DER intracellular domain indicates that Kek1 forms an heterodimer with DER in vivo. To characterize more precisely the mechanism underlying the Kek1/DER interaction, we used mammalian ErbB/EGFR cell-based assays. We show that Kek1 is capable of physically interacting with each of the known members of the mammalian ErbB receptor family and that the Kek1/EGFR interaction inhibits growth factor binding, receptor autophosphorylation and Erk1/2 activation in response to EGF. Finally, in vivo experiments show that Kek1 expression potently suppresses the growth of mouse mammary tumor cells derived from aberrant ErbB receptors activation, but does not interfere with the growth of tumor cells derived from activated Ras. Our results underscore the possibility that Kek1 may be used experimentally to inhibit ErbB receptors and point to the possibility that, as yet uncharacterized, mammalian transmembrane LRR proteins might act as modulators of growth factor signalling.
%B Development %V 130 %P 4483-93 %8 2003 Sep %G eng %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/12900463?dopt=Abstract %0 Journal Article %J Development %D 2003 %T neurotic, a novel maternal neurogenic gene, encodes an O-fucosyltransferase that is essential for Notch-Delta interactions. %A Sasamura, Takeshi %A Sasaki, Nobuo %A Miyashita, Fumiyasu %A Nakao, Shiho %A Ishikawa, Hiroyuki O %A Ito, Mikiko %A Kitagawa, Motoo %A Harigaya, Kenichi %A Spana, Eric %A Bilder, David %A Perrimon, Norbert %A Matsuno, Kenji %K Animals %K Drosophila %K Drosophila Proteins %K Fucosyltransferases %K Genes, Reporter %K Intracellular Signaling Peptides and Proteins %K Membrane Proteins %K Molecular Sequence Data %K Receptors, Notch %XNotch signalling, which is highly conserved from nematodes to mammals, plays crucial roles in many developmental processes. In the Drosophila embryo, deficiency in Notch signalling results in neural hyperplasia, commonly referred to as the neurogenic phenotype. We identify a novel maternal neurogenic gene, neurotic, and show that it is essential for Notch signalling. neurotic encodes a Drosophila homolog of mammalian GDP-fucose protein O-fucosyltransferase, which adds fucose sugar to epidermal growth factor-like repeats and is known to play a crucial role in Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. Further, we show that neurotic is required for Fringe activity, which encodes a fucose-specific beta1, 3 N-acetylglucosaminyltransferase, previously shown to modulate Notch receptor activity. Finally, Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. We present an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. Our results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions.
%B Development %V 130 %P 4785-95 %8 2003 Oct %G eng %N 20 %1 http://www.ncbi.nlm.nih.gov/pubmed/12917292?dopt=Abstract %R 10.1242/dev.00679 %0 Journal Article %J Development %D 2003 %T The retinoic-like juvenile hormone controls the looping of left-right asymmetric organs in Drosophila. %A Adám, Géza %A Perrimon, Norbert %A Noselli, Stéphane %K Alleles %K Animals %K Body Patterning %K Cell Adhesion Molecules, Neuronal %K Corpora Allata %K Drosophila %K Drosophila Proteins %K Female %K Genes, Insect %K Genitalia, Male %K Juvenile Hormones %K Male %K Microscopy, Electron, Scanning %K Models, Biological %K Mutation %K Pyridines %K Synapses %K Tretinoin %XIn vertebrate development, the establishment of left-right asymmetry is essential for sidedness and the directional looping of organs like the heart. Both the nodal pathway and retinoic acid play major and conserved regulatory roles in these processes. We carried out a novel screen in Drosophila to identify mutants that specifically affect the looping of left-right asymmetric organs. We report the isolation of spin, a novel mutant in which the looping of the genitalia and spermiduct are incomplete; under-rotation of the genitalia indicates that spin controls looping morphogenesis but not direction, thus uncoupling left-right asymmetry and looping morphogenesis. spin is a novel, rotation-specific allele of the fasciclin2 (Fas2) gene, which encodes a cell-adhesion protein involved in several aspects of neurogenesis. In spin mutants, the synapses connecting specific neurosecretory cells to the corpora allata are affected. The corpus allatum is part of the ring gland and is involved in the control of juvenile hormone titers during development. Our genetic and pharmacological results indicate that Fas2(spin) rotation defects are linked to an abnormal endocrine function and an elevated level of juvenile hormone. As juvenile hormone is an insect sesquiterpenoid related to retinoic acid, these results establish a new genetic model for studying organ looping and demonstrate an evolutionarily conserved role for terpenoids in this process.
%B Development %V 130 %P 2397-406 %8 2003 Jun %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/12702654?dopt=Abstract %0 Journal Article %J Development %D 2003 %T Roles of myosin phosphatase during Drosophila development. %A Tan, Change %A Stronach, Beth %A Perrimon, Norbert %K Animals %K Body Patterning %K Cloning, Molecular %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Eye %K Female %K Gene Expression Regulation, Developmental %K Membrane Proteins %K Mice %K Mutation %K Myosin Heavy Chains %K Myosin Type II %K Myosin-Light-Chain Phosphatase %K Oogenesis %K Ovary %K Phosphoprotein Phosphatases %K Phosphorylation %K Protein Subunits %K rac GTP-Binding Proteins %K rhoA GTP-Binding Protein %K Signal Transduction %XMyosins are a superfamily of actin-dependent molecular motor proteins, among which the bipolar filament forming myosins II have been the most studied. The activity of smooth muscle/non-muscle myosin II is regulated by phosphorylation of the regulatory light chains, that in turn is modulated by the antagonistic activity of myosin light chain kinase and myosin light chain phosphatase. The phosphatase activity is mainly regulated through phosphorylation of its myosin binding subunit MYPT. To identify the function of these phosphorylation events, we have molecularly characterized the Drosophila homologue of MYPT, and analyzed its mutant phenotypes. We find that Drosophila MYPT is required for cell sheet movement during dorsal closure, morphogenesis of the eye, and ring canal growth during oogenesis. Our results indicate that the regulation of the phosphorylation of myosin regulatory light chains, or dynamic activation and inactivation of myosin II, is essential for its various functions during many developmental processes.
%B Development %V 130 %P 671-81 %8 2003 Feb %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/12505998?dopt=Abstract %0 Journal Article %J Dev Cell %D 2003 %T Signaling role of hemocytes in Drosophila JAK/STAT-dependent response to septic injury. %A Agaisse, Hervé %A Petersen, Ulla Maja %A Boutros, Michael %A Mathey-Prevot, Bernard %A Perrimon, Norbert %K Adipocytes %K Animals %K Blotting, Northern %K Bridged Compounds %K Cloning, Molecular %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Fat Body %K Female %K Gene Expression Regulation, Developmental %K Hemocytes %K Immunohistochemistry %K In Situ Hybridization %K Infection %K Insect Proteins %K Male %K Membrane Proteins %K Protein-Tyrosine Kinases %K Receptors, Interleukin %K Reverse Transcriptase Polymerase Chain Reaction %K RNA, Messenger %K Sequence Analysis, Protein %K Signal Transduction %K Time Factors %K Trans-Activators %K Transcription Factors %K Transgenes %XTo characterize the features of JAK/STAT signaling in Drosophila immune response, we have identified totA as a gene that is regulated by the JAK/STAT pathway in response to septic injury. We show that septic injury triggers the hemocyte-specific expression of upd3, a gene encoding a novel Upd-like cytokine that is necessary for the JAK/STAT-dependent activation of totA in the Drosophila counterpart of the mammalian liver, the fat body. In addition, we demonstrate that totA activation also requires the NF-KB-like Relish pathway, indicating that fat body cells integrate the activity of NF-KB and JAK/STAT signaling pathways upon immune response. This study reveals that, in addition to the pattern recognition receptor-mediated NF-KB-dependent immune response, Drosophila undergoes a complex systemic response that is mediated by the production of cytokines in blood cells, a process that is similar to the acute phase response in mammals.
%B Dev Cell %V 5 %P 441-50 %8 2003 Sep %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/12967563?dopt=Abstract %0 Book Section %B Gastrulation: From cells to embryos %D 2003 %T Carbohydrate and Lipid Modifications of Developmental Signaling Molecules %A Nybakken, Kent %A Perrimon, Norbert %E Stern, C %B Gastrulation: From cells to embryos %I CSHL Press %P 667-676 %G eng %0 Book Section %B Handbook of Cell Signaling %D 2003 %T Developmental signaling: JNK pathway in Drosophila morphogenesis %A Stronach, Beth E. %A Perrimon, Norbert %E Bradshaw, Ralph A %E Dennis, Edward %B Handbook of Cell Signaling %I Academic Press %C San Diego, CA %V 2 %P 783-787 %G eng %0 Book Section %B Signal Transducers and Activators of Transcription (STATs): Activation and Biology %D 2003 %T Prime time for the Drosophila JAK/STAT pathway %A Bach, Erika A %A Perrimon, Norbert %E Segha, PB %E Levy, DE %E Hirano, T %B Signal Transducers and Activators of Transcription (STATs): Activation and Biology %I Kluwer Academic Publishers %P 87-104 %G eng %0 Journal Article %J J Biol %D 2003 %T A functional genomic analysis of cell morphology using RNA interference. %A Kiger, A A %A Baum, B %A Jones, S %A Jones, M R %A Coulson, A %A Echeverri, C %A Perrimon, N %K Animals %K Cell Line %K Cell Shape %K Cytoskeleton %K Drosophila %K Drosophila Proteins %K Genes, Insect %K Genome %K Genomics %K Microscopy, Fluorescence %K Mutation %K Phenotype %K Phosphoric Monoester Hydrolases %K PTEN Phosphohydrolase %K RNA Interference %K RNA, Double-Stranded %XBACKGROUND: The diversity of metazoan cell shapes is influenced by the dynamic cytoskeletal network. With the advent of RNA-interference (RNAi) technology, it is now possible to screen systematically for genes controlling specific cell-biological processes, including those required to generate distinct morphologies. RESULTS: We adapted existing RNAi technology in Drosophila cell culture for use in high-throughput screens to enable a comprehensive genetic dissection of cell morphogenesis. To identify genes responsible for the characteristic shape of two morphologically distinct cell lines, we performed RNAi screens in each line with a set of double-stranded RNAs (dsRNAs) targeting 994 predicted cell shape regulators. Using automated fluorescence microscopy to visualize actin filaments, microtubules and DNA, we detected morphological phenotypes for 160 genes, one-third of which have not been previously characterized in vivo. Genes with similar phenotypes corresponded to known components of pathways controlling cytoskeletal organization and cell shape, leading us to propose similar functions for previously uncharacterized genes. Furthermore, we were able to uncover genes acting within a specific pathway using a co-RNAi screen to identify dsRNA suppressors of a cell shape change induced by Pten dsRNA. CONCLUSIONS: Using RNAi, we identified genes that influence cytoskeletal organization and morphology in two distinct cell types. Some genes exhibited similar RNAi phenotypes in both cell types, while others appeared to have cell-type-specific functions, in part reflecting the different mechanisms used to generate a round or a flat cell morphology.
%B J Biol %V 2 %P 27 %8 2003 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/14527345?dopt=Abstract %R 10.1186/1475-4924-2-27 %0 Journal Article %J Genes Dev %D 2003 %T Retraction of the Drosophila germ band requires cell-matrix interaction. %A Schöck, Frieder %A Perrimon, Norbert %K Animals %K Body Patterning %K Cell Movement %K Cell-Matrix Junctions %K Drosophila %K Integrins %K Laminin %XIntegrins and laminins are important mediators of cell-matrix interactions in both vertebrates and invertebrates. Here, we show that germ-band retraction in the Drosophila embryo, during which the tail end of the embryo retracts to its final posterior position, allows the investigation of cell spreading and lamellipodia formation in real time in vivo. We demonstrate that alpha1, 2 laminin and alphaPS3betaPS integrin are required for the spreading of a small group of cells of the amnioserosa epithelium over the tail end of the germ band. We further implicate a role for this spreading in the process of germ-band retraction.
%B Genes Dev %V 17 %P 597-602 %8 2003 Mar 1 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/12629042?dopt=Abstract %R 10.1101/gad.1068403 %0 Journal Article %J Genetics %D 2003 %T A sensitized genetic screen to identify novel regulators and components of the Drosophila janus kinase/signal transducer and activator of transcription pathway. %A Bach, Erika A %A Vincent, Stephane %A Zeidler, Martin P %A Perrimon, Norbert %K Animals %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Flow Cytometry %K Microscopy, Electron, Scanning %K Polymerase Chain Reaction %K Protein-Tyrosine Kinases %K STAT Transcription Factors %K Trans-Activators %XThe JAK/STAT pathway exerts pleiotropic effects on a wide range of developmental processes in Drosophila. Four key components have been identified: Unpaired, a secreted ligand; Domeless, a cytokine-like receptor; Hopscotch, a JAK kinase; and Stat92E, a STAT transcription factor. The identification of additional components and regulators of this pathway remains an important issue. To this end, we have generated a transgenic line where we misexpress the upd ligand in the developing Drosophila eye. GMR-upd transgenic animals have dramatically enlarged eye-imaginal discs and compound eyes that are normally patterned. We demonstrate that the enlarged-eye phenotype is a result of an increase in cell number, and not cell volume, and arises from additional mitoses in larval eye discs. Thus, the GMR-upd line represents a system in which the proliferation and differentiation of eye precursor cells are separable. Removal of one copy of stat92E substantially reduces the enlarged-eye phenotype. We performed an F1 deficiency screen to identify dominant modifiers of the GMR-upd phenotype. We have identified 9 regions that enhance this eye phenotype and two specific enhancers: C-terminal binding protein and Daughters against dpp. We also identified 20 regions that suppress GMR-upd and 13 specific suppressors: zeste-white 13, pineapple eye, Dichaete, histone 2A variant, headcase, plexus, kohtalo, crumbs, hedgehog, decapentaplegic, thickveins, saxophone, and Mothers against dpp.
%B Genetics %V 165 %P 1149-66 %8 2003 Nov %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/14668372?dopt=Abstract %0 Journal Article %J EMBO J %D 2003 %T Slalom encodes an adenosine 3'-phosphate 5'-phosphosulfate transporter essential for development in Drosophila. %A Lüders, Florian %A Segawa, Hiroaki %A Stein, David %A Selva, Erica M %A Perrimon, Norbert %A Turco, Salvatore J %A Häcker, Udo %K Amino Acid Sequence %K Animals %K Biological Transport %K Body Patterning %K Cytosol %K Drosophila %K Drosophila Proteins %K Female %K Gene Expression %K Genes, Insect %K Golgi Apparatus %K Membrane Proteins %K Membrane Transport Proteins %K Molecular Sequence Data %K Mutation %K Ovary %K Phosphoadenosine Phosphosulfate %K Proteoglycans %K Sequence Homology, Amino Acid %K Signal Transduction %K Sulfates %K Wings, Animal %XSulfation of all macromolecules entering the secretory pathway in higher organisms occurs in the Golgi and requires the high-energy sulfate donor adenosine 3'-phosphate 5'-phosphosulfate. Here we report the first molecular identification of a gene that encodes a transmembrane protein required to transport adenosine 3'-phosphate 5'-phosphosulfate from the cytosol into the Golgi lumen. Mutations in this gene, which we call slalom, display defects in Wg and Hh signaling, which are likely due to the lack of sulfation of glycosaminoglycans by the sulfotransferase sulfateless. Analysis of mosaic mutant ovaries shows that sll function is also essential for dorsal-ventral axis determination, suggesting that sll transports the sulfate donor required for sulfotransferase activity of the dorsal-ventral determinant pipe.
%B EMBO J %V 22 %P 3635-44 %8 2003 Jul 15 %G eng %N 14 %1 http://www.ncbi.nlm.nih.gov/pubmed/12853478?dopt=Abstract %R 10.1093/emboj/cdg345 %0 Journal Article %J Glycoconj J %D 2002 %T Developmental roles of heparan sulfate proteoglycans in Drosophila. %A Lin, Xinhua %A Perrimon, Norbert %K Animals %K Body Patterning %K Drosophila %K Drosophila Proteins %K Evolution, Molecular %K Genes, Insect %K Hedgehog Proteins %K Heparan Sulfate Proteoglycans %K Models, Biological %K Mutation %K Proto-Oncogene Proteins %K Signal Transduction %K Wnt Proteins %K Zebrafish Proteins %XThe formation of complex patterns in multi-cellular organisms is regulated by a number of signaling pathways. In particular, the Wnt and Hedgehog (Hh) pathways have been identified as critical organizers of pattern in many tissues. Although extensive biochemical and genetic studies have elucidated the central components of the signal transduction pathways regulated by these secreted molecules, we still do not understand fully how they organize gradients of gene activities through field of cells. Studies in Drosophila have implicated a role for heparan sulfate proteoglycans (HSPGs) in regulating the signaling activities and distribution of both Wnt and Hh. Here we review these findings and discuss various models by which HSPGs regulate the distributions of Wnt and Hh morphogens.
%B Glycoconj J %V 19 %P 363-8 %8 2002 May-Jun %G eng %N 4-5 %1 http://www.ncbi.nlm.nih.gov/pubmed/12975617?dopt=Abstract %R 10.1023/A:1025329323438 %0 Journal Article %J Curr Opin Genet Dev %D 2002 %T Hedgehog signal transduction: recent findings. %A Nybakken, Kent %A Perrimon, Norbert %K Animals %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Hedgehog Proteins %K Kinesin %K Phosphorylation %K Protein-Serine-Threonine Kinases %K Signal Transduction %K Transcription Factors %K Zinc Fingers %XThe Hedgehog (Hh) family of signaling molecules are key agents in patterning numerous types of tissues. Mutations in Hh and its downstream signaling molecules are also associated with numerous oncogenic and disease states. Consequently, understanding the mechanisms by which Hh signals are transduced is important for understanding both development and disease. Recent studies have clarified several aspects of Hh signal transduction. Several new Sonic Hedgehog binding partners have been identified. Cholesterol and palmitic acid modifications of Hh and Sonic hedgehog have been examined in greater detail. Characterization of the trafficking patterns of the Patched and Smoothened proteins has demonstrated that these two proteins function very differently from the previously established models. The Fused kinase has been demonstrated to phosphorylate the kinesin-like protein Costal2 and the sites identified, while Cubitus interruptus has been shown to be phosphorylated in a hierarchical manner by three different kinases. Finally, the interactions, both genetic and physical, between Fused, Costal2, Cubitus interruptus, and Suppressor of Fused have been further elucidated.
%B Curr Opin Genet Dev %V 12 %P 503-11 %8 2002 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/12200154?dopt=Abstract %0 Journal Article %J Biochim Biophys Acta %D 2002 %T Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila. %A Nybakken, Kent %A Perrimon, Norbert %K Amidohydrolases %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Fibroblast Growth Factor 1 %K Hedgehog Proteins %K Heparan Sulfate Proteoglycans %K N-Acetylglucosaminyltransferases %K Receptor Protein-Tyrosine Kinases %K Receptor, Fibroblast Growth Factor, Type 2 %K Receptors, Fibroblast Growth Factor %K Signal Transduction %K Sulfotransferases %K Uridine Diphosphate Glucose Dehydrogenase %XHeparan sulphate proteoglycans (HSPG's) are cell surface proteins to which long, unbranched chains of modified sugars called heparan sulphate glycosaminoglycans have been covalently attached. Cell culture studies have demonstrated that HSPG's are required for optimal signal transduction by many secreted cell signaling molecules. Now, genetic studies in both Drosophila and vertebrates have illustrated that HSPG's play important roles in signal transduction in vivo and have also begun to reveal new roles for HSPG's in signaling events. In particular, HSPG's have been shown to be important in ligand sequestration of wingless, for the transport of the Hedgehog ligand, and for modulation of the Dpp morphogenetic gradient.
%B Biochim Biophys Acta %V 1573 %P 280-91 %8 2002 Dec 19 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/12417410?dopt=Abstract %0 Journal Article %J Dev Cell %D 2002 %T The Jak/STAT pathway in model organisms: emerging roles in cell movement. %A Hou, Steven X %A Zheng, Zhiyu %A Chen, Xiu %A Perrimon, Norbert %K Animals %K Cell Division %K Cell Lineage %K Cell Movement %K Cell Polarity %K DNA-Binding Proteins %K Eukaryotic Cells %K Hematopoiesis %K Humans %K Janus Kinase 1 %K Models, Animal %K Protein-Tyrosine Kinases %K Signal Transduction %K STAT1 Transcription Factor %K Trans-Activators %K Zebrafish Proteins %XThe JAK/STAT pathway was originally identified in mammals. Studies of this pathway in the mouse have revealed that JAK/STAT signaling plays a central role during hematopoeisis and other developmental processes. The role of JAK/STAT signaling in blood appears to be conserved throughout evolution, as it is also required during fly hematopoeisis. Studies in Dictyostelium, Drosophila, and zebrafish have shown that the JAK/STAT pathway is also required in an unusually broad set of developmental decisions, including cell proliferation, cell fate determination, cell migration, planar polarity, convergent extension, and immunity. There is increasing evidence that the versatility of this pathway relies on its cooperation with other signal transduction pathways. In this review, we discuss the components of the JAK/STAT pathway in model organisms and what is known about its requirement in cellular and developmental processes. In particular, we emphasize recent insights into the role that this pathway plays in the control of cell movement.
%B Dev Cell %V 3 %P 765-78 %8 2002 Dec %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/12479803?dopt=Abstract %0 Journal Article %J Annu Rev Cell Dev Biol %D 2002 %T Molecular mechanisms of epithelial morphogenesis. %A Schock, Frieder %A Perrimon, Norbert %K Animals %K Body Patterning %K Cell Adhesion %K Cell Communication %K Cell Differentiation %K Cytoskeleton %K Epithelial Cells %K Epithelium %K Extracellular Matrix %K Humans %XEpithelial morphogenesis comprises the various processes by which epithelia contribute to organ formation and body shape. These complex and diverse events play a central role in animal development and regeneration. Recently, the characterization of some of the molecular mechanisms involved in epithelial morphogenesis has provided an abundance of new information on the role and regulation of the cytoskeleton, cell-cell adhesion, and cell-matrix adhesion in these processes. In this review, we discuss our current understanding of the molecular mechanisms driving cell shape changes, cell intercalation, fusion of epithelia, ingression, egression, and cell migration. Our discussion is mostly focused on results from Drosophila and mammalian tissue culture but also draws on the insights gained from other organisms.
%B Annu Rev Cell Dev Biol %V 18 %P 463-93 %8 2002 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/12142280?dopt=Abstract %R 10.1146/annurev.cellbio.18.022602.131838 %0 Journal Article %J Science %D 2002 %T The promise and perils of Wnt signaling through beta-catenin. %A Moon, Randall T %A Bowerman, Bruce %A Boutros, Michael %A Perrimon, Norbert %K Animals %K beta Catenin %K Cell Nucleus %K Cell Polarity %K Cytoskeletal Proteins %K Cytoskeleton %K Drosophila Proteins %K Embryonic and Fetal Development %K Embryonic Development %K Gene Expression Regulation %K Humans %K Models, Biological %K Neoplasms %K Proto-Oncogene Proteins %K Signal Transduction %K Trans-Activators %K Transcription, Genetic %K Wnt Proteins %K Wnt1 Protein %K Zebrafish Proteins %XWnt pathways are involved in the control of gene expression, cell behavior, cell adhesion, and cell polarity. In addition, they often operate in combination with other signaling pathways. The Wnt/beta-catenin pathway is the best studied of the Wnt pathways and is highly conserved through evolution. In this pathway, Wnt signaling inhibits the degradation of beta-catenin, which can regulate transcription of a number of genes. Some of the genes regulated are those associated with cancer and other diseases (for example, colorectal cancer and melanomas). As a result, components of the Wnt/beta-catenin pathway are promising targets in the search for therapeutic agents. Information about Wnt pathways is available both in canonical terms and at the species level. In addition to the canonical Wnt/beta-catenin pathway, information is now available for Drosophila, Caenorhabditis elegans, and Xenopus. The STKE Connections Maps for these pathways provide an important tool in accessing this large body of complex information.
%B Science %V 296 %P 1644-6 %8 2002 May 31 %G eng %N 5573 %1 http://www.ncbi.nlm.nih.gov/pubmed/12040179?dopt=Abstract %R 10.1126/science.1071549 %0 Journal Article %J Nat Cell Biol %D 2002 %T Unconventional ways to travel. %A Schober, Markus %A Perrimon, Norbert %K Animals %K Cadherins %K Cell Movement %K Drosophila melanogaster %K Female %K Myosin Heavy Chains %K Ovary %B Nat Cell Biol %V 4 %P E211-2 %8 2002 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/12205482?dopt=Abstract %R 10.1038/ncb0902-e211 %0 Journal Article %J Genesis %D 2002 %T Analysis of twenty-four Gal4 lines in Drosophila melanogaster. %A Hrdlicka, Lori %A Gibson, Matthew %A Kiger, Amy %A Micchelli, Craig %A Schober, Markus %A Schöck, Frieder %A Perrimon, Norbert %K Animals %K Animals, Genetically Modified %K Chromosome Mapping %K DNA Transposable Elements %K DNA-Binding Proteins %K Drosophila melanogaster %K Enhancer Elements, Genetic %K Female %K Larva %K Male %K Organ Specificity %K Ovary %K Saccharomyces cerevisiae Proteins %K Testis %K Transcription Factors %B Genesis %V 34 %P 51-7 %8 2002 Sep-Oct %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/12324947?dopt=Abstract %R 10.1002/gene.10125 %0 Journal Article %J Dev Biol %D 2002 %T Cellular processes associated with germ band retraction in Drosophila. %A Schöck, Frieder %A Perrimon, Norbert %K Actomyosin %K Animals %K Cell Adhesion %K Cell Movement %K Drosophila %K Embryo, Nonmammalian %K Epithelium %K Gene Expression Regulation, Developmental %K Genes, Dominant %K Green Fluorescent Proteins %K Luminescent Proteins %K Phenotype %K Pseudopodia %K Time Factors %XLarge-scale movements of epithelial sheets are necessary for most embryonic and regenerative morphogenetic events. We have characterized the cellular processes associated with germ band retraction (GBR) in the Drosophila embryo. During GBR, the caudal end of the embryo retracts to its final posterior position. We show using time-lapse recordings that, in contrast to germ band extension, cells within the lateral germ band do not intercalate. In addition, the germ band and amnioserosa move as one coherent sheet, and the amnioserosa strongly shortens along its dorsal-ventral axis. Furthermore, during GBR, the amnioserosa adheres to and migrates over the caudal end of the germ band via lamellipodia. Expression of both dominant-negative and constitutively active RhoA in the amnioserosa disrupts GBR. As RhoA acts on both actomyosin contractility and cell-matrix adhesion, it suggests a role for such processes in the amnioserosa during GBR. The results establish the cellular movements and shape changes occurring during GBR and provide the basis for an analysis of the forces acting during GBR.
%B Dev Biol %V 248 %P 29-39 %8 2002 Aug 1 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/12142018?dopt=Abstract %0 Journal Article %J Neuron %D 2002 %T A Drosophila homolog of cyclase-associated proteins collaborates with the Abl tyrosine kinase to control midline axon pathfinding. %A Wills, Zachary %A Emerson, Mark %A Rusch, Jannette %A Bikoff, Jay %A Baum, Buzz %A Perrimon, Norbert %A Van Vactor, David %K Animals %K Cell Communication %K Cell Cycle Proteins %K Cell Differentiation %K Cells, Cultured %K Chemotaxis %K Cytoskeletal Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Gene Expression Regulation, Developmental %K Growth Cones %K Immunohistochemistry %K Male %K Microfilament Proteins %K Microscopy, Electron, Scanning %K Mutation %K Nerve Tissue Proteins %K Nervous System %K Protein-Tyrosine Kinases %K Receptors, Immunologic %XWe demonstrate that Drosophila capulet (capt), a homolog of the adenylyl cyclase-associated protein that binds and regulates actin in yeast, associates with Abl in Drosophila cells, suggesting a functional relationship in vivo. We find a robust and specific genetic interaction between capt and Abl at the midline choice point where the growth cone repellent Slit functions to restrict axon crossing. Genetic interactions between capt and slit support a model where Capt and Abl collaborate as part of the repellent response. Further support for this model is provided by genetic interactions that both capt and Abl display with multiple members of the Roundabout receptor family. These studies identify Capulet as part of an emerging pathway linking guidance signals to regulation of cytoskeletal dynamics and suggest that the Abl pathway mediates signals downstream of multiple Roundabout receptors.
%B Neuron %V 36 %P 611-22 %8 2002 Nov 14 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/12441051?dopt=Abstract %0 Journal Article %J Development %D 2002 %T Mechanism of activation of the Drosophila EGF Receptor by the TGFalpha ligand Gurken during oogenesis. %A Ghiglione, Christian %A Bach, Erika A %A Paraiso, Yolande %A Carraway, Kermit L %A Noselli, Stéphane %A Perrimon, Norbert %K Animals %K Drosophila %K Drosophila Proteins %K Female %K Insect Proteins %K Ligands %K Oogenesis %K Receptor, Epidermal Growth Factor %K Signal Transduction %K Transforming Growth Factor alpha %K Transforming Growth Factors %XWe have analyzed the mechanism of activation of the Epidermal growth factor receptor (Egfr) by the transforming growth factor (TGF) alpha-like molecule, Gurken (Grk). Grk is expressed in the oocyte and activates the Egfr in the surrounding follicle cells during oogenesis. We show that expression of either a membrane bound form of Grk (mbGrk), or a secreted form of Grk (secGrk), in either the follicle cells or in the germline, activates the Egfr. In tissue culture cells, both forms can bind to the Egfr; however, only the soluble form can trigger Egfr signaling, which is consistent with the observed cleavage of Grk in vivo. We find that the two transmembrane proteins Star and Brho potentiate the activity of mbGrk. These two proteins collaborate to promote an activating proteolytic cleavage and release of Grk. After cleavage, the extracellular domain of Grk is secreted from the oocyte to activate the Egfr in the follicular epithelium.
%B Development %V 129 %P 175-86 %8 2002 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/11782411?dopt=Abstract %0 Journal Article %J Development %D 2002 %T Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. %A Micchelli, Craig A %A The, Inge %A Selva, Erica %A Mogila, Vladic %A Perrimon, Norbert %K Acyltransferases %K Amino Acid Sequence %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Hedgehog Proteins %K Membrane Proteins %K Molecular Sequence Data %K Phenotype %K Proto-Oncogene Proteins %K Signal Transduction %K Transcriptional Activation %K Wnt1 Protein %XMembers of the Hedgehog (Hh) family encode secreted molecules that act as potent organizers during vertebrate and invertebrate development. Post-translational modification regulates both the range and efficacy of Hh protein. One such modification is the acylation of the N-terminal cysteine of Hh. In a screen for zygotic lethal mutations associated with maternal effects, we have identified rasp, a novel Drosophila segment polarity gene. Analysis of the rasp mutant phenotype, in both the embryo and wing imaginal disc demonstrates that rasp does not disrupt Wnt/Wingless signaling but is specifically required for Hh signaling. The requirement of rasp is restricted only to those cells that produce Hh; hh transcription, protein levels and distribution are not affected by the loss of rasp. Molecular analysis reveals that rasp encodes a multipass transmembrane protein that has homology to a family of membrane bound O-acyl transferases. Our results suggest that Rasp-dependent acylation is necessary to generate a fully active Hh protein.
%B Development %V 129 %P 843-51 %8 2002 Feb %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/11861468?dopt=Abstract %0 Journal Article %J Dev Cell %D 2002 %T Sequential activation of signaling pathways during innate immune responses in Drosophila. %A Boutros, Michael %A Agaisse, Hervé %A Perrimon, Norbert %K Animals %K Cell Line %K Drosophila melanogaster %K Gene Expression Profiling %K Genes, Insect %K Immunity, Innate %K JNK Mitogen-Activated Protein Kinases %K Lipopolysaccharides %K Mitogen-Activated Protein Kinases %K Receptors, Steroid %K Receptors, Thyroid Hormone %K Signal Transduction %K Transcription, Genetic %XInnate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. We found that these transcriptional patterns can be dissected into distinct groups. We demonstrate that, in addition to signaling through the Toll and Imd pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, our results may imply a close link between the control of tissue repair and antimicrobial processes.
%B Dev Cell %V 3 %P 711-22 %8 2002 Nov %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/12431377?dopt=Abstract %0 Journal Article %J Genes Dev %D 2002 %T Activation of the JNK pathway during dorsal closure in Drosophila requires the mixed lineage kinase, slipper. %A Stronach, Beth %A Perrimon, Norbert %K Amino Acid Sequence %K Animals %K Cell Polarity %K Cloning, Molecular %K Drosophila %K Drosophila Proteins %K Female %K Gene Expression Regulation, Developmental %K Humans %K JNK Mitogen-Activated Protein Kinases %K Male %K MAP Kinase Kinase 4 %K MAP Kinase Kinase 7 %K MAP Kinase Kinase Kinases %K Mitogen-Activated Protein Kinase Kinases %K Molecular Sequence Data %K Morphogenesis %K Mutation %K Sequence Homology, Amino Acid %K Signal Transduction %XThe Jun kinase (JNK) pathway has been characterized for its role in stimulating AP-1 activity and for modulating the balance between cell growth and death during development, inflammation, and cancer. Six families of mammalian kinases acting at the level of JNKKK have emerged as upstream regulators of JNK activity (MLK, LZK, TAK, ASK, MEKK, and TPL); however, the specificity underlying which kinase is utilized for transducing a distinct signal is poorly understood. In Drosophila, JNK signaling plays a central role in dorsal closure, controlling cell fate and cell sheet morphogenesis during embryogenesis. Notably, in the fly genome, there are single homologs of each of the mammalian JNKKK families. Here, we identify mutations in one of those, a mixed lineage kinase, named slipper (slpr), and show that it is required for JNK activation during dorsal closure. Furthermore, our results show that other putative JNKKKs cannot compensate for the loss of slpr function and, thus, may regulate other JNK or MAPK-dependent processes.
%B Genes Dev %V 16 %P 377-87 %8 2002 Feb 1 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/11825878?dopt=Abstract %R 10.1101/gad.953002 %0 Journal Article %J Mol Cell Biol %D 2002 %T CKA, a novel multidomain protein, regulates the JUN N-terminal kinase signal transduction pathway in Drosophila. %A Chen, Hua-Wei %A Marinissen, Maria Julia %A Oh, Su-Wan %A Chen, Xiu %A Melnick, Michael %A Perrimon, Norbert %A Gutkind, J Silvio %A Hou, Steven X %K 3T3 Cells %K Adaptor Proteins, Signal Transducing %K Animals %K Carrier Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Insect Proteins %K JNK Mitogen-Activated Protein Kinases %K Macromolecular Substances %K Mice %K Mitogen-Activated Protein Kinase Kinases %K Mitogen-Activated Protein Kinases %K Molecular Sequence Data %K Phosphorylation %K Protein Structure, Tertiary %K Protein-Tyrosine Kinases %K Sequence Homology, Amino Acid %K Signal Transduction %K Transcription Factor AP-1 %K Transcription Factors %K Transcription, Genetic %K Transfection %XThe Drosophila melanogaster JUN N-terminal kinase (DJNK) and DPP (decapentaplegic) signal transduction pathways coordinately regulate epithelial cell sheet movement during the process of dorsal closure in the embryo. By a genetic screen of mutations affecting dorsal closure in Drosophila, we have now identified a multidomain protein, connector of kinase to AP-1 (cka), that functions in the DJNK pathway and controls the localized expression of dpp in the leading-edge cells. We have also investigated how CKA acts. This unique molecule forms a complex with HEP (DJNKK), BSK (DJNK), DJUN, and DFOS. Complex formation activates BSK kinase, which in turn phosphorylates and activates DJUN and DFOS. These data suggest that CKA represents a novel molecule regulating AP-1 activity by organizing a molecular complex of kinases and transcription factors, thus coordinating the spatial-temporal expression of AP-1-regulated genes.
%B Mol Cell Biol %V 22 %P 1792-803 %8 2002 Mar %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/11865058?dopt=Abstract %0 Journal Article %J Development %D 2002 %T Differential requirement for STAT by gain-of-function and wild-type receptor tyrosine kinase Torso in Drosophila. %A Li, Willis X %A Agaisse, Herve %A Mathey-Prevot, Bernard %A Perrimon, Norbert %K Animals %K Base Sequence %K Consensus Sequence %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Gene Expression Regulation, Developmental %K MAP Kinase Signaling System %K Mutation %K Plasmids %K Polymerase Chain Reaction %K Receptor Protein-Tyrosine Kinases %K STAT Transcription Factors %K Trans-Activators %XMalignant transformation frequently involves aberrant signaling from receptor tyrosine kinases (RTKs). These receptors commonly activate Ras/Raf/MEK/MAPK signaling but when overactivated can also induce the JAK/STAT pathway, originally identified as the signaling cascade downstream of cytokine receptors. Inappropriate activation of STAT has been found in many human cancers. However, the contribution of the JAK/STAT pathway in RTK signaling remains unclear. We have investigated the requirement of the JAK/STAT pathway for signaling by wild-type and mutant forms of the RTK Torso (Tor) using a genetic approach in Drosophila. Our results indicate that the JAK/STAT pathway plays little or no role in signaling by wild-type Tor. In contrast, we find that STAT, encoded by marelle (mrl; DStat92E), is essential for the gain-of-function mutant Tor (Tor(GOF)) to activate ectopic gene expression. Our findings indicate that the Ras/Raf/MEK/MAPK signaling pathway is sufficient to mediate the normal functions of wild-type RTK, whereas the effects of gain-of-function mutant RTK additionally require STAT activation.
%B Development %V 129 %P 4241-8 %8 2002 Sep %G eng %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/12183376?dopt=Abstract %0 Journal Article %J Genetics %D 2002 %T The fruitless gene is required for the proper formation of axonal tracts in the embryonic central nervous system of Drosophila. %A Song, Ho-Juhn %A Billeter, Jean-Christophe %A Reynaud, Enrique %A Carlo, Troy %A Spana, Eric P %A Perrimon, Norbert %A Goodwin, Stephen F %A Baker, Bruce S %A Taylor, Barbara J %K Animals %K Animals, Genetically Modified %K Axons %K Base Sequence %K Body Patterning %K Central Nervous System %K Chromosome Mapping %K DNA, Complementary %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Gene Expression Regulation, Developmental %K Genes, Insect %K Heterozygote %K Homozygote %K Male %K Mutation %K Nerve Tissue Proteins %K Neuroglia %K Neurons %K Peripheral Nervous System %K Phenotype %K Promoter Regions, Genetic %K RNA, Messenger %K Transcription Factors %XThe fruitless (fru) gene in Drosophila melanogaster is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less is known about its sex-nonspecific functions. We have examined fru's sex-nonspecific role in embryonic neural development. fru transcripts from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis, and Fru proteins are present in both neurons and glia. In embryos that lack most or all fru function, FasII- and BP102-positive axons have defasciculation defects and grow along abnormal pathways in the CNS. These defects in axonal projections in fru mutants were rescued by the expression of specific UAS-fru transgenes under the control of a pan-neuronal scabrous-GAL4 driver. Our results suggest that one of fru's sex-nonspecific roles is to regulate the pathfinding ability of axons in the embryonic CNS.
%B Genetics %V 162 %P 1703-24 %8 2002 Dec %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/12524343?dopt=Abstract %0 Journal Article %J Curr Biol %D 2002 %T Recruitment of scribble to the synaptic scaffolding complex requires GUK-holder, a novel DLG binding protein. %A Mathew, Dennis %A Gramates, L Sian %A Packard, Mary %A Thomas, Ulrich %A Bilder, David %A Perrimon, Norbert %A Gorczyca, Michael %A Budnik, Vivian %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Guanylate Kinase %K Insect Proteins %K Membrane Proteins %K Nucleoside-Phosphate Kinase %K Synapses %K Tumor Suppressor Proteins %XBACKGROUND: Membrane-associated guanylate kinases (MAGUKs), such as Discs-Large (DLG), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between DLG and another PDZ scaffolding protein, SCRIBBLE (SCRIB), during the establishment of cell polarity in developing epithelia. A possible interaction between DLG and SCRIB at synaptic junctions has not yet been addressed. Likewise, the biochemical nature of this interaction remains elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated. RESULTS: Here we report the isolation of a new DLG-interacting protein, GUK-holder, that interacts with the GUK domain of DLG and which is dynamically expressed during synaptic bouton budding. We also show that at Drosophila synapses DLG colocalizes with SCRIB and that this colocalization is likely to be mediated by direct interactions between GUKH and the PDZ2 domain of SCRIB. We show that DLG, GUKH, and SCRIB form a tripartite complex at synapses, in which DLG and GUKH are required for the proper synaptic localization of SCRIB. CONCLUSIONS: Our results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse.
%B Curr Biol %V 12 %P 531-9 %8 2002 Apr 2 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/11937021?dopt=Abstract %0 Journal Article %J Semin Cell Dev Biol %D 2001 %T Cellular functions of proteoglycans--an overview. %A Perrimon, N %A Bernfield, M %K Animals %K Cell Physiological Phenomena %K Extracellular Space %K Humans %K Models, Biological %K Proteoglycans %B Semin Cell Dev Biol %V 12 %P 65-7 %8 2001 Apr %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/11292371?dopt=Abstract %R 10.1006/scdb.2000.0237 %0 Journal Article %J Cell %D 2001 %T Doublesex surprises. %A Vincent, S. %A Perkins, L A %A Perrimon, N %K Animals %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Female %K Genes, Homeobox %K Genes, Insect %K Genitalia, Female %K Genitalia, Male %K Insect Proteins %K Male %K Models, Biological %K Morphogenesis %K Sex Characteristics %K Sex Determination Processes %K Signal Transduction %B Cell %V 106 %P 399-402 %8 2001 Aug 24 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/11525726?dopt=Abstract %0 Journal Article %J Nature %D 2001 %T Fishing for morphogens. %A Vincent, S. %A Perrimon, N %K Activins %K Animals %K Body Patterning %K Drosophila %K Embryo, Nonmammalian %K Gene Expression Regulation, Developmental %K Inhibins %K Intracellular Signaling Peptides and Proteins %K Morphogenesis %K Nodal Signaling Ligands %K Signal Transduction %K Transforming Growth Factor beta %K Zebrafish %K Zebrafish Proteins %B Nature %V 411 %P 533, 535-6 %8 2001 May 31 %G eng %N 6837 %1 http://www.ncbi.nlm.nih.gov/pubmed/11385549?dopt=Abstract %R 10.1038/35079214 %0 Journal Article %J Adv Cancer Res %D 2001 %T Role of heparan sulfate proteoglycans in cell signaling and cancer. %A Selva, E M %A Perrimon, N %K Animals %K Drosophila %K Heparan Sulfate Proteoglycans %K Humans %K Models, Biological %K Neoplasms %K Protein Binding %K Signal Transduction %B Adv Cancer Res %V 83 %P 67-80 %8 2001 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/11665721?dopt=Abstract %0 Journal Article %J Development %D 2001 %T The cell adhesion molecule Echinoid defines a new pathway that antagonizes the Drosophila EGF receptor signaling pathway. %A Bai, J %A Chiu, W %A Wang, J. %A Tzeng, T %A Perrimon, N %A Hsu, J %K Alleles %K Amino Acid Sequence %K Animals %K Cell Adhesion Molecules %K Cell Membrane %K Cloning, Molecular %K Drosophila %K Drosophila Proteins %K Epistasis, Genetic %K Gene Expression Regulation, Developmental %K Genes, Reporter %K Histocytochemistry %K Immunoglobulins %K Insect Proteins %K Microscopy, Electron, Scanning %K Molecular Sequence Data %K Mutation %K Nuclear Proteins %K Phenotype %K Photoreceptor Cells, Invertebrate %K Protein Structure, Tertiary %K Receptor, Epidermal Growth Factor %K Repressor Proteins %K Sequence Alignment %K Signal Transduction %K Ubiquitin-Protein Ligases %K Wings, Animal %XPhotoreceptor and cone cells in the Drosophila eye are recruited following activation of the epidermal growth factor receptor (EGFR) pathway. We have identified echinoid (ed) as a novel putative cell adhesion molecule that negatively regulates EGFR signaling. The ed mutant phenotype is associated with extra photoreceptor and cone cells. Conversely, ectopic expression of ed in the eye leads to a reduction in the number of photoreceptor cells. ed expression is independent of EGFR signaling and ED is localized to the plasma membrane of every cells throughout the eye disc. We present evidence that ed acts nonautonomously to generate extra R7 cells by a mechanism that is sina-independent but upstream of Tramtrack (TTK88). Together, our results support a model whereby ED defines an independent pathway that antagonizes EGFR signaling by regulating the activity, but not the level, of the TTK88 transcriptional repressor.
%B Development %V 128 %P 591-601 %8 2001 Feb %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/11171342?dopt=Abstract %0 Journal Article %J Dev Biol %D 2001 %T The Drosophila JNK pathway controls the morphogenesis of the egg dorsal appendages and micropyle. %A Suzanne, M %A Perrimon, N %A Noselli, S %K Animals %K Cell Movement %K Chorion %K Drosophila %K Drosophila Proteins %K Female %K Galactosides %K Gene Expression Regulation, Developmental %K Immunohistochemistry %K Indoles %K Insect Proteins %K MAP Kinase Signaling System %K Microscopy, Fluorescence %K Mitogen-Activated Protein Kinase 8 %K Mitogen-Activated Protein Kinase Kinases %K Mitogen-Activated Protein Kinases %K Ovarian Follicle %K Ovum %K Phenotype %K Signal Transduction %K Tissue Distribution %XDuring Drosophila oogenesis, the formation of the egg respiratory appendages and the micropyle require the shaping of anterior and dorsal follicle cells. Prior to their morphogenesis, cells of the presumptive appendages are determined by integrating dorsal-ventral and anterior-posterior positional information provided by the epidermal growth factor receptor (EGFR) and Decapentaplegic (Dpp) pathways, respectively. We show here that another signaling pathway, the Drosophila Jun-N-terminal kinase (JNK) cascade, is essential for the correct morphogenesis of the dorsal appendages and the micropyle during oogenesis. Mutant follicle cell clones of members of the JNK pathway, including DJNKK/hemipterous (hep), DJNK/basket (bsk), and Djun, block dorsal appendage formation and affect the micropyle shape and size, suggesting a late requirement for the JNK pathway in anterior chorion morphogenesis. In support of this view, hep does not affect early follicle cell patterning as indicated by the normal expression of kekkon (kek) and Broad-Complex (BR-C), two of the targets of the EGFR pathway in dorsal follicle cells. Furthermore, the expression of the TGF-beta homolog dpp, which is under the control of hep in embryos, is not coupled to JNK activity during oogenesis. We show that hep controls the expression of puckered (puc) in the follicular epithelium in a cell-autonomous manner. Since puc overexpression in the egg follicular epithelium mimics JNK appendages and micropyle phenotypes, it indicates a negative role of puc in their morphogenesis. The role of the JNK pathway in the morphogenesis of follicle cells and other epithelia during development is discussed.
%B Dev Biol %V 237 %P 282-94 %8 2001 Sep 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/11543614?dopt=Abstract %R 10.1006/dbio.2001.0384 %0 Journal Article %J Nature %D 2001 %T Drosophila Stardust interacts with Crumbs to control polarity of epithelia but not neuroblasts. %A Hong, Y %A Stronach, B %A Perrimon, N %A Jan, L Y %A Jan, Y N %K Adherens Junctions %K Amino Acid Sequence %K Animals %K Carrier Proteins %K Cell Polarity %K Cloning, Molecular %K DNA, Complementary %K Drosophila %K Drosophila Proteins %K Epithelial Cells %K Guanylate Kinase %K Intracellular Signaling Peptides and Proteins %K Membrane Proteins %K Membrane Transport Proteins %K Molecular Sequence Data %K Mutation %K Neurons %K Nucleoside-Phosphate Kinase %K Protein Binding %K Protein Kinase C %K Proteins %XEstablishing cellular polarity is critical for tissue organization and function. Initially discovered in the landmark genetic screen for Drosophila developmental mutants, bazooka, crumbs, shotgun and stardust mutants exhibit severe disruption in apicobasal polarity in embryonic epithelia, resulting in multilayered epithelia, tissue disintegration, and defects in cuticle formation. Here we report that stardust encodes single PDZ domain MAGUK (membrane-associated guanylate kinase) proteins that are expressed in all primary embryonic epithelia from the onset of gastrulation. Stardust colocalizes with Crumbs at the apicolateral boundary, although their expression patterns in sensory organs differ. Stardust binds to the carboxy terminus of Crumbs in vitro, and Stardust and Crumbs are mutually dependent in their stability, localization and function in controlling the apicobasal polarity of epithelial cells. However, for the subset of ectodermal cells that delaminate and form neuroblasts, their polarity requires the function of Bazooka, but not of Stardust or Crumbs.
%B Nature %V 414 %P 634-8 %8 2001 Dec 6 %G eng %N 6864 %1 http://www.ncbi.nlm.nih.gov/pubmed/11740559?dopt=Abstract %R 10.1038/414634a %0 Journal Article %J Nat Cell Biol %D 2001 %T Dual role of the fringe connection gene in both heparan sulphate and fringe-dependent signalling events. %A Selva, E M %A Hong, K %A Baeg, G H %A Beverley, S M %A Turco, S J %A Perrimon, N %A Häcker, U %K Amino Acid Sequence %K Animals %K Cytoplasm %K Drosophila melanogaster %K Drosophila Proteins %K Endoplasmic Reticulum %K Glycosyltransferases %K Golgi Apparatus %K Heparitin Sulfate %K Humans %K Molecular Sequence Data %K Morphogenesis %K N-Acetylglucosaminyltransferases %K Phenotype %K Sequence Alignment %K Sequence Homology, Amino Acid %K Signal Transduction %K Uridine Diphosphate Glucuronic Acid %K Uridine Diphosphate N-Acetylglucosamine %K Uridine Diphosphate Xylose %K Wings, Animal %XThe precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. We describe a new gene, fringe connection (frc), that encodes a nucleotide-sugar transporter that transfers UDP-glucuronic acid, UDP-N-acetylglucosamine and possibly UDP-xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue.
%B Nat Cell Biol %V 3 %P 809-15 %8 2001 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/11533660?dopt=Abstract %R 10.1038/ncb0901-809 %0 Journal Article %J Development %D 2001 %T Heparan sulfate proteoglycans are critical for the organization of the extracellular distribution of Wingless. %A Baeg, G H %A Lin, X %A Khare, N %A Baumgartner, S %A Perrimon, N %K Amino Acid Sequence %K Animals %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation %K Genes, Insect %K Heparan Sulfate Proteoglycans %K Molecular Sequence Data %K Proto-Oncogene Proteins %K Repressor Proteins %K Signal Transduction %K Wnt1 Protein %XRecent studies in Drosophila have shown that heparan sulfate proteoglycans (HSPGs) are required for Wingless (Wg/Wnt) signaling. In addition, genetic and phenotypic analyses have implicated the glypican gene dally in this process. Here, we report the identification of another Drosophila glypican gene, dally-like (dly) and show that it is also involved in Wg signaling. Inhibition of dly gene activity implicates a function for DLY in Wg reception and we show that overexpression of DLY leads to an accumulation of extracellular Wg. We propose that DLY plays a role in the extracellular distribution of Wg. Consistent with this model, a dramatic decrease of extracellular Wg was detected in clones of cells that are deficient in proper glycosaminoglycan biosynthesis. We conclude that HSPGs play an important role in organizing the extracellular distribution of Wg.
%B Development %V 128 %P 87-94 %8 2001 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/11092814?dopt=Abstract %0 Journal Article %J Development %D 2001 %T Investigation of leading edge formation at the interface of amnioserosa and dorsal ectoderm in the Drosophila embryo. %A Stronach, B E %A Perrimon, N %K Animals %K Antigens, Differentiation %K Bone Morphogenetic Proteins %K Cell Differentiation %K Drosophila %K Ectoderm %K Embryo, Nonmammalian %K Morphogenesis %K Mutation %K Signal Transduction %XThe leading edge (LE) is a single row of cells in the Drosophila embryonic epidermis that marks the boundary between two fields of cells: the amnioserosa and the dorsal ectoderm. LE cells play a crucial role in the morphogenetic process of dorsal closure and eventually form the dorsal midline of the embryo. Mutations that block LE differentiation result in a failure of dorsal closure and embryonic lethality. How LE cells are specified remains unclear. To explore whether LE cells are specified in response to early dorsoventral patterning information or whether they arise secondarily, we have altered the extent of amnioserosa and dorsal ectoderm genetically, and assayed LE cell fate. We did not observe an expansion of LE fate in dorsalized or ventralized mutants. Furthermore, we observed that the LE fate arises as a single row of cells, wherever amnioserosa tissue and dorsal epidermis are physically juxtaposed. Taken together our data indicate that LE formation is a secondary consequence of early zygotic dorsal patterning signals. In particular, proper LE specification requires the function of genes such as u-shaped and hindsight, which are direct transcriptional targets of the early Decapentaplegic/Screw patterning gradient, to establish a competency zone from which LE arises. We propose that subsequent inductive signaling between amnioserosa and dorsal ectoderm restricts the formation of LE to a single row of cells.
%B Development %V 128 %P 2905-13 %8 2001 Aug %G eng %N 15 %1 http://www.ncbi.nlm.nih.gov/pubmed/11532914?dopt=Abstract %0 Journal Article %J Nat Cell Biol %D 2001 %T Spatial control of the actin cytoskeleton in Drosophila epithelial cells. %A Baum, B %A Perrimon, N %K Actin Cytoskeleton %K Actins %K Animals %K Cell Cycle Proteins %K Cell Polarity %K Cytoskeletal Proteins %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Epithelial Cells %K Female %K Genes, abl %K Insect Proteins %K Microfilament Proteins %K Microscopy, Fluorescence %K Models, Biological %K Ovarian Follicle %XThe actin cytoskeleton orders cellular space and transduces many of the forces required for morphogenesis. Here we combine genetics and cell biology to identify genes that control the polarized distribution of actin filaments within the Drosophila follicular epithelium. We find that profilin and cofilin regulate actin-filament formation throughout the cell cortex. In contrast, CAP-a Drosophila homologue of Adenylyl Cyclase Associated Proteins-functions specifically to limit actin-filament formation catalysed by Ena at apical cell junctions. The Abl tyrosine kinase also collaborates in this process. We therefore propose that CAP, Ena and Abl act in concert to modulate the subcellular distribution of actin filaments in Drosophila.
%B Nat Cell Biol %V 3 %P 883-90 %8 2001 Oct %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/11584269?dopt=Abstract %R 10.1038/ncb1001-883 %0 Journal Article %J Nat Cell Biol %D 2001 %T Response to "Problems with LAP nomenclature." %A Bilder, David %A Birnbaum, Daniel %A Borg, Jean-Paul %A Huibregtse, Jon %A Kennedy, Mary %A Labouesse, Michel %A Mechler, Bernard %A Perrimon, Norbert %K Leucine %K Membrane Proteins %K Nuclear Proteins %K Proteins %K Terminology as Topic %B Nat Cell Biol %V 3 %P E90 %8 2001 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/11283624?dopt=Abstract %R 10.1038/35070147 %0 Book Section %B The JAK-STAT Pathway in Hematopoeisis %D 2001 %T The role of the JAK-STAT in hematopoeisis and immune responses in Drosophila %A Bach, Erika A %A Perrimon, Norbert %E Ward, Alister C %B The JAK-STAT Pathway in Hematopoeisis %I Landes Bioscience %P 112-127 %G eng %0 Journal Article %J Medecine Sciences %D 2000 %T [Role des proteoglycanes dans la distribution du facteur secrete Hedgehog] (French) %A Bellaiche, Yohanns %A The, I %A Perrimon, Norbert %B Medecine Sciences %V 2 %P 250-252 %G eng %0 Journal Article %J Nat Cell Biol %D 2000 %T Drosophila genome takes flight. %A Boutros, M %A Perrimon, N %K Animals %K Chromosome Mapping %K Drosophila melanogaster %K Genome %K Molecular Biology %XIn the March 24 issue of Science, a flurry of papers report on the impending completion of the Drosophila melanogaster genome sequence. This historic achievement is the result of a unique collaboration between the Berkeley Drosophila Genome Project (BDGP), led by Gerry Rubin, and the genomics company Celera, headed by Craig Venter. With its genome almost completely sequenced ahead of schedule, Drosophila is another important model organism to enter the postgenomic age, and represents the largest genome sequenced to date.
%B Nat Cell Biol %V 2 %P E53-4 %8 2000 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/10783247?dopt=Abstract %R 10.1038/35008678 %0 Journal Article %J Curr Opin Cell Biol %D 2000 %T Functional binding of secreted molecules to heparan sulfate proteoglycans in Drosophila. %A Baeg, G H %A Perrimon, N %K Animals %K Drosophila %K Drosophila Proteins %K Fibroblast Growth Factors %K Hedgehog Proteins %K Heparan Sulfate Proteoglycans %K Insect Proteins %K Protein Binding %K Proto-Oncogene Proteins %K Signal Transduction %K Wnt1 Protein %XHeparan sulfate proteoglycans (HSPGs) are associated with the cell surface and covalently linked to a small number of long unbranched chains of repeating disaccharides. Numerous biochemical studies of these extracellular matrix molecules have implicated them in a variety of biological phenomena, in particular cell-cell interactions. Recent genetic studies in Drosophila have begun to clarify the function of HSPGs in vivo and recent findings have implicated HSPGs in Wnt, Hedgehog, fibroblast growth factor and transforming growth factor-beta signaling pathways during development.
%B Curr Opin Cell Biol %V 12 %P 575-80 %8 2000 Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/10978892?dopt=Abstract %0 Journal Article %J Nat Cell Biol %D 2000 %T Morphogen diffusion: the case of the wingless protein. %A The, I %A Perrimon, N %K Animals %K Biological Transport %K Diffusion %K Drosophila %K Drosophila Proteins %K Morphogenesis %K Proto-Oncogene Proteins %K Wnt1 Protein %B Nat Cell Biol %V 2 %P E79-82 %8 2000 May %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/10806491?dopt=Abstract %R 10.1038/35010511 %0 Journal Article %J Matrix Biol %D 2000 %T Role of heparan sulfate proteoglycans in cell-cell signaling in Drosophila. %A Lin, X %A Perrimon, N %K Animals %K Cell Communication %K Drosophila %K Drosophila Proteins %K Fibroblast Growth Factors %K Hedgehog Proteins %K Heparan Sulfate Proteoglycans %K Insect Proteins %K Membrane Proteins %K Proto-Oncogene Proteins %K Signal Transduction %K Wnt1 Protein %XHeparan sulfate proteoglycans (HSPGs) are abundant molecules associated with the cell surface and extracellular matrix, and consist of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. Although these molecules have been the focus of intense biochemical studies in vitro, their biological functions in vivo were unclear until recently. We have undertaken an in vivo functional study of HSPGs in Drosophila. Our studies, as well as others, demonstrate the critical roles of HSPGs in several major signaling pathways, including ibroblast growth factor (FGF), Wnt, Hedgehog (Hh) and TGF-beta. Our results also suggest that specific HS GAG chain modifications, as well as specific HSPG protein cores, are involved in specific signaling pathways.
%B Matrix Biol %V 19 %P 303-7 %8 2000 Aug %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/10963990?dopt=Abstract %0 Journal Article %J Oncogene %D 2000 %T The roles of the Drosophila JAK/STAT pathway. %A Zeidler, M P %A Bach, E A %A Perrimon, N %K Animals %K Body Patterning %K DNA-Binding Proteins %K Drosophila melanogaster %K Eye %K Hematopoiesis %K Janus Kinase 1 %K Larva %K Protein-Tyrosine Kinases %K Sex Determination Processes %K Signal Transduction %K STAT1 Transcription Factor %K Trans-Activators %XThe JAK/STAT signal transduction pathway has been conserved throughout evolution such that true structural and functional homologues of components originally identified in vertebrate systems are also present in the model genetic system Drosophila melanogaster. In addition to roles during larval hematopoiesis reminiscent of the requirement for this pathway in mammalian systems, the JAK/STAT pathway in Drosophila is also involved in a number of other developmental events. Recent data has demonstrated further roles for the JAK/STAT pathway in the establishment of sexual identity via the early embryonic expression of Sex lethal, the segmentation of the embryo via the control of pair rule genes including even skipped and the establishment of polarity within the adult compound eye via a mechanism that includes the four jointed gene. Use of the powerful genetics in the model organism Drosophila may identify new components of the JAK/STAT pathway, define new roles for this pathway, and provide insights into the function of this signal transduction system. Here we review the roles of STAT and its associated signaling pathway during both embryonic and adult stages of Drosophila development and discuss future prospects for the identification and characterization of novel pathway components and targets. Oncogene (2000).
%B Oncogene %V 19 %P 2598-606 %8 2000 May 15 %G eng %N 21 %1 http://www.ncbi.nlm.nih.gov/pubmed/10851058?dopt=Abstract %R 10.1038/sj.onc.1203482 %0 Journal Article %J Curr Biol %D 2000 %T Sex determination: co-opted signals determine gender. %A Zeidler, M P %A Perrimon, N %K Animals %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Gene Expression Regulation %K Glycoproteins %K Insect Hormones %K Male %K Protein-Tyrosine Kinases %K RNA-Binding Proteins %K Sex Chromosomes %K Sex Determination Processes %K Signal Transduction %K STAT1 Transcription Factor %K Trans-Activators %K Transcription Factors %XThe Drosophila JAK-STAT pathway and its ligand Unpaired are required for a wide range of developmental processes. Recent results have identified Unpaired as an activator of sex-lethal and revealed a new role for the JAK-STAT pathway in sex determination.
%B Curr Biol %V 10 %P R682-4 %8 2000 Sep 21 %G eng %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/10996811?dopt=Abstract %0 Journal Article %J Science %D 2000 %T Signal transduction. Are there close encounters between signaling pathways? %A Noselli, S %A Perrimon, N %K Animals %K Drosophila %K Gene Expression Profiling %K Humans %K Mutation %K Neoplasms %K Phenotype %K Receptor Cross-Talk %K Signal Transduction %XDo different signaling pathways inside the same cell talk to each other? Evidence suggests that in the worm and fly, signaling pathways exist as separate linear cassettes, whereas in mammalian cells there does appear to be cross talk between signaling pathways. However, as Noselli and Perrimon argue in their Perspective, most of the evidence in mammalian cells comes from tumor cells and overexpression assays. They suggest that true cross talk may not actually exist in mammalian cells under normal circumstances.
%B Science %V 290 %P 68-9 %8 2000 Oct 6 %G eng %N 5489 %1 http://www.ncbi.nlm.nih.gov/pubmed/11183153?dopt=Abstract %0 Journal Article %J Nature %D 2000 %T Specificities of heparan sulphate proteoglycans in developmental processes. %A Perrimon, N %A Bernfield, M %K Animals %K Body Patterning %K Heparan Sulfate Proteoglycans %K Humans %K Ligands %K Mutation %K Signal Transduction %XHeparan sulphate proteoglycans are abundant cell-surface molecules that consist of a protein core to which heparan sulphate glycosaminoglycan chains are attached. The functions of these molecules have remained mostly underappreciated by developmental biologists; however, the actions of important signalling molecules, for example Wnt and Hedgehog, depend on them. To understand both the mechanisms by which ligands involved in development interact with their receptors and how morphogens pattern tissues, biologists need to consider the functions of heparan sulphate proteoglycans in signalling and developmental patterning.
%B Nature %V 404 %P 725-8 %8 2000 Apr 13 %G eng %N 6779 %1 http://www.ncbi.nlm.nih.gov/pubmed/10783877?dopt=Abstract %R 10.1038/35008000 %0 Journal Article %J Science %D 2000 %T Cooperative regulation of cell polarity and growth by Drosophila tumor suppressors. %A Bilder, D %A Li, M %A Perrimon, N %K Animals %K Cell Division %K Cell Membrane %K Cell Polarity %K Cell Transformation, Neoplastic %K Cytoplasm %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Epidermis %K Epithelial Cells %K Female %K Genes, Insect %K Genes, Tumor Suppressor %K Insect Proteins %K Intercellular Junctions %K Membrane Proteins %K Morphogenesis %K Mutation %K Phenotype %K Tumor Suppressor Proteins %XLoss of cell polarity and tissue architecture are characteristics of malignant cancers derived from epithelial tissues. We provide evidence from Drosophila that a group of membrane-associated proteins act in concert to regulate both epithelial structure and cell proliferation. Scribble (Scrib) is a cell junction-localized protein required for polarization of embryonic and, as demonstrated here, imaginal disc and follicular epithelia. We show that the tumor suppressors lethal giant larvae (lgl) and discs-large (dlg) have identical effects on all three epithelia, and that scrib also acts as a tumor suppressor. Scrib and Dlg colocalize and overlap with Lgl in epithelia; activity of all three genes is required for cortical localization of Lgl and junctional localization of Scrib and Dlg. scrib, dlg, and lgl show strong genetic interactions. Our data indicate that the three tumor suppressors act together in a common pathway to regulate cell polarity and growth control.
%B Science %V 289 %P 113-6 %8 2000 Jul 7 %G eng %N 5476 %1 http://www.ncbi.nlm.nih.gov/pubmed/10884224?dopt=Abstract %0 Journal Article %J Curr Biol %D 2000 %T A cyclase-associated protein regulates actin and cell polarity during Drosophila oogenesis and in yeast. %A Baum, B %A W Li %A Perrimon, N %K Actins %K Adaptor Proteins, Signal Transducing %K Amino Acid Sequence %K Animals %K Cell Cycle Proteins %K Cell Polarity %K Cytoskeletal Proteins %K Cytoskeleton %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Gene Expression Regulation, Fungal %K Humans %K Microfilament Proteins %K Molecular Sequence Data %K Oocytes %K Oogenesis %K Saccharomyces cerevisiae %K Saccharomyces cerevisiae Proteins %XBACKGROUND: A polarised cytoskeleton is required to pattern cellular space, and for many aspects of cell behaviour. While the mechanisms ordering the actin cytoskeleton have been extensively studied in yeast, little is known about the analogous processes in other organisms. We have used Drosophila oogenesis as a model genetic system in which to investigate control of cytoskeletal organisation and cell polarity in multicellular eukaryotes. RESULTS: In a screen to identify genes required for Drosophila oocyte polarity, we isolated a Drosophila homologue of the yeast cyclase-associated protein, CAP. Here we show that CAP preferentially accumulates in the oocyte, where it inhibits actin polymerisation. CAP also has a role in oocyte polarity, as cap mutants fail to establish the proper, asymmetric distribution of mRNA determinants within the oocyte. Similarly in yeast, loss of CAP causes analogous polarity defects, altering the distribution of actin filaments and mRNA determinants. CONCLUSIONS: This study identifies CAP as a new effector of actin dynamics in Drosophila. As CAP controls the spatial distribution of actin filaments and mRNA determinants in both yeast and Drosophila, we conclude that CAP has an evolutionarily conserved function in the genesis of eukaryotic cell polarity.
%B Curr Biol %V 10 %P 964-73 %8 2000 Aug 24 %G eng %N 16 %1 http://www.ncbi.nlm.nih.gov/pubmed/10985383?dopt=Abstract %0 Journal Article %J Eur J Biochem %D 2000 %T The evolutionarily conserved porcupine gene family is involved in the processing of the Wnt family. %A Tanaka, K %A Okabayashi, K %A Asashima, M %A Perrimon, N %A Kadowaki, T %K Alternative Splicing %K Amino Acid Sequence %K Animals %K Base Sequence %K Biological Evolution %K Conserved Sequence %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Membrane Proteins %K Mice %K Molecular Sequence Data %K Molecular Weight %K Proto-Oncogene Proteins %K Wnt Proteins %K Wnt1 Protein %K Zebrafish Proteins %XThe Drosophila segment polarity gene product Porcupine (Porc) was first identified as being necessary for processing Wingless (Wg), a Drosophila Wnt (Wnt) family member. Mouse and Xenopus homologs of porc (Mporc and Xporc) were identified and found to encode endoplasmic reticulum (ER) proteins with multiple transmembrane domains. In contrast with porc, four different types of Mporc and Xporc mRNA (A-D) are generated from a single gene by alternative splicing. Mporc mRNA is differentially expressed during embryogenesis and in various adult tissues, demonstrating that the alternative splicing is regulated to synthesize the specific types of Mporc. In transfected mammalian cells, all Mporc types affect the processing of mouse Wnt 1, 3A, 4, 6, and 7B but not 5A. Furthermore, all Mporc types are co-immunoprecipitated with various Wnt proteins. These results suggest that Mporc may function as a chaperone-like molecule for Wnt. Interestingly, all Mporc types can substitute for Porc, as they are able to rescue the phenotypes of Drosophila porc embryos. Consistent with this observation, Mporc, like Porc, modifies the processing of Wg expressed in mammalian cells. These results demonstrate that the porc gene family encodes the multitransmembrane ER proteins, which are evolutionarily well conserved and involved in processing the Wnt family.
%B Eur J Biochem %V 267 %P 4300-11 %8 2000 Jul %G eng %N 13 %1 http://www.ncbi.nlm.nih.gov/pubmed/10866835?dopt=Abstract %0 Journal Article %J Nature %D 2000 %T Localization of apical epithelial determinants by the basolateral PDZ protein Scribble. %A Bilder, D %A Perrimon, N %K Amino Acid Sequence %K Animals %K Cell Polarity %K Cloning, Molecular %K Drosophila %K Drosophila Proteins %K Epithelial Cells %K Intercellular Junctions %K Membrane Proteins %K Molecular Sequence Data %K Mutation %K Open Reading Frames %K Protein Structure, Tertiary %K Tissue Distribution %XThe generation of membrane domains with distinct protein constituents is a hallmark of cell polarization. In epithelia, segregation of membrane proteins into apical and basolateral compartments is critical for cell morphology, tissue physiology and cell signalling. Drosophila proteins that confer apical membrane identity have been found, but the mechanisms that restrict these determinants to the apical cell surface are unknown. Here we show that a laterally localized protein is required for the apical confinement of polarity determinants. Mutations in Drosophila scribble (scrib), which encodes a multi-PDZ (PSD-95, Discs-large and ZO-1) and leucine-rich-repeat protein, cause aberrant cell shapes and loss of the monolayer organization of embryonic epithelia. Scrib is localized to the epithelial septate junction, the analogue of the vertebrate tight junction, at the boundary of the apical and basolateral cell surfaces. Loss of scrib function results in the misdistribution of apical proteins and adherens junctions to the basolateral cell surface, but basolateral protein localization remains intact. These phenotypes can be accounted for by mislocalization of the apical determinant Crumbs. Our results show that the lateral domain of epithelia, particularly the septate junction, functions in restricting apical membrane identity and correctly placing adherens junctions.
%B Nature %V 403 %P 676-80 %8 2000 Feb 10 %G eng %N 6770 %1 http://www.ncbi.nlm.nih.gov/pubmed/10688207?dopt=Abstract %R 10.1038/35001108 %0 Journal Article %J Dev Biol %D 2000 %T Multiple roles for four-jointed in planar polarity and limb patterning. %A Zeidler, M P %A Perrimon, N %A Strutt, D I %K Abdomen %K Animals %K Animals, Genetically Modified %K beta-Galactosidase %K Body Patterning %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Genes, Reporter %K Membrane Glycoproteins %K Pupa %K Wings, Animal %XInsect cuticles have been a model system for the study of planar polarity for many years and a number of genes required for this process have been identified. These genes organise the polarised arrangement of hairs on the legs, wings, thorax, and abdomen of adult Drosophila. It has previously been shown that four-jointed is involved in planar polarity decisions in the eye as well as proximal distal leg and wing development. We now present evidence that four-jointed is expressed in a gradient through the developing wing and show that it is required for planar polarity determination in both the wing and the abdomen. Clones of cells either lacking or ectopically expressing four-jointed cause both autonomous and nonautonomous repolarisation of hairs in these tissues. We propose that the inferred four-jointed expression gradient is important for planar polarity establishment and that local inversions of the gradient by the clones are the probable cause of the observed polarity phenotypes. In addition we observe defects in wing vein development. The subtle phenotypes of mutant flies, and the diverse patterning processes in which it is involved, suggest that four-jointed may act as a modifier of the activity of multiple other signalling factors.
%B Dev Biol %V 228 %P 181-96 %8 2000 Dec 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/11112323?dopt=Abstract %R 10.1006/dbio.2000.9940 %0 Journal Article %J Dev Biol %D 2000 %T Presenilin affects arm/beta-catenin localization and function in Drosophila. %A Noll, E %A Medina, M %A Hartley, D %A Zhou, J %A Perrimon, N %A Kosik, K S %K Animals %K Animals, Genetically Modified %K Armadillo Domain Proteins %K beta Catenin %K Catenins %K Cell Adhesion Molecules %K Cytoskeletal Proteins %K Drosophila %K Drosophila Proteins %K Female %K Gene Expression Regulation, Developmental %K Genes, Insect %K Humans %K In Vitro Techniques %K Insect Proteins %K Membrane Proteins %K Models, Biological %K Mutation %K Phenotype %K Phosphoproteins %K Presenilin-1 %K Receptors, Notch %K Trans-Activators %K Transcription Factors %XPresenilin is an essential gene for development that when disrupted leads to a neurogenic phenotype that closely resembles Notch loss of function in Drosophila. In humans, many naturally occurring mutations in Presenilin 1 or 2 cause early onset Alzheimer's disease. Both loss of expression and overexpression of Presenilin suggested a role for this protein in the localization of Armadillo/beta-catenin. In blastoderm stage Presenilin mutants, Arm is aberrantly distributed, often in Ubiquitin-immunoreactive cytoplasmic inclusions predominantly located basally in the cell. These inclusions were not observed in loss of function Notch mutants, suggesting that failure to process Notch is not the only consequence of the loss of Presenilin function. Human presenilin 1 expressed in Drosophila produces embryonic phenotypes resembling those associated with mutations in Armadillo and exhibited reduced Armadillo at the plasma membrane that is likely due to retention of Armadillo in a complex with Presenilin. The interaction between Armadillo/beta-catenin and Presenilin 1 requires a third protein which may be delta-catenin. Our results suggest that Presenilin may regulate the delivery of a multiprotein complex that regulates Armadillo trafficking between the adherens junction and the proteasome.
%B Dev Biol %V 227 %P 450-64 %8 2000 Nov 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/11071766?dopt=Abstract %R 10.1006/dbio.2000.9925 %0 Journal Article %J Nat Cell Biol %D 2000 %T Collective nomenclature for LAP proteins. %A Bilder, D %A Birnbaum, D %A Borg, J P %A Bryant, P %A Huigbretse, J %A Jansen, E %A Kennedy, M B %A Labouesse, M %A Legouis, R %A Mechler, B %A Perrimon, N %A Petit, M %A Sinha, P. %K Amino Acid Motifs %K Animals %K Humans %K Protein Structure, Tertiary %K Proteins %K Sequence Homology, Amino Acid %K Terminology as Topic %K TNF Receptor-Associated Factor 3 %B Nat Cell Biol %V 2 %P E114 %8 2000 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/10878817?dopt=Abstract %R 10.1038/35017119 %0 Journal Article %J Genetics %D 2000 %T Identification of autosomal regions involved in Drosophila Raf function. %A W Li %A Noll, E %A Perrimon, N %K Animals %K Chromosome Mapping %K Crosses, Genetic %K Drosophila melanogaster %K Enhancer Elements, Genetic %K Female %K Gene Deletion %K Gene Expression Regulation, Developmental %K Larva %K Male %K Mutagenesis %K Phenotype %K Proto-Oncogene Proteins c-raf %K Signal Transduction %K Transcription, Genetic %XRaf is an essential downstream effector of activated p21(Ras) (Ras) in transducing proliferation or differentiation signals. Following binding to Ras, Raf is translocated to the plasma membrane, where it is activated by a yet unidentified "Raf activator." In an attempt to identify the Raf activator or additional molecules involved in the Raf signaling pathway, we conducted a genetic screen to identify genomic regions that are required for the biological function of Drosophila Raf (Draf). We tested a collection of chromosomal deficiencies representing approximately 70% of the autosomal euchromatic genomic regions for their abilities to enhance the lethality associated with a hypomorphic viable allele of Draf, Draf(Su2). Of the 148 autosomal deficiencies tested, 23 behaved as dominant enhancers of Draf(Su2), causing lethality in Draf(Su2) hemizygous males. Four of these deficiencies identified genes known to be involved in the Drosophila Ras/Raf (Ras1/Draf) pathway: Ras1, rolled (rl, encoding a MAPK), 14-3-3epsilon, and bowel (bowl). Two additional deficiencies removed the Drosophila Tec and Src homologs, Tec29A and Src64B. We demonstrate that Src64B interacts genetically with Draf and that an activated form of Src64B, when overexpressed in early embryos, causes ectopic expression of the Torso (Tor) receptor tyrosine kinase-target gene tailless. In addition, we show that a mutation in Tec29A partially suppresses a gain-of-function mutation in tor. These results suggest that Tec29A and Src64B are involved in Tor signaling, raising the possibility that they function to activate Draf. Finally, we discovered a genetic interaction between Draf(Su2) and Df(3L)vin5 that revealed a novel role of Draf in limb development. We find that loss of Draf activity causes limb defects, including pattern duplications, consistent with a role for Draf in regulation of engrailed (en) expression in imaginal discs.
%B Genetics %V 156 %P 763-74 %8 2000 Oct %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/11014822?dopt=Abstract %0 Journal Article %J Nature %D 1999 %T Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling. %A Lin, X %A Perrimon, N %K Amidohydrolases %K Amino Acid Sequence %K Animals %K Body Patterning %K Drosophila %K Drosophila Proteins %K Female %K Frizzled Receptors %K Genes, Insect %K Heparin %K Insect Proteins %K Membrane Glycoproteins %K Molecular Sequence Data %K Mutation %K Proteoglycans %K Proto-Oncogene Proteins %K Receptors, G-Protein-Coupled %K Receptors, Neurotransmitter %K Sequence Homology, Amino Acid %K Signal Transduction %K Sulfotransferases %K Wings, Animal %K Wnt1 Protein %XThe Drosophila wingless gene (wg) encodes a protein of the Wnt family and is a critical regulator in many developmental processes. Biochemical studies have indicated that heparan sulphate proteoglycans, consisting of a protein core to which heparan sulphate glycosaminoglycans are attached, are important for Wg function. Here we show that, consistent with these findings, the Drosophila gene sulfateless (sfl), which encodes a homologue of vertebrate heparan sulphate N-deacetylase/N-sulphotransferase (an enzyme needed for the modification of heparan sulphate) is essential for Wg signalling. We have identified the product of division abnormally delayed (dally), a glycosyl-phosphatidyl inositol (GPI)-linked glypican, as a heparan sulphate proteoglycan molecule involved in Wg signalling. Our results indicate that Dally may act as a co-receptor for Wg, and that Dally, together with Drosophila Frizzled 2, modulates both short- and long-range activities of Wg.
%B Nature %V 400 %P 281-4 %8 1999 Jul 15 %G eng %N 6741 %1 http://www.ncbi.nlm.nih.gov/pubmed/10421372?dopt=Abstract %R 10.1038/22343 %0 Journal Article %J Curr Biol %D 1999 %T The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification. %A Zeidler, M P %A Perrimon, N %A Strutt, D I %K Animals %K Base Sequence %K Body Patterning %K Cloning, Molecular %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Eye %K Frizzled Receptors %K Insect Proteins %K Janus Kinase 3 %K Membrane Glycoproteins %K Membrane Proteins %K Microscopy, Confocal %K Molecular Sequence Data %K Mutation %K Polymerase Chain Reaction %K Protein-Tyrosine Kinases %K Proto-Oncogene Proteins %K Receptors, G-Protein-Coupled %K Receptors, Notch %K Signal Transduction %K STAT1 Transcription Factor %K Trans-Activators %K Wnt1 Protein %XBACKGROUND: The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signalling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signalling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. RESULTS: The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. We have analysed the function and regulation of four-jointed during eye patterning. Loss-of-function clones or ectopic expression of four-jointed resulted in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicated that localised four-jointed expression was required at the time during development when ommatidial polarity was being determined. In contrast, complete removal of four-jointed function resulted in only a mild ommatidial polarity defect. Finally, we found that four-jointed expression was regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. CONCLUSIONS: The clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal.
%B Curr Biol %V 9 %P 1363-72 %8 1999 Dec 2 %G eng %N 23 %1 http://www.ncbi.nlm.nih.gov/pubmed/10607560?dopt=Abstract %0 Journal Article %J Mol Cell %D 1999 %T Hedgehog movement is regulated through tout velu-dependent synthesis of a heparan sulfate proteoglycan. %A The, I %A Bellaiche, Y %A Perrimon, N %K Animals %K Biomarkers %K Drosophila %K Drosophila Proteins %K Fibroblast Growth Factors %K Gene Expression Regulation %K Hedgehog Proteins %K Heparan Sulfate Proteoglycans %K Immunohistochemistry %K Insect Proteins %K Membrane Proteins %K Proto-Oncogene Proteins %K Signal Transduction %K Wnt1 Protein %XHedgehog (Hh) molecules play critical roles during development as a morphogen, and therefore their distribution must be regulated. Hh proteins undergo several modifications that tether them to the membrane. We have previously identified tout velu (ttv), a homolog of the mammalian EXT tumor suppressor gene family, as a gene required for movement of Hh. In this paper, we present in vivo evidence that ttv is involved in heparan sulfate proteoglycan (HSPG) biosynthesis, suggesting that HSPGs control Hh distribution. In contrast to mutants in other HSPG biosynthesis genes, the activity of the HSPG-dependent FGF and Wingless signaling pathways are not affected in ttv mutants. This demonstrates an unexpected level of specificity in the regulation of the distribution of extracellular signals by HSPGs.
%B Mol Cell %V 4 %P 633-9 %8 1999 Oct %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/10549295?dopt=Abstract %0 Journal Article %J Development %D 1999 %T Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development. %A Lin, X %A Buff, E M %A Perrimon, N %A Michelson, A M %K Amidohydrolases %K Animals %K Drosophila %K Drosophila Proteins %K Fibroblast Growth Factors %K Gene Expression %K Genes, Insect %K Heparan Sulfate Proteoglycans %K Insect Proteins %K Mesoderm %K Mitogen-Activated Protein Kinases %K Mutation %K Phenotype %K Protein-Tyrosine Kinases %K Receptors, Fibroblast Growth Factor %K Signal Transduction %K Sulfotransferases %K Trachea %XThe Drosophila sugarless and sulfateless genes encode enzymes required for the biosynthesis of heparan sulfate glycosaminoglycans. Biochemical studies have shown that heparan sulfate glycosaminoglycans are involved in signaling by fibroblast growth factor receptors, but evidence for such a requirement in an intact organism has not been available. We now demonstrate that sugarless and sulfateless mutant embryos have phenotypes similar to those lacking the functions of two Drosophila fibroblast growth factor receptors, Heartless and Breathless. Moreover, both Heartless- and Breathless-dependent MAPK activation is significantly reduced in embryos which fail to synthesize heparan sulfate glycosaminoglycans. Consistent with an involvement of Sulfateless and Sugarless in fibroblast growth factor receptor signaling, a constitutively activated form of Heartless partially rescues sugarless and sulfateless mutants, and dosage-sensitive interactions occur between heartless and the heparan sulfate glycosaminoglycan biosynthetic enzyme genes. We also find that overexpression of Branchless, the Breathless ligand, can partially overcome the requirement of Sugarless and Sulfateless for Breathless activity. These results provide the first genetic evidence that heparan sulfate glycosaminoglycans are essential for fibroblast growth factor receptor signaling in a well defined developmental context, and support a model in which heparan sulfate glycosaminoglycans facilitate fibroblast growth factor ligand and/or ligand-receptor oligomerization.
%B Development %V 126 %P 3715-23 %8 1999 Sep %G eng %N 17 %1 http://www.ncbi.nlm.nih.gov/pubmed/10433902?dopt=Abstract %0 Journal Article %J Dev Biol %D 1999 %T Quantitative variations in the level of MAPK activity control patterning of the embryonic termini in Drosophila. %A Ghiglione, C %A Perrimon, N %A Perkins, L A %K Animals %K Body Patterning %K Calcium-Calmodulin-Dependent Protein Kinases %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Gene Expression Regulation, Developmental %K Insect Hormones %K Morphogenesis %K Protein Tyrosine Phosphatases %K Protein Tyrosine Phosphatases, Non-Receptor %K Proto-Oncogene Proteins c-raf %K Receptor Protein-Tyrosine Kinases %K Repressor Proteins %K Signal Transduction %K Transcription, Genetic %K Zinc Fingers %XWe have examined the role in patterning of quantitative variations of MAPK activity in signaling from the Drosophila Torso (Tor) receptor tyrosine kinase (RTK). Activation of Tor at the embryonic termini leads to differential expression of the genes tailless and huckebein. We demonstrate, using a series of mutations in the signal transducers Corkscrew/SHP-2 and D-Raf, that quantitative variations in the magnitude of MAPK activity trigger both qualitatively and quantitatively distinct transcriptional responses. We also demonstrate that two chimeric receptors, Torextracellular-Egfrcytoplasmic and Torextracellular-Sevcytoplasmic, cannot fully functionally replace the wild-type Tor receptor, revealing that the precise activation of MAPK involves not only the number of activated RTK molecules but also the magnitude of the signal generated by the RTK cytoplasmic domain. Altogether, our results illustrate how a gradient of MAPK activity controls differential gene expression and, thus, the establishment of various cell fates. We discuss the roles of quantitative mechanisms in defining RTK specificity.
%B Dev Biol %V 205 %P 181-93 %8 1999 Jan 1 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/9882506?dopt=Abstract %R 10.1006/dbio.1998.9102 %0 Journal Article %J Proc Natl Acad Sci U S A %D 1999 %T Synergistic activities of multiple phosphotyrosine residues mediate full signaling from the Drosophila Torso receptor tyrosine kinase. %A Gayko, U %A Cleghon, V %A Copeland, T %A Morrison, D K %A Perrimon, N %K Animals %K Animals, Genetically Modified %K Drosophila %K Drosophila Proteins %K Embryonic Development %K Insect Proteins %K Mutation %K Phosphopeptides %K Phosphorylation %K Phosphotyrosine %K Receptor Protein-Tyrosine Kinases %K Recombinant Proteins %K Signal Transduction %K Structure-Activity Relationship %XHere, we identify four tyrosine residues (Y644, Y698, Y767, and Y772) that become phosphorylated after activation of the Torso (Tor) receptor tyrosine kinase. Previously, we characterized phosphotyrosine sites (P-Y630 and P-Y918). Of the six P-Y sites identified, three (Y630, Y644, and Y698) are located in the kinase domain insert region, one (Y918) is located in the C-terminal tail region, and two (Y767 and Y772) are located in the activation loop of the kinase domain. To investigate the function of each P-Y residue in Tor signaling, we have generated transgenic Drosophila embryos expressing mutant Tor receptors containing either single or multiple tyrosine to phenylalanine substitutions. Single P-Y mutations were found to have either positive, negative, or no effect on the signaling activity of the receptor. Elimination of all P-Y sites within the kinase insert region resulted in the complete loss of receptor function, indicating that some combination of these sites is necessary for Tor signaling. Mutation of the C-terminal P-Y918 site revealed that this site is responsible for negative signaling or down-regulation of receptor activity. Mutation of the P-Y sites in the kinase domain activation loop demonstrated that these sites are essential for enzymatic activity. Our analysis provides a detailed in vivo example of the extent of cooperativity between P-Y residues in transducing the signal received by a receptor tyrosine kinase and in vivo data demonstrating the function of P-Y residues in the activation loop of the kinase domain.
%B Proc Natl Acad Sci U S A %V 96 %P 523-8 %8 1999 Jan 19 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/9892666?dopt=Abstract %0 Journal Article %J Development %D 1999 %T A temporal switch in DER signaling controls the specification and differentiation of veins and interveins in the Drosophila wing. %A Martín-Blanco, E %A Roch, F %A Noll, E %A Baonza, A %A Duffy, JB %A Perrimon, N %K Animals %K Cell Differentiation %K Drosophila melanogaster %K Drosophila Proteins %K Eye Proteins %K Gene Expression %K Insect Proteins %K Membrane Proteins %K Mitogen-Activated Protein Kinases %K Nerve Tissue Proteins %K Protein Kinases %K Proto-Oncogene Proteins c-raf %K Pupa %K Receptor, Epidermal Growth Factor %K Receptors, Invertebrate Peptide %K Signal Transduction %K Wings, Animal %XThe Drosophila EGF receptor (DER) is required for the specification of diverse cell fates throughout development. We have examined how the activation of DER controls the development of vein and intervein cells in the Drosophila wing. The data presented here indicate that two distinct events are involved in the determination and differentiation of wing cells. (1) The establishment of a positive feedback amplification loop, which drives DER signaling in larval stages. At this time, rhomboid (rho), in combination with vein, initiates and amplifies the activity of DER in vein cells. (2) The late downregulation of DER activity. At this point, the inactivation of MAPK in vein cells is necessary for the maintenance of the expression of decapentaplegic (dpp) and becomes essential for vein differentiation. Together, these temporal and spatial changes in the activity of DER constitute an autoregulatory network that controls the definition of vein and intervein cell types.
%B Development %V 126 %P 5739-47 %8 1999 Dec %G eng %N 24 %1 http://www.ncbi.nlm.nih.gov/pubmed/10572049?dopt=Abstract %0 Journal Article %J Cell %D 1999 %T The transmembrane molecule kekkon 1 acts in a feedback loop to negatively regulate the activity of the Drosophila EGF receptor during oogenesis. %A Ghiglione, C %A Carraway, K L %A Amundadottir, L T %A Boswell, R E %A Perrimon, N %A Duffy, JB %K Animals %K Binding Sites %K Calcium-Calmodulin-Dependent Protein Kinases %K Drosophila %K Drosophila Proteins %K Feedback %K Gene Expression %K Insect Proteins %K MAP Kinase Kinase 1 %K Membrane Proteins %K Mitogen-Activated Protein Kinase Kinases %K Nerve Tissue Proteins %K Oogenesis %K Protein Tyrosine Phosphatases %K Protein-Serine-Threonine Kinases %K Protein-Tyrosine Kinases %K Proto-Oncogene Proteins c-raf %K ras Proteins %K Receptor, Epidermal Growth Factor %K Signal Transduction %K Transforming Growth Factor alpha %K Transforming Growth Factors %XWe have identified the Drosophila transmembrane molecule kekkon 1 (kek1) as an inhibitor of the epidermal growth factor receptor (EGFR) and demonstrate that it acts in a negative feedback loop to modulate the activity of the EGFR tyrosine kinase. During oogenesis, kek1 is expressed in response to the Gurken/EGFR signaling pathway, and loss of kek1 activity is associated with an increase in EGFR signaling. Consistent with our loss-of-function studies, we demonstrate that ectopic overexpression of kek1 mimics a loss of EGFR activity. We show that the extracellular and transmembrane domains of Kek1 can inhibit and physically associate with the EGFR, suggesting potential models for this inhibitory mechanism.
%B Cell %V 96 %P 847-56 %8 1999 Mar 19 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/10102272?dopt=Abstract %0 Journal Article %J Trends Genet %D 1999 %T The latest in signal transduction, 1999. Specificity in Signal Transduction, Keystone, Colorado, USA, 9-14 April 1999. %A Spana, E %A Perrimon, N %K Animals %K Drosophila %K Signal Transduction %B Trends Genet %V 15 %P 301-2 %8 1999 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/10475855?dopt=Abstract %0 Journal Article %J Cell %D 1999 %T Negative feedback mechanisms and their roles during pattern formation. %A Perrimon, N %A McMahon, A.P. %K Animals %K Body Patterning %K Feedback %B Cell %V 97 %P 13-6 %8 1999 Apr 2 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/10199398?dopt=Abstract %0 Journal Article %J Curr Opin Genet Dev %D 1999 %T Pattern formation and developmental mechanisms unresolved issues of pattern formation %A Perrimon, N %A Stern, C %B Curr Opin Genet Dev %V 9 %P 387-9 %8 1999 Aug %G Eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/10449360?dopt=Abstract %0 Journal Article %J Oncogene %D 1999 %T Stress signaling in Drosophila. %A Stronach, B E %A Perrimon, N %K Animals %K Body Patterning %K Drosophila melanogaster %K Drosophila Proteins %K GTP Phosphohydrolases %K Immunity %K Intercellular Junctions %K JNK Mitogen-Activated Protein Kinases %K MAP Kinase Kinase 4 %K MAP Kinase Signaling System %K Membrane Proteins %K Mitogen-Activated Protein Kinase Kinases %K Mitogen-Activated Protein Kinases %K p38 Mitogen-Activated Protein Kinases %K Receptors, Notch %K Receptors, Tumor Necrosis Factor %XCells commonly use multiprotein kinase cascades to signal information from the cell membrane to the nucleus. Several conserved signaling pathways related to the mitogen activated protein kinase (MAPK) pathway allow cells to respond to normal developmental signals as well as signals produced under stressful conditions. Genetic and molecular studies in Drosophila melanogaster over the last several years have related that components of stress signaling pathways, namely the Jun kinase (JNK) and p38 kinase signaling modules, are functionally conserved and participate in numerous processes during normal development. Specifically, the JNK pathway is required for morphogenetic movements in embryogenesis and generation of tissue polarity in the adult. The role of the p38 pathway in generation of axial polarity during oogenesis has been inferred from phenotypic analysis of mutations in the Drosophila homolog of DMKK3. In addition to their requirement for normal development, cell culture and genetic investigations point to a role for both the JNK and p38 pathways in regulation of the immune response in the fly. This review details the known components of stress signaling pathways in Drosophila and recent insights into how these pathways are used and regulated during development and homeostasis.
%B Oncogene %V 18 %P 6172-82 %8 1999 Nov 1 %G eng %N 45 %1 http://www.ncbi.nlm.nih.gov/pubmed/10557109?dopt=Abstract %R 10.1038/sj.onc.1203125 %0 Book Section %B Cell Surface Proteoglycans in Signalling and Development %D 1999 %T Role of HSPG in growth-factor signalling pathways in Drosophila %A Perrimon, Norbert %B Cell Surface Proteoglycans in Signalling and Development %I Human Frontiers Science Program Workshop VI %P 157-162 %G eng %0 Journal Article %J Genetics %D 1999 %T I-SceI endonuclease, a new tool for studying DNA double-strand break repair mechanisms in Drosophila. %A Bellaiche, Y %A Mogila, V %A Perrimon, N %K Animals %K Crosses, Genetic %K Deoxyribonucleases, Type II Site-Specific %K DNA %K DNA Repair %K Drosophila %K Female %K Genes, Reporter %K Male %K Models, Genetic %K Molecular Biology %K Saccharomyces cerevisiae Proteins %XAs a step toward the development of a homologous recombination system in Drosophila, we have developed a methodology to target double-strand breaks (DSBs) to a specific position in the Drosophila genome. This method uses the mitochondrial endonuclease I-SceI that recognizes and cuts an 18-bp restriction site. We find that >6% of the progeny derived from males that carry a marker gene bordered by two I-SceI sites and that express I-SceI in their germ line lose the marker gene. Southern blot analysis and sequencing of the regions surrounding the I-SceI sites revealed that in the majority of the cases, the introduction of DSBs at the I-SceI sites resulted in the complete deletion of the marker gene; the other events were associated with partial deletion of the marker gene. We discuss a number of applications for this novel technique, in particular its use to study DSB repair mechanisms.
%B Genetics %V 152 %P 1037-44 %8 1999 Jul %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/10388822?dopt=Abstract %0 Journal Article %J Genes Dev %D 1999 %T Polarity determination in the Drosophila eye: a novel role for unpaired and JAK/STAT signaling. %A Zeidler, M P %A Perrimon, N %A Strutt, D I %K Animals %K Body Patterning %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Eye %K Glycoproteins %K Janus Kinase 1 %K Janus Kinases %K Models, Biological %K Protein-Tyrosine Kinases %K Signal Transduction %K STAT Transcription Factors %K STAT1 Transcription Factor %K Trans-Activators %K Transcription Factors %XThe JAK/STAT signaling pathway is required for many processes including cytokine signaling, hematopoiesis, gliagenesis, and Drosophila segmentation. In this report we present evidence demonstrating that the JAK/STAT pathway is also central to the establishment of planar polarity during Drosophila eye development. We show that a localized source of the pathway ligand, Unpaired, is present at the midline of the developing eye, which is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror.
%B Genes Dev %V 13 %P 1342-53 %8 1999 May 15 %G eng %N 10 %1 http://www.ncbi.nlm.nih.gov/pubmed/10346822?dopt=Abstract %0 Journal Article %J Genes Dev %D 1998 %T Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways. %A Axelrod, J D %A Miller, J R %A Shulman, J M %A Moon, R T %A Perrimon, N %K Adaptor Proteins, Signal Transducing %K Amino Acid Sequence %K Animals %K Biological Transport %K Cell Membrane %K Cell Polarity %K Cloning, Molecular %K Cytoplasm %K Drosophila %K Drosophila Proteins %K Frizzled Receptors %K Membrane Proteins %K Molecular Sequence Data %K Mutation %K Phosphoproteins %K Proto-Oncogene Proteins %K Receptors, G-Protein-Coupled %K Signal Transduction %K Wnt1 Protein %K Xenopus %XIn Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. Using a heterologous system, we show that Dsh is recruited selectively to the membrane by Fz but not DFz2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. We propose that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation.
%B Genes Dev %V 12 %P 2610-22 %8 1998 Aug 15 %G eng %N 16 %1 http://www.ncbi.nlm.nih.gov/pubmed/9716412?dopt=Abstract %0 Journal Article %J Genes Dev %D 1998 %T DRhoGEF2 encodes a member of the Dbl family of oncogenes and controls cell shape changes during gastrulation in Drosophila. %A Häcker, U %A Perrimon, N %K Amino Acid Sequence %K Animals %K Cell Size %K Drosophila %K Embryonic Development %K Eukaryotic Initiation Factor-2 %K Guanine Nucleotide Exchange Factors %K Immunohistochemistry %K In Situ Hybridization %K Microscopy, Electron, Scanning %K Molecular Sequence Data %K Morphogenesis %K Mutation %K Proteins %K Proto-Oncogene Proteins %K Sequence Analysis %XWe have identified a gene, DRhoGEF2, which encodes a putative guanine nucleotide exchange factor belonging to the Dbl family of oncogenes. DRhoGEF2 function is essential for the coordination of cell shape changes during gastrulation. In the absence of maternal DRhoGEF2 gene activity, mesodermal and endodermal primordia fail to invaginate. The phenotype seen in DRhoGEF2 mutants is more severe than the defects associated with mutations in two previously identified gastrulation genes, folded gastrulation and concertina, suggesting that DRhoGEF2 acts in a signaling pathway independent of these genes. Expression of dominant-negative DRhoA during gastrulation results in phenocopies of the DRhoGEF2 mutant, suggesting that a signaling cascade involving DRhoGEF2 and the small GTPase DRhoA is responsible for the regulation of cell shape changes during early Drosophila morphogenesis.
%B Genes Dev %V 12 %P 274-84 %8 1998 Jan 15 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/9436986?dopt=Abstract %0 Journal Article %J Genes Dev %D 1998 %T Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway. %A Harrison, D A %A McCoon, P E %A Binari, R %A Gilman, M %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Pairing %K Base Sequence %K Cytokines %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Extracellular Matrix Proteins %K Gene Expression Regulation, Developmental %K Genes, Insect %K Glycoproteins %K Insect Proteins %K Janus Kinases %K Molecular Sequence Data %K Protein-Tyrosine Kinases %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %K STAT Transcription Factors %K Trans-Activators %K Transcription Factors %K Transcription, Genetic %XIn vertebrates, many cytokines and growth factors have been identified as activators of the JAK/STAT signaling pathway. In Drosophila, JAK and STAT molecules have been isolated, but no ligands or receptors capable of activating the pathway have been described. We have characterized the unpaired (upd) gene, which displays the same distinctive embryonic mutant defects as mutations in the Drosophila JAK (hopscotch) and STAT (stat92E) genes. Upd is a secreted protein, associated with the extracellular matrix, that activates the JAK pathway. We propose that Upd is a ligand that relies on JAK signaling to stimulate transcription of pair-rule genes in a segmentally restricted manner in the early Drosophila embryo.
%B Genes Dev %V 12 %P 3252-63 %8 1998 Oct 15 %G eng %N 20 %1 http://www.ncbi.nlm.nih.gov/pubmed/9784499?dopt=Abstract %0 Journal Article %J Development %D 1998 %T Dual function of Ras in Raf activation. %A W Li %A Melnick, M %A Perrimon, N %K Animals %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Enzyme Activation %K Gene Expression Regulation %K In Situ Hybridization %K Microinjections %K Models, Biological %K Mutation %K Protein Binding %K Proto-Oncogene Proteins c-raf %K Proto-Oncogene Proteins p21(ras) %K Repressor Proteins %K RNA, Messenger %XThe small guanine nucleotide binding protein p21(Ras) plays an important role in the activation of the Raf kinase. However, the precise mechanism by which Raf is activated remains unclear. It has been proposed that the sole function of p21(Ras )in Raf activation is to recruit Raf to the plasma membrane. We have used Drosophila embryos to examine the mechanism of Raf (Draf) activation in the complete absence of p21(Ras) (Ras1). We demonstrate that the role of Ras1 in Draf activation is not limited to the translocation of Draf to the membrane through a Ras1-Draf association. In addition, Ras1 is essential for the activation of an additional factor which in turn activates Draf.
%B Development %V 125 %P 4999-5008 %8 1998 Dec %G eng %N 24 %1 http://www.ncbi.nlm.nih.gov/pubmed/9811584?dopt=Abstract %0 Journal Article %J Methods %D 1998 %T Editorial %A Perrimon, Norbert %B Methods %V 14 %P 353 %G eng %N 4 %0 Journal Article %J Development %D 1998 %T Identifying loci required for follicular patterning using directed mosaics. %A Duffy, JB %A Harrison, D A %A Perrimon, N %K Animals %K Body Patterning %K DNA Nucleotidyltransferases %K DNA-Binding Proteins %K Drosophila melanogaster %K Female %K Genes, Insect %K Mitosis %K Mosaicism %K Oogenesis %K Ovarian Follicle %K Phenotype %K Receptor Protein-Tyrosine Kinases %K Receptor, Epidermal Growth Factor %K Recombination, Genetic %K Saccharomyces cerevisiae Proteins %K Signal Transduction %K Transcription Factors %XWe have developed a 'directed mosaic' system in Drosophila by using the GAL4 system to control the expression of the yeast recombinase, FLP, in a spatial and temporal fashion. By directing FLP expression, we show that it is possible to efficiently and specifically target loss-of-function studies for vital loci to the developmental pathway of interest. A simple F1 adult phenotypic screen demonstrated that most adult tissues can be analyzed with this approach. Using GAL4 lines expressed during oogenesis, we have refined the system to examine the roles of vital loci in the development of the follicular epithelium. We have identified essential genes involved in egg chamber organization, cell migration and cell shape. Further, we have used this technique to gain insights into the role of the Drosophila EGF receptor pathway in establishing the egg axes. Finally, using different UAS-FLP, GAL4 and existing FRT lines, we have built stocks that permit the analysis of approximately 95% of the genome in follicular mosaics.
%B Development %V 125 %P 2263-71 %8 1998 Jun %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/9584125?dopt=Abstract %0 Journal Article %J Mol Cell %D 1998 %T Opposing actions of CSW and RasGAP modulate the strength of Torso RTK signaling in the Drosophila terminal pathway. %A Cleghon, V %A Feldmann, P %A Ghiglione, C %A Copeland, T D %A Perrimon, N %A Hughes, D A %A Morrison, D K %K Animals %K Binding Sites %K Drosophila %K Drosophila Proteins %K Insect Proteins %K Phosphorylation %K Phosphotyrosine %K Protein Binding %K Protein Tyrosine Phosphatases %K Protein Tyrosine Phosphatases, Non-Receptor %K Proteins %K ras GTPase-Activating Proteins %K Receptor Protein-Tyrosine Kinases %K Repressor Proteins %K Signal Transduction %K Substrate Specificity %K Tyrosine %XIn Drosophila, specification of embryonic terminal cells is controlled by the Torso receptor tyrosine kinase. Here, we analyze the molecular basis of positive (Y630) and negative (Y918) phosphotyrosine (pY) signaling sites on Torso. We find that the Drosophila homolog of RasGAP associates with pY918 and is a negative effector of Torso signaling. Further, we show that the tyrosine phosphatase Corkscrew (CSW), which associates with pY630, specifically dephosphorylates the negative pY918 Torso signaling site, thus identifying Torso to be a substrate of CSW in the terminal pathway. CSW also serves as an adaptor protein for DRK binding, physically linking Torso to Ras activation. The opposing actions of CSW and RasGAP modulate the strength of the Torso signal, contributing to the establishment of precise boundaries for terminal structure development.
%B Mol Cell %V 2 %P 719-27 %8 1998 Dec %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/9885560?dopt=Abstract %0 Journal Article %J Nature %D 1998 %T Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. %A Bellaiche, Y %A The, I %A Perrimon, N %K Amino Acid Sequence %K Animals %K Body Patterning %K Cloning, Molecular %K Drosophila %K Drosophila Proteins %K Genes, Insect %K Genes, Tumor Suppressor %K Hedgehog Proteins %K Humans %K Insect Proteins %K Membrane Proteins %K Molecular Sequence Data %K Mutation %K N-Acetylglucosaminyltransferases %K Proteins %K Signal Transduction %XHedgehog (Hh) proteins act through both short-range and long-range signalling to pattern tissues during invertebrate and vertebrate development. The mechanisms allowing Hedgehog to diffuse over a long distance and to exert its long-range effects are not understood. Here we identify a new Drosophila gene, named tout-velu, that is required for diffusion of Hedgehog. Characterization of tout-velu shows that it encodes an integral membrane protein that belongs to the EXT gene family. Members of this family are involved in the human multiple exostoses syndrome, which affects bone morphogenesis. Our results, together with the previous characterization of the role of Indian Hedgehog in bone morphogenesis, lead us to propose that the multiple exostoses syndrome is associated with abnormal diffusion of Hedgehog proteins. These results show the existence of a new conserved mechanism required for diffusion of Hedgehog.
%B Nature %V 394 %P 85-8 %8 1998 Jul 2 %G eng %N 6688 %1 http://www.ncbi.nlm.nih.gov/pubmed/9665133?dopt=Abstract %R 10.1038/27932 %0 Journal Article %J Int J Dev Biol %D 1998 %T Creating mosaics in Drosophila. %A Perrimon, N %K Animals %K Drosophila %K Female %K Gene Expression %K Genes, Insect %K Genetic Techniques %K Germ-Line Mutation %K Molecular Biology %K Mosaicism %XThe ability to create mosaic animals allows the phenotypic analysis of patches of groups of genetically different cells that develop in a wild type environment. In Drosophila, a variety of techniques have been developed over the years to generate mosaics, and in this chapter, I review the techniques that our laboratory has developed. These include the "Dominant Female Sterile" technique which allows the analysis of gene functions to oogenesis and embryogenesis; the "Gal4-UAS" technique which allows the control of where and when specific genes are expressed; and, the "Positive Marked Mutant Lineages" technique which allows clones of cells to express a specific reporter gene.
%B Int J Dev Biol %V 42 %P 243-7 %8 1998 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/9654004?dopt=Abstract %0 Journal Article %J Trends Genet %D 1998 %T Frizzled signaling and the developmental control of cell polarity. %A Shulman, J M %A Perrimon, N %A Axelrod, J D %K Animals %K Caenorhabditis elegans %K Cell Polarity %K Drosophila %K Drosophila Proteins %K Frizzled Receptors %K Gene Expression Regulation, Developmental %K Hair %K Membrane Proteins %K Organ Specificity %K Photoreceptor Cells, Invertebrate %K Receptors, G-Protein-Coupled %K Signal Transduction %K Xenopus laevis %XWithin the last three years, Frizzled receptors have risen from obscurity to celebrity status owing to their functional identification as receptors for the ubiquitous family of secreted WNT signaling factors. However, the founding member of the Frizzled family, Drosophila Frizzled (FZ), was cloned almost a decade ago because of its role in regulating cell polarity within the plane of an epithelium. In this review, we consider the role of FZ in this intriguing context. We discuss recent progress towards elucidating mechanisms for the intracellular specification of planar polarity, and further review evidence for models of global polarity regulation at the tissue level. The data suggest that a genetic 'cassette', encoding a set of core signaling components, could pattern hair, bristle and ommatidial planar polarity in Drosophila, and that additional tissue-specific factors might explain the diversity of signal responses. Recently described examples from the nematode and frog suggest that the developmental control of cell polarity by FZ receptors might represent a functionally conserved signaling mechanism.
%B Trends Genet %V 14 %P 452-8 %8 1998 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/9825673?dopt=Abstract %0 Journal Article %J Biochim Biophys Acta %D 1998 %T Highlights of the 1998 Wnt meeting, Cambridge, MA, January 9-11. %A Perrimon, N %A Nusse, R %K Animals %K Armadillos %K Calcium-Calmodulin-Dependent Protein Kinases %K Glycogen Synthase Kinase 3 %K Humans %K Proto-Oncogene Proteins %K Signal Transduction %K Wnt Proteins %K Wnt4 Protein %B Biochim Biophys Acta %V 1377 %P R45-9 %8 1998 Jun 19 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/9659404?dopt=Abstract %0 Journal Article %J Cell %D 1998 %T Mammalian and Drosophila blood: JAK of all trades? %A Mathey-Prevot, B %A Perrimon, N %K Animals %K Blood Physiological Phenomena %K Drosophila %K Hematopoiesis %K Humans %K Janus Kinase 1 %K Mammals %K Protein-Tyrosine Kinases %K Signal Transduction %B Cell %V 92 %P 697-700 %8 1998 Mar 20 %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/9529244?dopt=Abstract %0 Journal Article %J Proc Natl Acad Sci U S A %D 1998 %T New advances in Drosophila provide opportunities to study gene functions. %A Perrimon, N %K Animals %K Body Patterning %K DNA, Complementary %K DNA-Binding Proteins %K Drosophila %K Fungal Proteins %K Gene Expression Regulation, Developmental %K Genes, Insect %K Mutation %K Phenotype %K Saccharomyces cerevisiae Proteins %K Transcription Factors %B Proc Natl Acad Sci U S A %V 95 %P 9716-7 %8 1998 Aug 18 %G eng %N 17 %1 http://www.ncbi.nlm.nih.gov/pubmed/9707540?dopt=Abstract %0 Journal Article %J Nature %D 1998 %T Sending all the right signals. %A Perrimon, N %A Duffy, JB %K Animals %K Biological Evolution %K Cell Lineage %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Insect Proteins %K Oogenesis %K Ovarian Follicle %K Receptor, Epidermal Growth Factor %K Signal Transduction %K Transforming Growth Factor alpha %K Transforming Growth Factors %B Nature %V 396 %P 18-9 %8 1998 Nov 5 %G eng %N 6706 %1 http://www.ncbi.nlm.nih.gov/pubmed/9817197?dopt=Abstract %R 10.1038/23815 %0 Book Section %B Symposia on Quantitative Biology %D 1998 %T Brainiac and Fringe are pioneer proteins that impart specificity to Notch signals during Drosophila development %A Goode, S %A Perrimon, Norbert %B Symposia on Quantitative Biology %I Cold Spring Harbor %V LXII %P 177-184 %G eng %0 Book Section %B DNA Repair Protocols: Eukaryotic Systems (Methods in Molecular Biology) %D 1998 %T Expression of I-Sce.I in Drosophila to study Double Stand Breaks %A Mogila, Vladic %A Bellaiche, Yohanns %A Perrimon, Norbert %B DNA Repair Protocols: Eukaryotic Systems (Methods in Molecular Biology) %I Humana Press %P 439-445 %G eng %0 Journal Article %J Development %D 1997 %T The Drosophila 14-3-3 protein Leonardo enhances Torso signaling through D-Raf in a Ras 1-dependent manner. %A W Li %A Skoulakis, E M %A Davis, R L %A Perrimon, N %K 14-3-3 Proteins %K Animals %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Genes, Insect %K Proteins %K Proto-Oncogene Proteins c-raf %K ras Proteins %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %K Tyrosine 3-Monooxygenase %X14-3-3 proteins have been shown to interact with Raf-1 and cause its activation when overexpressed. However, their precise role in Raf-1 activation is still enigmatic, as they are ubiquitously present in cells and found to associate with Raf-1 in vivo regardless of its activation state. We have analyzed the function of the Drosophila 14-3-3 gene leonardo (leo) in the Torso (Tor) receptor tyrosine kinase (RTK) pathway. In the syncytial blastoderm embryo, activation of Tor triggers the Ras/Raf/MEK pathway that controls the transcription of tailless (tll). We find that, in the absence of Tor, overexpression of leo is sufficient to activate tll expression. The effect of leo requires D-Raf and Ras1 activities but not KSR or DOS, two recently identified essential components of Drosophila RTK signaling pathways. Tor signaling is impaired in embryos derived from females lacking maternal expression of leo. We propose that binding to 14-3-3 by Raf is necessary but not sufficient for the activation of Raf and that overexpressed Drosophila 14-3-3 requires Ras1 to activate D-Raf.
%B Development %V 124 %P 4163-71 %8 1997 Oct %G eng %N 20 %1 http://www.ncbi.nlm.nih.gov/pubmed/9374412?dopt=Abstract %0 Journal Article %J Genes Dev %D 1997 %T Drosophila Jun relays the Jun amino-terminal kinase signal transduction pathway to the Decapentaplegic signal transduction pathway in regulating epithelial cell sheet movement. %A Hou, X S %A Goldstein, E S %A Perrimon, N %K Animals %K Calcium-Calmodulin-Dependent Protein Kinases %K Cell Differentiation %K Drosophila %K Drosophila Proteins %K Epithelial Cells %K Eye Proteins %K Gene Expression Regulation, Developmental %K Genes, Insect %K Insect Proteins %K JNK Mitogen-Activated Protein Kinases %K Membrane Glycoproteins %K Mitogen-Activated Protein Kinases %K Mutation %K Photoreceptor Cells, Invertebrate %K Proto-Oncogene Proteins c-jun %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %XWe have characterized mutations in the Drosophila homolog of the mammalian proto-oncogene c-Jun gene (Djun). We demonstrate that DJUN in the embryo is a downstream target of the JNK signal transduction pathway during dorsal closure formation, and that the function of the JNK/DJUN pathway is to control the localized expression of decapentalegic (dpp), a member of the TGF-beta growth factor family. In contrast to previous observations, we find that both in the embryo and during photoreceptor cell determination, DJUN is not regulated by a pathway that involves MAPK.
%B Genes Dev %V 11 %P 1728-37 %8 1997 Jul 1 %G eng %N 13 %1 http://www.ncbi.nlm.nih.gov/pubmed/9224721?dopt=Abstract %0 Journal Article %J Development %D 1997 %T The Drosophila sugarless gene modulates Wingless signaling and encodes an enzyme involved in polysaccharide biosynthesis. %A Häcker, U %A Lin, X %A Perrimon, N %K Amino Acid Sequence %K Animals %K Body Patterning %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Epidermis %K Genes, Insect %K Hedgehog Proteins %K Homeodomain Proteins %K Insect Proteins %K Molecular Sequence Data %K Nervous System %K Proteoglycans %K Proto-Oncogene Proteins %K RNA, Messenger %K Signal Transduction %K Transcription Factors %K Uridine Diphosphate Glucose Dehydrogenase %K Wnt1 Protein %XWe have identified and characterized a Drosophila gene, which we have named sugarless, that encodes a homologue of vertebrate UDP-glucose dehydrogenase. This enzyme is essential for the biosynthesis of various proteoglycans, and we find that in the absence of both maternal and zygotic activities of this gene, mutant embryos develop with segment polarity phenotypes reminiscent to loss of either Wingless or Hedgehog signaling. To analyze the function of Sugarless in cell-cell interaction processes, we have focused our analysis on its requirement for Wingless signaling in different tissues. We report that sugarless mutations impair signaling by Wingless, suggesting that proteoglycans contribute to the reception of Wingless. We demonstrate that overexpression of Wingless can bypass the requirement for sugarless, suggesting that proteoglycans modulate signaling by Wingless, possibly by limiting its diffusion and thereby facilitating the binding of Wingless to its receptor. We discuss the possibility that tissue-specific regulation of proteoglycans may be involved in regulating both Wingless short- or long-range effects.
%B Development %V 124 %P 3565-73 %8 1997 Sep %G eng %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/9342049?dopt=Abstract %0 Journal Article %J Proc Natl Acad Sci U S A %D 1997 %T A genetic screen for mutations that disrupt an auditory response in Drosophila melanogaster. %A Eberl, D F %A Duyk, G M %A Perrimon, N %K Animals %K Auditory Perception %K Behavior, Animal %K Drosophila melanogaster %K Genes, Insect %K Mutation %XHearing is one of the last sensory modalities to be subjected to genetic analysis in Drosophila melanogaster. We describe a behavioral assay for auditory function involving courtship among groups of males triggered by the pulse component of the courtship song. In a mutagenesis screen for mutations that disrupt the auditory response, we have recovered 15 mutations that either reduce or abolish this response. Mutant audiograms indicate that seven mutants reduced the amplitude of the response at all intensities. Another seven abolished the response altogether. The other mutant, 5L3, responded only at high sound intensities, indicating that the threshold was shifted in this mutant. Six mutants were characterized in greater detail. 5L3 had a general courtship defect; courtship of females by 5L3 males also was affected strongly. 5P1 males courted females normally but had reduced success at copulation. 5P1 and 5N18 showed a significant decrement in olfactory response, indicating that the defects in these mutations are not specific to the auditory pathway. Two other mutants, 5M8 and 5N30, produced amotile sperm although in 5N30 this phenotype was genetically separable from the auditory phenotype. Finally, a new adult circling behavior phenotype, the pirouette phenotype, associated with massive neurodegeneration in the brain, was discovered in two mutants, 5G10 and 5N18. This study provides the basis for a genetic and molecular dissection of auditory mechanosensation and auditory behavior.
%B Proc Natl Acad Sci U S A %V 94 %P 14837-42 %8 1997 Dec 23 %G eng %N 26 %1 http://www.ncbi.nlm.nih.gov/pubmed/9405700?dopt=Abstract %0 Journal Article %J Genes Dev %D 1997 %T Inhibition of patterned cell shape change and cell invasion by Discs large during Drosophila oogenesis. %A Goode, S %A Perrimon, N %K Animals %K Cell Movement %K Cell Size %K Clone Cells %K Drosophila %K Drosophila Proteins %K Female %K Genes, Insect %K Genes, Reporter %K Genes, Tumor Suppressor %K Insect Proteins %K Membrane Proteins %K Microscopy, Confocal %K Mutation %K Nerve Tissue Proteins %K Oocytes %K Oogenesis %K Ovary %K Phenotype %K Protein Tyrosine Phosphatases %K Tumor Suppressor Proteins %XDrosophila Discs large (Dlg) is a tumor suppressor gene whose loss in epithelial tissues causes disrupted cell polarity and increased cell proliferation. A human Dlg homolog, hDlg, has been implicated in tumorigenic processes via its association with the product of the Adenomatous Polyposis Coli (APC) gene. We show for the first time that Drosophila Dlg is required to block cell invasion. Loss of dlg activity during oogenesis causes follicle cells to change shape and invade in a pattern similar to border cells, a small population of cells that break from the post-mitotic follicular epithelium during wild-type oogenesis, yet dlg mutant cells have not adopted a border cell fate. Both functional and morphological evidence indicates that cooperation between germ cell and follicle cell Dlg, probably mediated by Dlg PDZ domains, is crucial for regulating cell mixing, suggesting a novel developmental mechanism and mode of action for the Dlg family of molecules. These findings suggest that Dlg does not simply inhibit individual cell behaviors during oogenesis, but rather acts in a developmental pathway essential for blocking cell proliferation and migration in a spatio-temporally defined manner. A model for Dlg action in blocking cell invasion is presented.
%B Genes Dev %V 11 %P 2532-44 %8 1997 Oct 1 %G eng %N 19 %1 http://www.ncbi.nlm.nih.gov/pubmed/9334318?dopt=Abstract %0 Journal Article %J Trends Genet %D 1997 %T The JAK-STAT pathway in Drosophila. %A Hou, X S %A Perrimon, N %K Animals %K Cell Division %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Female %K Gene Expression Regulation, Developmental %K Janus Kinases %K Larva %K Milk Proteins %K Neoplasms %K Protein-Tyrosine Kinases %K Signal Transduction %K STAT Transcription Factors %K STAT5 Transcription Factor %K Trans-Activators %K Transcription Factors %XRecent studies in Drosophila have identified a single JAK and a single STAT protein. Genetic and biochemical analyses reveal that these two proteins operate in the same signal transduction pathway. Phenotypic analyses of JAK and STAT mutants implicate this pathway in a number of developmental decisions, in particular the regulation of pair-rule genes and fly hematopoiesis.
%B Trends Genet %V 13 %P 105-10 %8 1997 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/9066269?dopt=Abstract %0 Journal Article %J Medecine Sciences %D 1997 %T [La voie de signalisation Wingless chez la Drosophile] (French) %A Bellaiche, Yohanns %A Perrimon, Norbert %B Medecine Sciences %V 13 %P 165-174 %G eng %0 Journal Article %J Genetics %D 1997 %T A new enhancer of position-effect variegation in Drosophila melanogaster encodes a putative RNA helicase that binds chromosomes and is regulated by the cell cycle. %A Eberl, D F %A Lorenz, L J %A Melnick, M B %A Sood, V %A Lasko, P %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Cell Cycle %K Cell Nucleus %K Chromosomes %K Cloning, Molecular %K DEAD-box RNA Helicases %K Drosophila melanogaster %K Drosophila Proteins %K Enhancer Elements, Genetic %K Female %K Gene Expression Regulation, Enzymologic %K Genes, Insect %K Male %K Mitosis %K Molecular Sequence Data %K Oogenesis %K RNA Helicases %K RNA Nucleotidyltransferases %K Sequence Homology, Amino Acid %XIn Drosophila melanogaster, position-effect variegation of the white gene has been a useful phenomenon by which to study chromosome structure and the genes that modify it. We have identified a new enhancer of variegation locus, Dmrnahel (hel). Deletion of mutation of hel enhances white variegation, and this can be reversed by a transformed copy of hel+. In the presence of two endogenous copies, the transformed hel+ behaves as a suppressor of variegation. hel is an essential gene and functions both maternally and zygotically. The HEL protein is similar to known RNA helicases, but contains an unusual variant (DECD) of the DEAD motif common to these proteins. Potential HEL homologues have been found in mammals, yeast and worms. HEL protein associates with salivary gland chromosomes and locates to nuclei of embryos and ovaries, but disappears in mitotic domains of embryos as chromosomes condense. We propose that the HEL protein promotes an open chromatin structure that favors transcription during development by regulating the spread of heterochromatin, and that HEL is regulated by, and may have a role in, the mitotic cell cycle during embryogenesis.
%B Genetics %V 146 %P 951-63 %8 1997 Jul %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/9215899?dopt=Abstract %0 Journal Article %J Nature %D 1997 %T The nuclear hormone receptor Ftz-F1 is a cofactor for the Drosophila homeodomain protein Ftz. %A Yu, Y. %A W Li %A Su, K %A Yussa, M %A Han, W. %A Perrimon, N %A Pick, L %K Animals %K Base Sequence %K Binding Sites %K Cloning, Molecular %K DNA %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Female %K Fushi Tarazu Transcription Factors %K Gene Expression Regulation, Developmental %K Homeodomain Proteins %K Insect Proteins %K Male %K Molecular Sequence Data %K Mutation %K Protein Binding %K Receptors, Cytoplasmic and Nuclear %K Saccharomyces cerevisiae %K Steroidogenic Factor 1 %K Transcription Factors %XHomeobox genes specify cell fate and positional identity in embryos throughout the animal kingdom. Paradoxically, although each has a specific function in vivo, the in vitro DNA-binding specificities of homeodomain proteins are overlapping and relatively weak. A current model is that homeodomain proteins interact with cofactors that increase specificity in vivo. Here we use a native binding site for the homeodomain protein Fushi tarazu (Ftz) to isolate Ftz-F1, a protein of the nuclear hormone-receptor superfamily and a new Ftz cofactor. Ftz and Ftz-F1 are present in a complex in Drosophila embryos. Ftz-F1 facilitates the binding of Ftz to DNA, allowing interactions with weak-affinity sites at concentrations of Ftz that alone bind only high-affinity sites. Embryos lacking Ftz-F1 display ftz-like pair-rule cuticular defects. This phenotype is a result of abnormal ftz function because it is expressed but fails to activate downstream target genes. Cooperative interaction between homeodomain proteins and cofactors of different classes may serve as a general mechanism to increase HOX protein specificity and to broaden the range of target sites they regulate.
%B Nature %V 385 %P 552-5 %8 1997 Feb 6 %G eng %N 6616 %1 http://www.ncbi.nlm.nih.gov/pubmed/9020364?dopt=Abstract %R 10.1038/385552a0 %0 Book Section %B Curr Top Dev Biol %D 1997 %T Paradigms to study signal transduction pathways in Drosophila. %A Engstrom, L %A Noll, E %A Perrimon, N %K Animals %K Biological Evolution %K Body Patterning %K Drosophila melanogaster %K Embryonic Development %K Gene Expression Regulation, Developmental %K Oogenesis %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %B Curr Top Dev Biol %V 35 %P 229-61 %8 1997 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/9292272?dopt=Abstract %0 Journal Article %J Cell %D 1997 %T There must be 50 ways to rule the signal: the case of the Drosophila EGF receptor. %A Perrimon, N %A Perkins, L A %K Animals %K Drosophila %K Receptor, Epidermal Growth Factor %K Signal Transduction %B Cell %V 89 %P 13-6 %8 1997 Apr 4 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/9094709?dopt=Abstract %0 Book Section %B Cytoskeletal-Membrane and Signal Transduction %D 1997 %T Components of the Wnt signaling pathway %A Häcker, Udo %A Perrimon, Norbert %E Codwin, P %E Klymkowsky, M %B Cytoskeletal-Membrane and Signal Transduction %I Landes Bioscience %P 61-72 %G eng %0 Book Section %B Genetic Engineering: Principles and Methods %D 1997 %T Specificity of receptor tyrosine signaling pathways: Lessons from Drosophila %A Li, Willis %A Perrimon, Norbert %B Genetic Engineering: Principles and Methods %I Plenum Press %V 19 %P 167-182 %G eng %0 Journal Article %J Genetics %D 1996 %T The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. %A Chou, T B %A Perrimon, N %K Alleles %K Animals %K Drosophila melanogaster %K Female %K Genes, Dominant %K Genes, Insect %K Genetic Techniques %K Germ-Line Mutation %K Male %XThe production of female germline chimeras is invaluable for analyzing the tissue specificity of recessive female sterile mutations as well as detecting the maternal effect of recessive zygotic lethal mutations. Previously, we developed the "FLP-DFS" technique to efficiently generate germline clones. This technique uses the X-linked germline-dependent dominant female sterile mutation ovoD1 as a selection for the detection of germline recombination events, and the FLP-FRT recombination system to promote site-specific chromosomal exchange. This method allows the efficient production of germline mosaics only on the X chromosome. In this paper we have built chromosomes that allow the use of this technique to the autosomes. We describe the various steps involved in the development of this technique as well as the properties of the chromosomes utilized.
%B Genetics %V 144 %P 1673-9 %8 1996 Dec %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/8978054?dopt=Abstract %0 Journal Article %J Mech Dev %D 1996 %T Conservation of dishevelled structure and function between flies and mice: isolation and characterization of Dvl2. %A Klingensmith, J %A Yang, Y. %A Axelrod, J D %A Beier, D R %A Perrimon, N %A Sussman, D J %K Adaptor Proteins, Signal Transducing %K Amino Acid Sequence %K Animals %K Chromosome Mapping %K Conserved Sequence %K Drosophila %K Embryo, Mammalian %K Embryo, Nonmammalian %K In Situ Hybridization %K Mice %K Molecular Sequence Data %K Phosphoproteins %K Proteins %K RNA, Messenger %K Sequence Homology, Amino Acid %K Tissue Distribution %XThe segment polarity gene dishevelled (dsh) of Drosophila is required for pattern formation of the embryonic segments and the adult imaginal discs. dsh encodes the earliest-acting and most specific known component of the signal transduction pathway of Wingless, an extracellular signal homologous to Wnt1 in mice. We have previously described the isolation and characterization of the Dvl1 mouse dsh homolog. We report here the isolation of a second mouse dsh homolog, Dvl2, which maps to chromosome 11. The Dvl2 amino acid sequence is equally related to the dsh sequence as is that of Dvl1, but Dvl2 is most similar to the Xenopus homolog Xdsh. However, unlike the other vertebrate dsh homologs. Like the other genes, Dvl2 is ubiquitously expressed throughout most of embryogenesis and is expressed in many adult organs. We have developed an assay for dsh function in fly embryos, and show that Dvl2 can partially rescue the segmentation defects of embryos devoid of dsh. Thus, Dvl2 encodes a mammalian homolog of dsh which can transduce the Wingless signal.
%B Mech Dev %V 58 %P 15-26 %8 1996 Aug %G eng %N 1-2 %1 http://www.ncbi.nlm.nih.gov/pubmed/8887313?dopt=Abstract %0 Journal Article %J Dev Biol %D 1996 %T The Drosophila kekkon genes: novel members of both the leucine-rich repeat and immunoglobulin superfamilies expressed in the CNS. %A Musacchio, M %A Perrimon, N %K Amino Acid Sequence %K Animals %K Central Nervous System %K Cloning, Molecular %K Drosophila %K Drosophila Proteins %K Epithelium %K Female %K Gene Expression Regulation, Developmental %K Genes, Insect %K Immunoglobulins %K Leucine %K Membrane Proteins %K Molecular Sequence Data %K Mutation %K Nerve Tissue Proteins %K Organ Specificity %K Ovary %K Protein Tyrosine Phosphatases %K Repetitive Sequences, Nucleic Acid %K Restriction Mapping %K RNA, Messenger %K Sequence Analysis, DNA %XWe have identified two members of a novel class of genes in Drosophila that encode putative transmembrane proteins with six leucine-rich repeats and a single immunoglobulin loop. These two molecules, Kek1 and Kek2, show striking conservation in their extracellular domains and have large and more divergent intracellular regions. Both genes are expressed in neurons as they differentiate in the embryonic central nervous system (CNS). kek1 is also expressed in other patterned epithelia, such as the follicle cells of the developing egg chamber, where it is found in a dorsal-ventral gradient around the oocyte. The homology of the kek genes to other known adhesion and signaling molecules, together with their expression patterns, suggests that both genes are involved in interactions at the cell surface. Genetic analysis reveals that deletion of the kek1 gene causes no obvious developmental defects. The coexpression of kek2 in the CNS leads us to suggest that Kek1 is part of a family of cell surface proteins with redundant function.
%B Dev Biol %V 178 %P 63-76 %8 1996 Aug 25 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/8812109?dopt=Abstract %R 10.1006/dbio.1996.0198 %0 Journal Article %J Genes Dev %D 1996 %T Drosophila terminal structure development is regulated by the compensatory activities of positive and negative phosphotyrosine signaling sites on the Torso RTK. %A Cleghon, V %A Gayko, U %A Copeland, T D %A Perkins, L A %A Perrimon, N %A Morrison, D K %K Amino Acids %K Animals %K Baculoviridae %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Mutation %K Phosphopeptides %K Phosphorylation %K Precipitin Tests %K Protein Binding %K Protein Tyrosine Phosphatases %K Protein Tyrosine Phosphatases, Non-Receptor %K Receptor Protein-Tyrosine Kinases %K Recombinant Proteins %K Repressor Proteins %K Sequence Analysis %K Signal Transduction %K src Homology Domains %K Substrate Specificity %K Tyrosine %XSpecification of cell fates in the nonsegmented terminal regions of developing Drosophila embryos is under the control of a signal transduction pathway mediated by the receptor tyrosine kinase Torso (Tor). Here, we identify tyrosines (Y) 630 and 918 as the major sites of Tor autophosphorylation. We demonstrate that mutation of Y630, a site required for association with and tyrosine phosphorylation of the tyrosine phosphatase Corkscrew, decreases the efficiency of Tor signaling. In contrast, mutation of Y918, a site capable of binding mammalian rasGAP and PLC-gammal, increases Tor signaling. Interestingly, when receptors contain mutations in both the Y630 and Y918 sites, Tor signaling is restored to wild-type levels. These results identify a novel mechanism whereby Tor function is regulated using compensatory signals generated from distinct autophosphorylation sites and reveal an underlying signaling pathway for terminal development.
%B Genes Dev %V 10 %P 566-77 %8 1996 Mar 1 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/8598287?dopt=Abstract %0 Journal Article %J Science %D 1996 %T Interaction between Wingless and Notch signaling pathways mediated by dishevelled. %A Axelrod, J D %A Matsuno, K %A Artavanis-Tsakonas, S %A Perrimon, N %K Adaptor Proteins, Signal Transducing %K Amino Acid Sequence %K Animals %K Clone Cells %K Drosophila %K Drosophila Proteins %K Genes, Insect %K Intracellular Signaling Peptides and Proteins %K Membrane Proteins %K Molecular Sequence Data %K Mutation %K Phenotype %K Phosphoproteins %K Proteins %K Proto-Oncogene Proteins %K Pupa %K Receptors, Notch %K Signal Transduction %K Wings, Animal %K Wnt1 Protein %XIn Drosophila, the Wingless and Notch signaling pathways function in m any of the same developmental patterning events. Genetic analysis demonstrates that the dishevelled gene, which encodes a molecule previously implicated in implementation of the Winglass signal, interacts antagonistically with Notch and one of its known ligands, Delta. A direct physical interaction between Dishevelled and the Notch carboxyl terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction. It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross talk observed between these pathways.
%B Science %V 271 %P 1826-32 %8 1996 Mar 29 %G eng %N 5257 %1 http://www.ncbi.nlm.nih.gov/pubmed/8596950?dopt=Abstract %0 Journal Article %J Cell %D 1996 %T Marelle acts downstream of the Drosophila HOP/JAK kinase and encodes a protein similar to the mammalian STATs. %A Hou, X S %A Melnick, M B %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Cloning, Molecular %K DNA %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Female %K Genes, Insect %K Janus Kinases %K Male %K Models, Genetic %K Molecular Sequence Data %K Mutation %K Phenotype %K Protein-Tyrosine Kinases %K Sequence Homology, Amino Acid %K Signal Transduction %K Suppression, Genetic %K Trans-Activators %K Transcription Factors %XWe have identified a putative Drosophila STAT protein named Marelle that exhibits mutant phenotypes identical to mutations in the Hopscotch/JAK kinase. We show that a reduction in the amount of marelle gene activity suppresses the phenotype associated with a gain-of-function mutation in hopscotch and enhances the phenotype associated with a weak hopscotch mutation. We propose that Hopscotch activates Marelle to regulate transcription of target genes such as the pair rule gene even-skipped. Our results demonstrate the existence of an invertebrate JAK/STAT system.
%B Cell %V 84 %P 411-9 %8 1996 Feb 9 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/8608595?dopt=Abstract %0 Journal Article %J Development %D 1996 %T The neurogenic genes egghead and brainiac define a novel signaling pathway essential for epithelial morphogenesis during Drosophila oogenesis. %A Goode, S %A Melnick, M %A Chou, T B %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Cell Adhesion %K Cell Movement %K Drosophila %K Drosophila Proteins %K Epithelium %K Genes, Insect %K Membrane Proteins %K Models, Biological %K Molecular Sequence Data %K Morphogenesis %K Mutation %K Nervous System %K Oogenesis %K RNA, Messenger %K Sequence Homology, Amino Acid %K Signal Transduction %XNotch (N) and other neurogenic genes have been implicated in two fundamental processes, lateral specification of cell fates, and epithelial development. Previous studies have suggested that the neurogenic gene brainiac (brn) is specifically required for epithelial development (Goode, S., Morgan, M., Liang, Y-P. and Mahowald, A. P. (1996). Dev. Biol. 178, 35-50). In this report we show that egghead (egh), a gene with phenotypes identical to brn, encodes for a novel, putative secreted or transmembrane protein. We describe the role of egh and brn germline function in the morphogenesis of the follicular epithelium from the time it is born through the time that it migrates towards the oocyte late in oogenesis. By comparing the function of germline egh and brn to N during oogenesis, we have obtained direct evidence for the involvement of Notch in maintenance of the follicle cell epithelium, and the specificity of brn and egh in epithelial development during oogenesis. The most striking phenotype observed for all three genes is a loss of apical-basal polarity and accumulation of follicular epithelial cells in multiple layers around the oocyte. The spatiotemporal onset of this adenoma-like phenotype correlates with the differential accumulation of egh transcripts in the oocyte at stage 4 of oogenesis. In contrast to N, we find that brn and egh are essential for the organization, but not specification, of stalk and polar cells. The expression patterns and functional requirements of brn, egh, and N lead us to propose that these genes mediate follicular morphogenesis by regulating germline-follicle cell adhesion. This proposal offers explanations for (1) the involvement of egh and brn in N-mediated epithelial development, but not lateral specification, (2) why brn and egh embryonic neurogenic phenotypes are not as severe as N phenotypes, and (3) how egh and brn influence Egfr-mediated processes. The correlation between the differential expression of egh in the oocyte and the differential requirement for brn, egh, and N in maintaining the follicular epithelium around the oocyte, suggests that Egghead is a critical component of a differential oocyte-follicle cell adhesive system.
%B Development %V 122 %P 3863-79 %8 1996 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/9012507?dopt=Abstract %0 Journal Article %J Dev Biol %D 1996 %T The nonreceptor protein tyrosine phosphatase corkscrew functions in multiple receptor tyrosine kinase pathways in Drosophila. %A Perkins, L A %A Johnson, M R %A Melnick, M B %A Perrimon, N %K Alleles %K Animals %K Blastoderm %K Drosophila %K Drosophila Proteins %K Embryo, Nonmammalian %K Female %K Gene Expression Regulation, Developmental %K Male %K Mammals %K Mutagenesis %K Nervous System %K Phenotype %K Protein Tyrosine Phosphatases %K Protein Tyrosine Phosphatases, Non-Receptor %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %K Wings, Animal %XCorkscrew (csw) encodes a nonreceptor protein tyrosine phosphatase (PTPase) that has been implicated in signaling from the Torso receptor tyrosine kinase (RTK). csw mutations, unlike tor mutations, are associated with zygotic lethality, indicating that Csw plays additional roles during development. We have conducted a detailed phenotypic analysis of csw mutations to identify these additional functions of Csw. Our results indicate that Csw operates positively downstream of other Drosophila RTKs such as the Drosophila epidermal growth factor receptor (DER), the fibroblast growth factor receptor (Breathless), and likely other RTKs. This model is substantiated by specific dosage interactions between csw and DER. It is proposed that Csw is part of the evolutionarily conserved "signaling cassette" that operates downstream of all RTKs. In support of this hypothesis, we demonstrate that SHP-2, a vertebrate PTPase similar to Csw and previously implicated in RTK signaling, encodes the functional vertebrate homologue of Csw.
%B Dev Biol %V 180 %P 63-81 %8 1996 Nov 25 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/8948575?dopt=Abstract %R 10.1006/dbio.1996.0285 %0 Journal Article %J Genes Dev %D 1996 %T The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing. %A Kadowaki, T %A Wilder, E %A Klingensmith, J %A Zachary, K %A Perrimon, N %K Amino Acid Sequence %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Endoplasmic Reticulum %K Extremities %K Female %K Gene Expression Regulation, Developmental %K Genes, Insect %K Hedgehog Proteins %K Insect Proteins %K Male %K Membrane Proteins %K Molecular Sequence Data %K Polymorphism, Restriction Fragment Length %K Protein Processing, Post-Translational %K Proteins %K Proto-Oncogene Proteins %K Sequence Alignment %K Solubility %K Wings, Animal %K Wnt1 Protein %XThe Wnt protein Wingless (Wg) functions as a signal in patterning of both the Drosophila embryo and imaginal discs. Lack of porcupine (porc) activity is associated with mutant phenotypes similar to those of wg mutations. In porc mutant embryos, Wg protein is confined to the cells that produce it, suggesting that Porc plays a role in processing or secretion of Wg. porc encodes a novel transmembrane protein that appears to be concentrated at the endoplasmic reticulum. We present both genetic and in vitro evidence demonstrating that porc is involved specifically in the processing of Wg. We identified a human sequence related to Porc suggesting the existence of a family of proteins involved in processing of Wnts.
%B Genes Dev %V 10 %P 3116-28 %8 1996 Dec 15 %G eng %N 24 %1 http://www.ncbi.nlm.nih.gov/pubmed/8985181?dopt=Abstract %0 Journal Article %J Nature %D 1996 %T wingless refines its own expression domain on the Drosophila wing margin. %A Rulifson, E J %A Micchelli, C A %A Axelrod, J D %A Perrimon, N %A Blair, S S %K Adaptor Proteins, Signal Transducing %K Animals %K Cell Lineage %K Drosophila %K Drosophila Proteins %K Embryonic Induction %K Gene Expression Regulation, Developmental %K Membrane Proteins %K Phosphoproteins %K Proteins %K Proto-Oncogene Proteins %K Receptors, Notch %K Signal Transduction %K Wings, Animal %K Wnt1 Protein %XThe imaginal discs of Drosophila, which give rise to the adult appendages, are patterned during a period of intense cell proliferation. The specification of differing regions occurs in some cases by subdividing the disc epithelium into lineage compartments. However, in most cases precise boundaries are formed between different cell types without early compartmentalization. One such boundary occurs between the wingless (wg)-expressing cells of the wing margin and the adjacent proneural cells, which give rise to margin sensory bristles. Here we show that this boundary arises in part by a mechanism of 'self-refinement', by which wingless protein (Wg) represses wg expression in adjacent cells. Cells unable to receive the Wg signal do not resolve the boundary between wg-expressing and proneural cells.
%B Nature %V 384 %P 72-4 %8 1996 Nov 7 %G eng %N 6604 %1 http://www.ncbi.nlm.nih.gov/pubmed/8900280?dopt=Abstract %R 10.1038/384072a0 %0 Journal Article %J Genetics %D 1996 %T Zygotic lethal mutations with maternal effect phenotypes in Drosophila melanogaster. II. Loci on the second and third chromosomes identified by P-element-induced mutations. %A Perrimon, N %A A. Lanjuin %A Arnold, C %A Noll, E %K Animals %K Chromosome Mapping %K DNA Transposable Elements %K Drosophila melanogaster %K Genetic Techniques %K Mutation %K Phenotype %XScreens for zygotic lethal mutations that are associated with specific maternal effect lethal phenotypes have only been conducted for the X chromosome. To identify loci on the autosomes, which represent four-fifths of the Drosophila genome, we have used the autosomal "FLP-DFS" technique to screen a collection of 496 P element-induced mutations established by the Berkeley Drosophila Genome Project. We have identified 64 new loci whose gene products are required for proper egg formation or normal embryonic development.
%B Genetics %V 144 %P 1681-92 %8 1996 Dec %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/8978055?dopt=Abstract %0 Journal Article %J Curr Opin Cell Biol %D 1996 %T Recent advances in understanding signal transduction pathways in worms and flies. %A Duffy, JB %A Perrimon, N %K Animals %K Caenorhabditis elegans %K Drosophila melanogaster %K Drosophila Proteins %K Female %K Forecasting %K Humans %K Membrane Proteins %K Oocytes %K Protein Kinases %K Receptor, Epidermal Growth Factor %K Receptors, Invertebrate Peptide %K Receptors, Notch %K Signal Transduction %XOne major challenge in the fields of signal transduction and pattern formation is to understand how multiple signals are integrated to determine cell fates. Two developmental systems, vulval development in Caenorhabditis elegans and axis formation during Drosophila melanogaster oogenesis, require the epidermal growth factor receptor tyrosine kinase and the NOTCH signaling pathways to specify cell fates. Current work in both systems has provided new opportunities to investigate the potential for the cross-talk between these different signaling pathways.
%B Curr Opin Cell Biol %V 8 %P 231-8 %8 1996 Apr %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/8791421?dopt=Abstract %0 Journal Article %J Cell %D 1996 %T Serpentine proteins slither into the wingless and hedgehog fields. %A Perrimon, N %K Animals %K DNA-Binding Proteins %K Drosophila melanogaster %K Drosophila Proteins %K Frizzled Receptors %K Hedgehog Proteins %K Insect Hormones %K Membrane Proteins %K Morphogenesis %K Phosphorylation %K Proteins %K Proto-Oncogene Proteins %K Receptors, Cell Surface %K Receptors, G-Protein-Coupled %K Signal Transduction %K Transcription Factors %K Wnt1 Protein %B Cell %V 86 %P 513-6 %8 1996 Aug 23 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/8752205?dopt=Abstract %0 Journal Article %J EMBO J %D 1995 %T Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects. %A Harrison, D A %A Binari, R %A Nahreini, T S %A Gilman, M %A Perrimon, N %K Animals %K Base Sequence %K Cell Line %K Cell Transformation, Neoplastic %K DNA Mutational Analysis %K Drosophila melanogaster %K Drosophila Proteins %K Enzyme Activation %K Gene Expression Regulation, Developmental %K Gene Expression Regulation, Enzymologic %K Genes, Insect %K Genes, Lethal %K Janus Kinases %K Larva %K Lymphatic System %K Molecular Sequence Data %K Phosphorylation %K Point Mutation %K Protein-Tyrosine Kinases %K Recombinant Fusion Proteins %K Transcription Factors %K Tyrosine %XIn mammals, many cytokines and growth factors stimulate members of the Janus kinase (JAK) family to transduce signals for the proliferation and differentiation of various cell types, particularly in hematopoietic lineages. Mutations in the Drosophila hopscotch (hop) gene, which encodes a JAK, also cause proliferative defects. Loss-of-function alleles result in lethality and underproliferation of diploid tissues of the larva. A dominant gain-of-function allele, Tumorous-lethal (hopTum-l), leads to formation of melanotic tumors and hypertrophy of the larval lymph glands, the hematopoietic organs. We show that a single amino acid change in Hop is associated with the hopTum-l mutation. Overexpression of either wild-type hop or hopTum-l in the larval lymph glands causes melanotic tumors and lymph gland hypertrophy indistinguishable from the original hopTum-l mutation. In addition, overexpression of Hop in other tissues of the larva leads to pattern defects in the adult or to lethality. Finally, overexpression of either hop or hopTum-l in Drosophila cell culture results in tyrosine phosphorylation of Hop protein. However, overexpression of hopTum-l results in greater phosphorylation than overexpression of the wild-type. We conclude that hopTum-l encodes a hyperactive Hop kinase and that overactivity of Hop in lymph glands causes malignant neoplasia of Drosophila blood cells.
%B EMBO J %V 14 %P 2857-65 %8 1995 Jun 15 %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/7796812?dopt=Abstract %0 Journal Article %J Nature %D 1995 %T Activation of posterior gap gene expression in the Drosophila blastoderm. %A Rivera-Pomar, R %A Lu, X. %A Perrimon, N %A Taubert, H %A Jäckle, H %K Animals %K Base Sequence %K Blastoderm %K DNA %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Embryonic Development %K Female %K Gene Expression Regulation, Developmental %K Genes, Reporter %K Homeodomain Proteins %K Insect Hormones %K Kruppel-Like Transcription Factors %K Male %K Molecular Sequence Data %K Repressor Proteins %K Trans-Activators %K Transcription Factors %XThe process of body prepatterning during Drosophila blastoderm formation relies on the localized activities of zygotic segmentation genes, which are controlled by asymmetrically distributed maternal determinants. The anterior determinant bicoid, a homeodomain transcription factor, forms an anterior-to-posterior concentration gradient. It interacts with the maternal transcription factor hunchback to activate the anterior zygotic patterning genes, including the central gap gene Krüppel (Kr). In contrast, the posterior maternal system does not provide such a decisive transcription factor, but rather prevents the repressor hunchback from acting in the posterior half so that the gap genes giant (gt) and knirps (kni) are activated by an as yet unknown transcription factor. Here we show that caudal, a conserved homeodomain protein that forms a posterior-to-anterior concentration gradient, and the anterior determinant bicoid cooperate to form a partly redundant activator system in the posterior region of the embryo.
%B Nature %V 376 %P 253-6 %8 1995 Jul 20 %G eng %N 6537 %1 http://www.ncbi.nlm.nih.gov/pubmed/7617036?dopt=Abstract %R 10.1038/376253a0 %0 Journal Article %J Development %D 1995 %T Dorsalizing and neuralizing properties of Xdsh, a maternally expressed Xenopus homolog of dishevelled. %A Sokol, S Y %A Klingensmith, J %A Perrimon, N %A Itoh, K %K Adaptor Proteins, Signal Transducing %K Amino Acid Sequence %K Animals %K Blotting, Northern %K Drosophila %K Ectoderm %K Embryonic Induction %K Gene Transfer Techniques %K Mesoderm %K Mice %K Molecular Sequence Data %K Nervous System %K Phosphoproteins %K Proteins %K Proto-Oncogene Proteins %K Sequence Homology, Amino Acid %K Signal Transduction %K Wnt Proteins %K Wnt1 Protein %K Xenopus laevis %K Xenopus Proteins %K Zebrafish Proteins %XSignaling factors of the Wnt proto-oncogene family are implicated in dorsal axis formation during vertebrate development, but the molecular mechanism of this process is not known. Studies in Drosophila have indicated that the dishevelled gene product is required for wingless (Wnt1 homolog) signal transduction. We demonstrate that injection of mRNA encoding a Xenopus homolog of dishevelled (Xdsh) into prospective ventral mesodermal cells triggers a complete dorsal axis formation in Xenopus embryos. Lineage tracing experiments show that cells derived from the injected blastomere contribute to anterior and dorsal structures of the induced axis. In contrast to its effect on mesoderm, overexpression of Xdsh mRNA in prospective ectodermal cells triggers anterior neural tissue differentiation. These studies suggest that Wnt signal transduction pathway is conserved between Drosophila and vertebrates and point to a role for maternal Xdsh product in dorsal axis formation and in neural induction.
%B Development %V 121 %P 1637-47 %8 1995 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/7600981?dopt=Abstract %0 Journal Article %J Mol Cell Biol %D 1995 %T A Drosophila CREB/CREM homolog encodes multiple isoforms, including a cyclic AMP-dependent protein kinase-responsive transcriptional activator and antagonist. %A Yin, J C %A Wallach, J S %A Wilder, E L %A Klingensmith, J %A Dang, D %A Perrimon, N %A Zhou, Y,H. %A Tully, T %A Quinn, W G %K Alternative Splicing %K Amino Acid Sequence %K Animals %K Base Sequence %K Biological Evolution %K Blotting, Northern %K Cloning, Molecular %K Conserved Sequence %K Cyclic AMP Response Element Modulator %K Cyclic AMP Response Element-Binding Protein %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Genes, Insect %K Head %K In Situ Hybridization %K Molecular Sequence Data %K Protein Binding %K Repressor Proteins %K Sequence Homology, Amino Acid %K Tissue Distribution %K Trans-Activators %K Transcription, Genetic %XWe have characterized a Drosophila gene that is a highly conserved homolog of the mammalian cyclic AMP (cAMP)-responsive transcription factors CREB and CREM. Uniquely among Drosophila genes characterized to date, it codes for a cAMP-responsive transcriptional activator. An alternatively spliced product of the same gene is a specific antagonist of cAMP-inducible transcription. Analysis of the splicing pattern of the gene suggests that the gene may be the predecessor of the mammalian CREB and CREM genes.
%B Mol Cell Biol %V 15 %P 5123-30 %8 1995 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/7651429?dopt=Abstract %0 Journal Article %J Development %D 1995 %T Dual functions of wingless in the Drosophila leg imaginal disc. %A Wilder, E L %A Perrimon, N %K Animals %K Base Sequence %K Bromodeoxyuridine %K DNA Primers %K Drosophila %K Drosophila Proteins %K Embryonic Induction %K Gene Expression %K Immunohistochemistry %K Male %K Models, Genetic %K Molecular Sequence Data %K Morphogenesis %K Phenotype %K Proto-Oncogene Proteins %K Wnt1 Protein %XThe Drosophila gene wingless is a member of the Wnt gene family, a group of genes that are involved in embryonic development and the regulation of cell proliferation. wingless encodes a secreted glycoprotein that plays a role in embryogenesis as well as in the development of adult structures. In the primordia of the adult limbs, the imaginal discs, wingless is expressed in an anterior ventral sector and is required for specification of ventral fate. Ectopic expression of low levels of Wingless in the leg discs leads to partial ventralization and outgrowths of the proximodistal axis. Wingless has thus been proposed to specify ventral fate in a concentration dependent manner (i.e., as a morphogen) and to organize the proximodistal axis. We have extended the analysis of Wingless function in the leg primordium through targeted ectopic expression. We find that Wingless has two functions in the leg disc. In the specification of ventral fate, our data indicate that Wingless does not function as a morphogen but instead appears to collaborate with other factors. In addition to its role in ventral fate specification, Wingless inhibits the commitment of dorsal cells toward a determined state and influences the regulation of proliferation. We propose a model in which Wingless achieves separate functions via spatially regulated mechanisms and discuss the significance of these functions during axial patterning and organization.
%B Development %V 121 %P 477-88 %8 1995 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/7768188?dopt=Abstract %0 Journal Article %J Dev Biol %D 1995 %T Evidence for engrailed-independent wingless autoregulation in Drosophila. %A Yoffe, K B %A Manoukian, A S %A Wilder, E L %A Brand, A H %A Perrimon, N %K Animals %K Base Sequence %K DNA Primers %K Drosophila %K Female %K Gene Expression Regulation, Developmental %K Genes, Insect %K Homeostasis %K Male %K Models, Genetic %K Molecular Sequence Data %K Phenotype %K Polymerase Chain Reaction %XProper spatial expression of the wingless (wg) gene in the Drosophila embryonic epidermis is crucial to intrasegmental patterning. Single cell wide wg expression is initiated at the blastoderm stage in response to combinatorial regulation by the pair rule genes. Later, during gastrulation, when the epidermal expression of the pair rule genes has disappeared, wg becomes regulated by the activity of the segment polarity genes. The segment polarity gene engrailed (en) is expressed in cells adjacent to the wg-expressing cells and is required to maintain wg transcription. Since wg is in turn required to maintain en expression, wg appears to autoregulate its own expression through an endependent paracrine feedback loop. In this paper, we demonstrate that wild-type wg expression requires wg activity during stage 9, prior to its requirement for en maintenance, indicating that wg has an autoregulatory role that is distinct from its paracrine feedback loop through en. In addition, by misexpressing Wg and En in distinct spatial patterns in the epidermis, we find that En is capable of inducing expression from the endogenous wg gene only in immediate adjacent cells which have been exposed to Wg. Furthermore, exogenous Wg expression enables maintenance of endogenous wg transcription in both wg and en mutant embryos. Our results support the model that in the wild-type embryo, wg has an autoregulatory function which is distinct and separable from paracrine regulation via en. We also provide evidence that late, localized Wg expression is crucial for the asymmetric patterning of epidermal cell types as reflected in the larval cuticle.
%B Dev Biol %V 170 %P 636-50 %8 1995 Aug %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/7649390?dopt=Abstract %R 10.1006/dbio.1995.1243 %0 Journal Article %J Development %D 1995 %T The porcupine gene is required for wingless autoregulation in Drosophila. %A Manoukian, A S %A Yoffe, K B %A Wilder, E L %A Perrimon, N %K Animals %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Genes, Insect %K Larva %K Phenotype %K Proteins %K Proto-Oncogene Proteins %K Signal Transduction %K Wnt1 Protein %XThe Drosophila segment polarity gene wingless (wg) is required in the regulation of engrailed (en) expression and the determination of cell fates in neighboring cells. This paracrine wg activity also regulates transcription of wg itself, through a positive feedback loop including en activity. In addition, wg has a second, more direct autoregulatory requirement that is distinct from the en-dependent feedback loop. Four gene products, encoded by armadillo (arm), dishevelled (dsh), porcupine (porc) and zeste-white 3 (zw3), have been previously implicated as components of wg paracrine signaling. Here we have used three different assays to assess the requirements of these genes in the more direct wg autoregulatory pathway. While the activities of dsh, zw3 and arm appear to be specific to the paracrine feedback pathway, the more direct autoregulatory pathway requires porc.
%B Development %V 121 %P 4037-44 %8 1995 Dec %G eng %N 12 %1 http://www.ncbi.nlm.nih.gov/pubmed/8575304?dopt=Abstract %0 Journal Article %J Cell %D 1995 %T The torso receptor tyrosine kinase can activate Raf in a Ras-independent pathway. %A Hou, X S %A Chou, T B %A Melnick, M B %A Perrimon, N %K Animals %K DNA-Binding Proteins %K Drosophila %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Germ-Line Mutation %K Insect Hormones %K Membrane Proteins %K Models, Biological %K Mosaicism %K Phenotype %K Protein-Serine-Threonine Kinases %K Protein-Tyrosine Kinases %K Proto-Oncogene Proteins %K Proto-Oncogene Proteins c-raf %K Proto-Oncogene Proteins p21(ras) %K Receptor Protein-Tyrosine Kinases %K Repressor Proteins %K Signal Transduction %K Son of Sevenless Proteins %XActivation of the receptor tyrosine kinase (RTK) torso defines the spatial domains of expression of the transcription factors tailless and huckebein. Previous analyses have demonstrated that Ras1 (p21ras) operates upstream of the D-Raf (Raf1) serine/threonine kinase in this signaling pathway. By using a recently developed technique of germline mosaics, we find that D-Raf can be activated by torso in the complete absence of Ras1. This result is supported by analysis of D-Raf activation in the absence of either the exchange factor Son of sevenless (Sos) or the adaptor protein drk (Grb2), as well as by the phenotype of a D-Raf mutation that abolishes binding of Ras1 to D-Raf. Our study provides in vivo evidence that Raf can be activated by an RTK in a Ras-independent pathway.
%B Cell %V 81 %P 63-71 %8 1995 Apr 7 %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/7720074?dopt=Abstract %0 Journal Article %J Mol Reprod Dev %D 1995 %T Dissection of the Torso signal transduction pathway in Drosophila. %A Perrimon, N %A Lu, X. %A Hou, X S %A Hsu, J C %A Melnick, M B %A Chou, T B %A Perkins, L A %K Animals %K Drosophila %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %XCell fate choice at the anterior and posterior embryonic termini of the Drosophila embryo requires the activation of a signal transduction pathway regulated by the receptor tyrosine kinase Torso. When Torso, which is uniformly distributed in the egg cell membrane, becomes activated locally at the termini, it triggers a phosphorylation cascade that culminates with localized expression of the transcription factors, tailless and huckebein. Expression of tailless and huckebein in turn determines terminal cell fates. Several genes have been characterized which encode proteins that are involved in Torso signaling: the adaptor protein Drk, the GTP-binding protein Ras1, the guanine nucleotide exchange factor Son of sevenless, and the kinases D-Raf and D-Mek. Genetic and molecular evidence supports a model in which these proteins lie in the same biochemical pathway. When activated by its ligand the membrane-bound receptor tyrosine kinase Torso initiates a signal transduction pathway mediated by Drk, Sos, and Ras1, which in turn activates a phosphorylation cascade mediated by the kinases D-Raf and D-Mek, which ultimately control the localized expression of the transcription factors tailless and huckebein. Recently, we found that D-Raf can be partially activated by Torso in the absence of Ras1, a finding supported by the phenotype of embryos lacking either Drk or Sos activity, as well as by the phenotype of a D-raf mutation that abolishes binding of Ras1 to D-Raf. These findings indicate that full D-Raf activation requires input not only from Ras1 but also from an as yet uncharacterized Ras1-independent pathway. In addition to these molecules we have characterized the putative protein tyrosine phosphatase Corkscrew as a positive transducer downstream of Torso.
%B Mol Reprod Dev %V 42 %P 515-22 %8 1995 Dec %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/8607984?dopt=Abstract %R 10.1002/mrd.1080420421 %0 Journal Article %J Cell %D 1995 %T Hedgehog and beyond. %A Perrimon, N %K Animals %K Cell Communication %K Cyclic AMP-Dependent Protein Kinases %K Drosophila %K Drosophila Proteins %K Embryonic Induction %K Extremities %K Feedback %K Hedgehog Proteins %K Proteins %K Signal Transduction %K Vertebrates %B Cell %V 80 %P 517-20 %8 1995 Feb 24 %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/7867057?dopt=Abstract %0 Book Section %B The Protein Kinase FactsBook: Protein-Serine Kinases %D 1995 %T DmRaf %A Melnick, M %A Perrimon, Norbert %E Hardie, Grahame %E Hanks, Steven %B The Protein Kinase FactsBook: Protein-Serine Kinases %I Academic Press %C Orlando, FL %P 331-332 %G eng %0 Book Section %B Metamorphosis: Post-embryonic Re-programming of Gene Expression in Amphibian and Insect Cells %D 1995 %T Genes involved in post-embryonic cell proliferation in Drosophila %A Wilder, Elizabeth L %A Perrimon, Norbert %E Gilbert, LI %E Atkinson, BG %E Tata, JR %B Metamorphosis: Post-embryonic Re-programming of Gene Expression in Amphibian and Insect Cells %I Elsevier Inc. %P 363-400 %G eng %0 Journal Article %J Nature %D 1994 %T Components of wingless signalling in Drosophila. %A Siegfried, E %A Wilder, E L %A Perrimon, N %K Adaptor Proteins, Signal Transducing %K Animals %K Armadillo Domain Proteins %K Cell Polarity %K Crosses, Genetic %K Drosophila %K Drosophila Proteins %K Female %K Gene Expression Regulation %K Genotype %K Insect Hormones %K Larva %K Male %K Mutation %K Phosphoproteins %K Proteins %K Proto-Oncogene Proteins %K Signal Transduction %K Trans-Activators %K Transcription Factors %K Wnt1 Protein %XThe determination of specific cell fates and polarity within each segmental unit of the Drosophila embryo involves the products of the segment polarity genes. One of these, wingless (wg), encodes a secreted protein that is homologous to the mammalian proto-oncogene Wnt-1 (refs 4, 5). In the embryonic epidermis, wg is expressed in a single row of cells within each segmental unit, although its activity is required for the correct patterning of most of the epidermis. Initially Wg signals to adjacent posterior cells, maintaining engrailed (en) expression. Later during embryogenesis, wg specifies the differentiation of naked cuticle. Wg signalling functions by inactivating or antagonizing the activity of zestewhite 3 (zw3). We have investigated the requirement in the Wg signal transduction pathway for the three genes armadillo (arm), dishevelled (dsh) and porcupine (porc), all of which have embryonic mutant phenotypes similar to wg. Our results indicate that dsh and porc act upstream of zw3, and arm acts downstream of zw3.
%B Nature %V 367 %P 76-80 %8 1994 Jan 6 %G eng %N 6458 %1 http://www.ncbi.nlm.nih.gov/pubmed/8107779?dopt=Abstract %R 10.1038/367076a0 %0 Journal Article %J Nature %D 1994 %T dishevelled and armadillo act in the wingless signalling pathway in Drosophila. %A Noordermeer, J %A Klingensmith, J %A Perrimon, N %A Nusse, R %K Adaptor Proteins, Signal Transducing %K Animals %K Armadillo Domain Proteins %K Cell Polarity %K Crosses, Genetic %K Drosophila %K Drosophila Proteins %K Female %K Genes, Homeobox %K Homeodomain Proteins %K Insect Hormones %K Male %K Phosphoproteins %K Proteins %K Proto-Oncogene Proteins %K Signal Transduction %K Trans-Activators %K Transcription Factors %K Wnt1 Protein %XThe Wnt genes encode conserved secreted proteins that play a role in normal development and tumorigenesis. Little is known about the signal transduction pathways of Wnt gene products. One of the best characterized Wnt family members is the Drosophila segment polarity gene wingless. We have investigated whether segment polarity genes with a wingless-like phenotype mediate the wingless signal. We used a wingless transgene controlled by a heat-shock promoter for genetic epistasis experiments. We show that wingless acts through dishevelled and armadillo to affect the expression of the homeobox gene engrailed and cuticle differentiation.
%B Nature %V 367 %P 80-3 %8 1994 Jan 6 %G eng %N 6458 %1 http://www.ncbi.nlm.nih.gov/pubmed/7906389?dopt=Abstract %R 10.1038/367080a0 %0 Journal Article %J Genes Dev %D 1994 %T The Drosophila segment polarity gene dishevelled encodes a novel protein required for response to the wingless signal. %A Klingensmith, J %A Nusse, R %A Perrimon, N %K Adaptor Proteins, Signal Transducing %K Amino Acid Sequence %K Animals %K Base Sequence %K Cell Polarity %K DNA %K Drosophila %K Drosophila Proteins %K Embryonic Induction %K Female %K Male %K Molecular Sequence Data %K Phenotype %K Phosphoproteins %K Proteins %K Proto-Oncogene Proteins %K Restriction Mapping %K Sequence Homology, Amino Acid %K Signal Transduction %K Transcription, Genetic %K Wnt1 Protein %XThe Drosophila Wnt-1 homolog, wingless (wg), is involved in the signaling of patterning information in several contexts. In the embryonic epidermis, Wg protein is secreted and taken up by neighboring cells, in which it is required for maintenance of engrailed transcription and accumulation of Armadillo protein. The dishevelled (dsh) gene mediates these signaling events as well as wg-dependent induction across tissue layers in the embryonic midgut. dsh is also required for the development processes in which wg functions in adult development. Overall, cells lacking dsh are unable to adopt fates specified by Wg. dsh functions cell autonomously, indicating that it is involved in the response of target cells to the Wg signal. dsh is expressed uniformly in the embryo and encodes a novel protein with no known catalytic motifs, although it shares a domain of homology with several junction-associated proteins. Our results demonstrate that dsh encodes a specific component of Wg signaling and illustrate that Wnt proteins may utilize a novel mechanism of extracellular signal transduction.
%B Genes Dev %V 8 %P 118-30 %8 1994 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/8288125?dopt=Abstract %0 Journal Article %J EMBO J %D 1994 %T Genetic and molecular analyses of mutations involved in Drosophila raf signal transduction. %A Lu, X. %A Melnick, M B %A Hsu, J C %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Binding Sites %K Chromosome Mapping %K Crosses, Genetic %K Drosophila %K Eye %K Female %K Genes, Insect %K Male %K Molecular Sequence Data %K Mutagenesis, Site-Directed %K Mutation %K Oogenesis %K Protein-Serine-Threonine Kinases %K Protein-Tyrosine Kinases %K Receptor Protein-Tyrosine Kinases %K Receptor, Epidermal Growth Factor %K Signal Transduction %K Suppression, Genetic %XWe have identified dominant mutations that suppress the lethality associated with an R217-->L mutation in the GTP.Ras binding region (CR1) of the Drosophila raf (D-raf) serine/threonine kinase. Four intragenic and seven extragenic suppressors were recovered. Each of the four intragenic mutations contains one compensatory amino acid change located in either the CR1 or the kinase domain of D-raf. The seven extragenic suppressors represent at least four genetic loci whose effects strongly suggest that they participate in both the sevenless and Drosophila EGF receptor (DER) signaling pathways. One of these mutations, Su(D-raf)34B, is an allele of D-mek which encodes the known signaling molecule MAPK kinase (MEK). A D83V mutation in D-MEK is identified and shown to be sufficient to confer the dominant activity of Su(D-raf)34B.
%B EMBO J %V 13 %P 2592-9 %8 1994 Jun 1 %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/8013459?dopt=Abstract %0 Journal Article %J Dev Biol %D 1994 %T Isolation and characterization of a mouse homolog of the Drosophila segment polarity gene dishevelled. %A Sussman, D J %A Klingensmith, J %A Salinas, P %A Adams, P S %A Nusse, R %A Perrimon, N %K Adaptor Proteins, Signal Transducing %K Aging %K Amino Acid Sequence %K Animals %K Base Sequence %K Brain %K DNA %K DNA Primers %K Drosophila %K Embryo, Nonmammalian %K Embryonic and Fetal Development %K Gene Expression %K Gestational Age %K Humans %K Male %K Mice %K Molecular Sequence Data %K Organ Specificity %K Phosphoproteins %K Polymerase Chain Reaction %K Protein Biosynthesis %K Proteins %K Restriction Mapping %K RNA, Messenger %K Sequence Homology, Amino Acid %K Species Specificity %K Spinal Cord %K Testis %XIn the Drosophila embryo dishevelled (dsh) function is required by target cells in order to respond to wingless (wg, the homolog of Wnt-1), demonstrating a role for dsh in Wnt signal transduction. We have isolated a mouse homolog of the Drosophila dsh segment polarity gene. The 695-amino-acid protein encoded by the mouse dishevelled gene (Dvl-1) shares 50% identity (65% similarity) with dsh. Similarity searches of protein and DNA data bases revealed that Dvl-1 encodes an otherwise novel polypeptide. While no functional motifs were identified, one region of Dvl-1 was found to be similar to a domain of discs large-1 (dlg), a Drosophila tumor suppressor gene. In the embryo, Dvl-1 is expressed in most tissues, with uniformly high levels in the central nervous system. From 7.5 days postcoitum Dvl-1 is expressed throughout the developing brain and spinal cord, including those regions expressing Wnt-1 and En. Expression of Dvl-1 in adult mice was found to be widespread, with brain and testis exhibiting the highest levels. The majority of Dvl-1 expression in the adult cerebellum is in the granular cell layer, similar to the pattern seen for engrailed-2 (En-2). Throughout postnatal development of the brain Dvl-1 is highly expressed in areas of high neuronal cell density.
%B Dev Biol %V 166 %P 73-86 %8 1994 Nov %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/7958461?dopt=Abstract %R 10.1006/dbio.1994.1297 %0 Journal Article %J Genes Dev %D 1994 %T Raf acts downstream of the EGF receptor to determine dorsoventral polarity during Drosophila oogenesis. %A Brand, A H %A Perrimon, N %K Animals %K Drosophila %K Female %K Gene Expression %K Genes, Insect %K Oogenesis %K Ovary %K Protein-Serine-Threonine Kinases %K Proto-Oncogene Proteins %K Proto-Oncogene Proteins c-raf %K Receptor, Epidermal Growth Factor %K Signal Transduction %XIn Drosophila, as in mammalian cells, the Raf serine/threonine kinase appears to act as a common transducer of signals from several different receptor tyrosine kinases. We describe a new role for Raf in Drosophila development, showing that Raf acts in the somatic follicle cells to specify the dorsoventral polarity of the egg. Targeted expression of activated Raf (Rafgof) within follicle cells is sufficient to dorsalize both the eggshell and the embryo, whereas reduced Raf activity ventralizes the eggshell. We show that Raf functions downstream of the EGF receptor to instruct the dorsal follicle cell fate. In this assay, human and Drosophila Rafgof are functionally similar, in that either can induce ventral follicle cells to assume a dorsal fate.
%B Genes Dev %V 8 %P 629-39 %8 1994 Mar 1 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/7926754?dopt=Abstract %0 Journal Article %J Genes Dev %D 1994 %T Stripe-specific regulation of pair-rule genes by hopscotch, a putative Jak family tyrosine kinase in Drosophila. %A Binari, R %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Female %K Gene Expression Regulation, Enzymologic %K Genes, Insect %K Genes, Lethal %K Janus Kinase 2 %K Janus Kinases %K Larva %K Male %K Molecular Sequence Data %K Promoter Regions, Genetic %K Protein-Tyrosine Kinases %K Proto-Oncogene Proteins %K Sequence Analysis, DNA %K Transcription Factors %K X Chromosome %XWe describe the characterization of the Drosophila gene, hopscotch (hop), which is required maternally for the establishment of the normal array of embryonic segments. In hop embryos, although expression of the gap genes appears normal, there are defects in the expression patterns of the pair-rule genes even-skipped, runt, and fushi tarazu, as well as the segment-polarity genes engrailed and wingless. We demonstrate that the effect of hop on the expression of these genes is stripe-specific. The hop gene encodes a putative nonreceptor tyrosine kinase of the Janus kinase family, based on an internal duplication of the catalytic domain. We present a model in which the Hop tyrosine kinase is involved in the control of pair-rule gene transcription in a stripe-specific manner. Our results provide the first evidence for stripe-specific regulation of pair-rule genes by a tyrosine kinase.
%B Genes Dev %V 8 %P 300-12 %8 1994 Feb 1 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/8314084?dopt=Abstract %0 Journal Article %J Genes Dev %D 1994 %T A temperature-sensitive MEK mutation demonstrates the conservation of the signaling pathways activated by receptor tyrosine kinases. %A Hsu, J C %A Perrimon, N %K Alleles %K Amino Acid Sequence %K Animals %K Base Sequence %K Conserved Sequence %K DNA Primers %K Drosophila %K Female %K Gene Expression Regulation, Developmental %K Gene Expression Regulation, Enzymologic %K Genes, Insect %K Male %K MAP Kinase Kinase 1 %K Mitogen-Activated Protein Kinase Kinases %K Molecular Sequence Data %K Mutagenesis, Site-Directed %K Mutation %K Protein-Serine-Threonine Kinases %K Protein-Tyrosine Kinases %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %K Species Specificity %K Temperature %XMEK, a dual specificity threonine/tyrosine kinase, has been postulated to be a convergent point for signaling from receptor protein tyrosine kinases (RTKs) and G-protein-coupled receptors. In contrast to yeast and mammalian cells where several MEKs have been isolated, only one Drosophila MEK (D-Mek) has been characterized to date. Previous studies have shown that D-Mek acts in the Torso RTK signaling pathway. To demonstrate that D-Mek also operates downstream of other RTKs, we generated a temperature-sensitive allele of D-mek (D-mekts) by site-directed mutagenesis based on the amino acid change of a yeast cdc2ts mutation. Using D-mekts, we show that in addition to its role in Torso signaling, D-Mek operates in the Sevenless and in the Drosophila epidermal growth factor RTK pathways. Because loss-of-function mutations in D-mek and the upstream receptors give rise to similar phenotypes, it suggests that D-mek is the only MEK activated by Drosophila RTKs. In addition, we demonstrate that different RTK pathways respond differently to alteration in D-Mek activity.
%B Genes Dev %V 8 %P 2176-87 %8 1994 Sep 15 %G eng %N 18 %1 http://www.ncbi.nlm.nih.gov/pubmed/7958887?dopt=Abstract %0 Journal Article %J Bioessays %D 1994 %T Drosophila wingless: a paradigm for the function and mechanism of Wnt signaling. %A Siegfried, E %A Perrimon, N %K Animals %K Cell Differentiation %K Cell Transformation, Neoplastic %K Drosophila melanogaster %K Drosophila Proteins %K Embryonic Induction %K Gene Expression Regulation %K Metamorphosis, Biological %K Mice %K Morphogenesis %K Multigene Family %K Nervous System %K Protein-Tyrosine Kinases %K Proto-Oncogene Proteins %K Proto-Oncogenes %K Sequence Homology %K Signal Transduction %K Species Specificity %K Wings, Animal %K Wnt Proteins %K Wnt1 Protein %K Xenopus %K Zebrafish Proteins %XThe link between oncogenesis and normal development is well illustrated by the study of the Wnt family of proteins. The first Wnt gene (int-1) was identified over a decade ago as a proto-oncogene, activated in response to proviral insertion of a mouse mammary tumor virus. Subsequently, the discovery that Drosophila wingless, a developmentally important gene, is homologous to int-1 supported the notion that int-1 may have a role in normal development. In the last few years it has been recognized that int-1 and Wingless belong to a large family of related glyco-proteins found in vertebrates and invertebrates. In recognition of this, members of this family have been renamed Wnts, an amalgam of int and Wingless. Investigation of Wnt genes in Xenopus and mouse indicates that Wnts have a role in cell proliferation, differentiation and body axis formation. Further analysis in Drosophila has revealed that Wingless function is required in several developmental processes in the embryo and imaginal discs. In addition, a genetic approach has identified some of the molecules required for the transmission and reception of the Wingless signal. We will review recent data which have contributed to our growing understanding of the function and mechanism of Drosophila Wingless signaling in cell fate determination, growth and specification of pattern.
%B Bioessays %V 16 %P 395-404 %8 1994 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/8080429?dopt=Abstract %R 10.1002/bies.950160607 %0 Journal Article %J Cell %D 1994 %T The genetic basis of patterned baldness in Drosophila. %A Perrimon, N %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Epistasis, Genetic %K Genes, Homeobox %K Genes, Insect %K Hedgehog Proteins %K Morphogenesis %K Proteins %K Proto-Oncogene Proteins %K Signal Transduction %K Wings, Animal %K Wnt1 Protein %B Cell %V 76 %P 781-4 %8 1994 Mar 11 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/7907277?dopt=Abstract %0 Journal Article %J Trends Biochem Sci %D 1994 %T Signal transduction in the early Drosophila embryo: when genetics meets biochemistry. %A Perrimon, N %A Desplan, C %K Animals %K Drosophila %K Embryo, Nonmammalian %K Gestational Age %K Signal Transduction %K Transcription, Genetic %XAn elegant combination of genetic and biochemical approaches has been used to investigate a variety of signal transduction pathways in developmental processes. Here, we describe the 'terminal' signaling system in the Drosophila embryo, which is responsible for pattern formation in the polar regions of the embryo. This pathway involves a membrane-bound receptor tyrosine kinase (RTK) that is similar to other Drosophila RTKs, such as sevenless, and the mammalian RTKs, such as the epidermal growth factor or platelet-derived growth factor receptors.
%B Trends Biochem Sci %V 19 %P 509-13 %8 1994 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/7855897?dopt=Abstract %0 Journal Article %J Curr Opin Cell Biol %D 1994 %T Signalling pathways initiated by receptor protein tyrosine kinases in Drosophila. %A Perrimon, N %K Animals %K Biological Evolution %K Drosophila %K Embryo, Nonmammalian %K Models, Structural %K Mutation %K Photoreceptor Cells, Invertebrate %K Protein Structure, Secondary %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %XThe isolation and characterization of Drosophila mutations in receptor protein tyrosine kinases (RPTKs) have allowed a detailed analysis of the cellular processes regulated by these proteins. Recent investigations have identified a number of putative ligands involved in the activation of the receptors, and have demonstrated that these RPTKs trigger an evolutionarily conserved biochemical pathway. In addition to molecules previously identified from vertebrate studies, i.e. Grb2, Sos, Ras-Gap, p21ras, Raf, MEK and MAPK, genetic studies have suggested that two novel proteins, the protein tyrosine phosphatase (PTPase) Csw and the transmembrane protein Rho, are involved in RPTK signalling.
%B Curr Opin Cell Biol %V 6 %P 260-6 %8 1994 Apr %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/8024818?dopt=Abstract %0 Journal Article %J Dev Biol %D 1994 %T The torso pathway in Drosophila: lessons on receptor tyrosine kinase signaling and pattern formation. %A Duffy, JB %A Perrimon, N %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Gene Expression Regulation, Developmental %K Genes, Insect %K Morphogenesis %K Oogenesis %K Protein-Tyrosine Kinases %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %XPattern formation at the anterior and posterior termini of the Drosophila embryo involves intercellular communication via the Torso receptor tyrosine kinase (RTK). Recent advances in the understanding of Torso signaling has provided further support for the conservation of a signal transduction cassette downstream of RTKs. In addition, the analysis of the Torso pathway has begun to reveal general molecular mechanisms by which cells may impart patterning information to their neighbors through the use of RTKs.
%B Dev Biol %V 166 %P 380-95 %8 1994 Dec %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/7813764?dopt=Abstract %R 10.1006/dbio.1994.1324 %0 Book Section %B Methods in Cell Biology %D 1994 %T Ectopic Expression in Drosophila %A Brand, Andrea %A Manoukian, Armen S %A Perrimon, Norbert %E Goldstein, Lawrence S B %E Fryberg, Eric A %B Methods in Cell Biology %I Academic Press %C Orlando, FL %V 44 %P 635-654 %G eng %0 Journal Article %J Development %D 1993 %T Autosomal P[ovoD1] dominant female-sterile insertions in Drosophila and their use in generating germ-line chimeras. %A Chou, T B %A Noll, E %A Perrimon, N %K Animals %K Chimera %K Cloning, Molecular %K Drosophila %K Female %K Gene Transfer Techniques %K Genes, Dominant %K Genetic Vectors %K In Situ Hybridization %K Infertility, Female %K Mutagenesis, Insertional %K Oocytes %K Ovarian Follicle %K Phenotype %XThe 'dominant female-sterile' technique used to generate germ-line mosaics in Drosophila is a powerful tool to determine the tissue specificity (germ line versus somatic) of recessive female-sterile mutations as well as to analyze the maternal effect of recessive zygotic lethal mutations. This technique requires the availability of germ-line-dependent, dominant female-sterile (DFS) mutations that block egg laying but do not affect viability. To date only one X-linked mutation, ovoD1 has been isolated that completely fulfills these criteria. Thus the 'DFS technique' has been largely limited to the X-chromosome. To extend this technique to the autosomes, we have cloned the ovoD1 mutation into a P-element vector and recovered fully expressed P[ovoD1] insertions on each autosomal arm. We describe the generation of these P[ovoD1] strains as well as demonstrate their use in generating germ-line chimeras. Specifically, we show that the Gap1 gene, which encodes a Drosophila homologue of mammalian GTPase-activating protein, is required in somatic follicle cells for embryonic dorsoventral polarity determination.
%B Development %V 119 %P 1359-69 %8 1993 Dec %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/8306893?dopt=Abstract %0 Journal Article %J Genes Dev %D 1993 %T Control of cell fate determination by p21ras/Ras1, an essential component of torso signaling in Drosophila. %A Lu, X. %A Chou, T B %A Williams, N G %A Roberts,T. %A Perrimon, N %K Animals %K Cell Differentiation %K Cleavage Stage, Ovum %K Drosophila %K Drosophila Proteins %K Enzyme Activation %K Female %K Genes, Insect %K Genes, ras %K Insect Hormones %K Male %K Membrane Proteins %K Morphogenesis %K Protein Tyrosine Phosphatases %K Protein-Serine-Threonine Kinases %K Protein-Tyrosine Kinases %K Proto-Oncogene Proteins %K Proto-Oncogene Proteins c-raf %K Proto-Oncogene Proteins p21(ras) %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %K Son of Sevenless Proteins %XDetermination of cell fate at the posterior termini of the Drosophila embryo is specified by the activation of the torso (tor) receptor tyrosine kinase. This signaling pathway is mediated by the serine/threonine kinase D-raf and a protein tyrosine phosphatase corkscrew (csw). We found that expression of an activated form of Ras1 during oogenesis resulted in embryos with tor gain-of-function phenotypes. To demonstrate that p21ras/Ras1 mediates tor signaling, we injected mammalian p21ras variants into early Drosophila embryos. We found that the injection of activated p21v-ras rescued the maternal-effect phenotypes of both tor and csw null mutations. These rescuing effects of p21v-ras are dependent on the presence of maternally derived D-raf activity. In addition, wild-type embryos show a terminal-class phenotype resembling csw when injected with p21rasN17, a dominant-negative form of p21ras. Furthermore, we have analyzed the maternal-effect phenotype of Son of sevenless (Sos), a positive regulator of Ras1, and showed that embryos derived from germ cells lacking Sos+ activity exhibit a terminal-class phenotype. Our study demonstrates that the Drosophila p21ras, encoded by Ras1, is an intrinsic component of the tor signaling pathway, where it is both necessary and sufficient in specifying posterior terminal cell fates. p21ras/Ras1 operates upstream of the D-raf kinase in this signaling pathway.
%B Genes Dev %V 7 %P 621-32 %8 1993 Apr %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/8458578?dopt=Abstract %0 Journal Article %J Development %D 1993 %T Developmental and molecular characterization of mutations in the Drosophila-raf serine/threonine protein kinase. %A Melnick, M B %A Perkins, L A %A M. Lee %A Ambrosio, L %A Perrimon, N %K Amino Acid Sequence %K Animals %K Blastocyst %K Drosophila %K Eye %K In Situ Hybridization %K Microscopy, Electron, Scanning %K Molecular Sequence Data %K Morphogenesis %K Mutation %K Phenotype %K Protein-Serine-Threonine Kinases %K Signal Transduction %XFormation of the tail region of the Drosophila larva requires the activities of the terminal class genes. Genetic and molecular analyses of these genes suggests that localized activation of the receptor tyrosine kinase torso at the posterior egg pole triggers a signal transduction pathway. This pathway, mediated through the serine/threonine protein kinase D-raf and the protein tyrosine phosphatase corkscrew, controls the domains of expression of the transcription factors tailless and huckebein. In this paper, we report the molecular and developmental characterization of mutations in the D-raf gene. We show that mutations that alter conserved residues known to be necessary for kinase activity are associated with a null phenotype, demonstrating that D-raf kinase activity is required for its role in torso signaling. Another mutation, D-rafPB26, which prematurely truncates the kinase domain shows a weaker maternal effect phenotype that is strikingly similar to the corkscrew maternal effect phenotype, suggesting that a lower amount of kinase activity decreases the terminal signaling pathway. Finally, molecular and developmental characterization of two mutations that affect the late D-raf zygotic function(s) implies a novel role for D-raf in cell fate establishment in the eye. One of these mutations, D-rafC110, is associated with a single amino acid change within the putative D-raf regulatory region, while the other, D-rafHM-7, most likely reduces the wild-type amount of D-raf protein.
%B Development %V 118 %P 127-38 %8 1993 May %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/8375330?dopt=Abstract %0 Journal Article %J Genetics %D 1993 %T The Drosophila stubarista phenotype is associated with a dosage effect of the putative ribosome-associated protein D-p40 on spineless. %A Melnick, M B %A Noll, E %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Conserved Sequence %K Crosses, Genetic %K DNA %K DNA, Complementary %K Drosophila melanogaster %K Drosophila Proteins %K Embryo, Nonmammalian %K Female %K Gene Expression %K Genes, Lethal %K Genomic Library %K Humans %K Hydra %K In Situ Hybridization %K Insect Proteins %K Male %K Molecular Sequence Data %K Mutagenesis, Insertional %K Mutation %K Phenotype %K Proteins %K Restriction Mapping %K Sequence Homology, Amino Acid %K Transcription, Genetic %K X Chromosome %XWe describe the molecular characterization of the Drosophila melanogaster gene stubarista (sta) that encodes the highly conserved putative ribosome-associated protein D-p40. sta maps to cytological position 2A3-B2 on the X chromosome and encodes a protein (D-p40) of 270 amino acids. D-p40 shares 63% identity with the human p40 ribosomal protein. P element-mediated transformation of a 4.4-kb genomic fragment encompassing the 1-kb transcript corresponding to D-p40 was used to rescue both a lethal (sta2) and a viable (sta1) mutation at the stubarista (sta) locus. Developmental analysis of the sta2 mutation implicates a requirement for D-p40 during oogenesis and imaginal development, which is consistent with the expression of sta throughout development. In addition, we have analyzed the basis of the sta1 visible phenotype which consists of shortened antennae and bristles. sta1 is a translocation of the 1E1-2 to 2B3-4 region of the X chromosome onto the third chromosome at 89B21-C4. We provide genetic evidence that Dp(1;3)sta1 is mutant at the spineless (ss) locus and that it is associated with partial D-p40 activity. We demonstrate that sta1 acts as a recessive enhancer of ss; reduction in the amount of D-p40 provided by the transposed X chromosomal region of sta1 reveals a haplo-insufficient phenotype of the otherwise recessive ss mutations. This phenomenon is reminiscent of the enhancing effect observed with Minute mutations, one of which, rp49, has previously been shown to encode a ribosomal protein.
%B Genetics %V 135 %P 553-64 %8 1993 Oct %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/7916731?dopt=Abstract %0 Journal Article %J EMBO J %D 1993 %T Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein. %A van den Heuvel, M %A Harryman-Samos, C %A Klingensmith, J %A Perrimon, N %A Nusse, R %K Alleles %K Amino Acid Sequence %K Animals %K Base Sequence %K Cloning, Molecular %K Drosophila melanogaster %K Drosophila Proteins %K Gene Expression Regulation %K Genes, Insect %K Genes, Regulator %K Molecular Sequence Data %K Mutation %K Precipitin Tests %K Proto-Oncogene Proteins %K RNA, Messenger %K Wnt1 Protein %XWe have characterized the molecular nature of mutations in wingless (wg), a segment polarity gene acting during various stages of Drosophila development. Embryo-lethal alleles have undergone mutations in the protein-encoding domain of the gene, including deletions and point mutations of conserved residues. In a temperature sensitive mutation, a conserved cysteine residue is replaced by a serine. In embryo-viable alleles, the wg transcriptional unit is not affected. Immunostaining of mutant embryos shows that the embryo-lethal alleles produce either no wg antigen or a form of the protein that is retained within cells. Interestingly, embryos mutant for the segment polarity gene porcupine show a similar retention of the wg antigen. We have also transfected wild type wg alleles into Drosophila tissue culture cells, which then display wg protein on the cell surface and in the extracellular matrix. In similar experiments with mutant alleles, the proteins are retained in intracellular compartments and appear not to be secreted. These data provide further evidence that wg acts as a secreted factor and suggest that porcupine provides an accessory function for wg protein secretion or transport.
%B EMBO J %V 12 %P 5293-302 %8 1993 Dec 15 %G eng %N 13 %1 http://www.ncbi.nlm.nih.gov/pubmed/8262072?dopt=Abstract %0 Journal Article %J Curr Biol %D 1993 %T Simple and efficient generation of marked clones in Drosophila. %A Harrison, D A %A Perrimon, N %XBACKGROUND: Cell lineage analysis and mosaic analysis of mutations are important techniques that are used to study the development of many organisms. Unfortunately, the methods employed for such analyses are usually inefficient, technically demanding or labor intensive. In Drosophila, the most common methodology used for the generation of mosaic animals is mitotic recombination, which is induced by X-rays. Although this technique is simple, it has the undesirable characteristics of a low efficiency and a high rate of cell death. Furthermore, although a large number of marker systems has been employed to detect mitotic recombinants, none allows easy identification of clones for all cell types. RESULTS: A system is described here that allows a highly efficient generation of clones with the concomitant expression of an easily detectable cellular marker. This method can be applied to cell lineage and mosaic analysis in Drosophila. The site-specific yeast FLP recombinase, under the control of a heat shock-inducible promoter, efficiently catalyses mitotic recombination specifically at the site of a FLP recombination target (FRT). In this system, recombination fuses the alpha-tubulin promoter to the lacZ gene, allowing transcription of the marker. Recombinant cells and their progeny can, therefore, be detected by standard assays for beta-galactosidase. Of particular importance is the fact that only the cells of interest stain, thus allowing their simple detection in any tissue. CONCLUSIONS: We demonstrate that, by intermolecular recombination, we can use FLIP recombinase to generate marked clones efficiently in embryonic, larval and adult tissues. This simple and efficient technique is well suited to cell-lineage analysis and can be easily extended to the generation and detection of mutant clones in mosaic animals.
%B Curr Biol %V 3 %P 424-33 %8 1993 Jul 1 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/15335709?dopt=Abstract %0 Journal Article %J Development %D 1993 %T Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. %A Brand, A H %A Perrimon, N %K Animals %K Base Sequence %K Drosophila %K Enhancer Elements, Genetic %K Eye %K Gene Expression Regulation %K Genes, Dominant %K Microscopy, Electron, Scanning %K Molecular Sequence Data %K Mutagenesis, Insertional %K Phenotype %XWe have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.
%B Development %V 118 %P 401-15 %8 1993 Jun %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/8223268?dopt=Abstract %0 Journal Article %J J Neurobiol %D 1993 %T Approaches to identify genes involved in Drosophila embryonic CNS development. %A Noll, E %A Perkins, L A %A Mahowald, A P %A Perrimon, N %K Animals %K Cells, Cultured %K Central Nervous System %K Drosophila %K Genetic Techniques %XMany of the steps involved in formation of the Drosophila embryonic central nervous system (CNS) have been identified by both descriptive and experimental studies. In this review we will describe the various approaches that have been used to identify molecules involved in CNS development and the advantages and disadvantages of each of them. Our discussion will by no means be exhaustive; but rather we will discuss our experiences with each approach and provide an overview of what has been learned by using these methodologies. Finally, we will discuss methods that have been recently developed and how they are likely to provide further insight into CNS development.
%B J Neurobiol %V 24 %P 701-22 %8 1993 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/8331336?dopt=Abstract %R 10.1002/neu.480240603 %0 Journal Article %J Dev Suppl %D 1993 %T Cell patterning in the Drosophila segment: engrailed and wingless antigen distributions in segment polarity mutant embryos. %A van den Heuvel, M %A Klingensmith, J %A Perrimon, N %A Nusse, R %K Animals %K Drosophila melanogaster %K Drosophila Proteins %K Genes, Insect %K Homeodomain Proteins %K Insect Hormones %K Morphogenesis %K Mutation %K Proto-Oncogene Proteins %K Transcription Factors %K Wnt1 Protein %XBy a complex and little understood mechanism, segment polarity genes control patterning in each segment of the Drosophila embryo. During this process, cell to cell communication plays a pivotal role and is under direct control of the products of segment polarity genes. Many of the cloned segment polarity genes have been found to be highly conserved in evolution, providing a model system for cellular interactions in other organisms. In Drosophila, two of these genes, engrailed and wingless, are expressed on either side of the parasegment border. wingless encodes a secreted molecule and engrailed a nuclear protein with a homeobox. Maintenance of engrailed expression is dependent on wingless and vice versa. To investigate the role of other segment polarity genes in the mutual control between these two genes, we have examined wingless and engrailed protein distribution in embryos mutant for each of the segment polarity genes. In embryos mutant for armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in wingless mutant embryos, suggesting that their gene products act in the wingless pathway. In embryos mutant for hedgehog, fused, cubitus interruptus Dominant and gooseberry, expression of engrailed is affected to varying degrees. However wingless expression in the latter group decays in a similar way earlier than engrailed expression, indicating that these gene products might function in the maintenance of wingless expression. Using double mutant embryos, epistatic relationships between some segment polarity genes have been established. We present a model showing a current view of segment polarity gene interactions.
%B Dev Suppl %P 105-14 %8 1993 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/8049466?dopt=Abstract %0 Journal Article %J Dev Suppl %D 1993 %T The torso pathway in Drosophila: a model system to study receptor tyrosine kinase signal transduction. %A Lu, X. %A Perkins, L A %A Perrimon, N %K Animals %K Drosophila %K Drosophila Proteins %K Embryonic Induction %K Models, Biological %K Morphogenesis %K Protein-Tyrosine Kinases %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %XIn the Drosophila embryo, specification of terminal cell fates that result in the formation of both the head (acron) and tail (telson) regions is under the control of the torso (tor) receptor tyrosine kinase. The current knowledge suggests that activation of tor at the egg pole initiates a signal transduction pathway that is mediated sequentially by the guanine nucleotide releasing factor son of sevenless (Sos), the p21Ras1 GTPase, the serine/threonine kinase D-raf and the tyrosine/threonine kinase MAPKK (Dsor1). Subsequently, it is postulated that activation, possibly by phosphorylation, of a transcription factor at the egg poles activates the transcription of the terminal gap genes tailless and huckebein. These gap genes, which encode putative transcription factors, then control the expression of more downstream factors that ultimately result in head and tail differentiation. Also involved in tor signaling is the non-receptor protein tyrosine phosphatase corkscrew (csw). Here, we review the current model and discuss future research directions in this field.
%B Dev Suppl %P 47-56 %8 1993 %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/8049487?dopt=Abstract %0 Journal Article %J Cell %D 1993 %T The torso receptor protein-tyrosine kinase signaling pathway: an endless story. %A Perrimon, N %K Animals %K Drosophila %K Drosophila Proteins %K Enzyme Activation %K Protein-Tyrosine Kinases %K Receptor Protein-Tyrosine Kinases %K Signal Transduction %B Cell %V 74 %P 219-22 %8 1993 Jul 30 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/8343949?dopt=Abstract %0 Journal Article %J Cell %D 1992 %T corkscrew encodes a putative protein tyrosine phosphatase that functions to transduce the terminal signal from the receptor tyrosine kinase torso. %A Perkins, L A %A Larsen, I %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Drosophila %K Gene Expression %K Models, Genetic %K Molecular Sequence Data %K Open Reading Frames %K Protein Tyrosine Phosphatases %K Protein-Tyrosine Kinases %K Sequence Alignment %K Signal Transduction %K Transduction, Genetic %XWe describe the characterization of the Drosophila gene, corkscrew (csw), which is maternally required for normal determination of cell fates at the termini of the embryo. Determination of terminal cell fates is mediated by a signal transduction pathway that involves a receptor tyrosine kinase, torso, a serine/threonine kinase, D-raf, and the transcription factors, tailless and huckebein. Double mutant and cellular analyses between csw, torso, D-raf, and tailless indicate that csw acts downstream of torso and in concert with D-raf to positively transduce the torso signal via tailless, to downstream terminal genes. The csw gene encodes a putative nonreceptor protein tyrosine phosphatase covalently linked to two N-terminal SH2 domains, which is similar to the mammalian PTP1C protein.
%B Cell %V 70 %P 225-36 %8 1992 Jul 24 %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/1638629?dopt=Abstract %0 Journal Article %J Genes Dev %D 1992 %T The Drosophila spitz gene encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis. %A Rutledge, B J %A Zhang, K %A Bier, E %A Jan, Y N %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Blotting, Northern %K Cloning, Molecular %K Drosophila %K Drosophila Proteins %K Epidermal Growth Factor %K Larva %K Membrane Proteins %K Molecular Sequence Data %K Mutation %K Nucleic Acid Hybridization %K Recombinant Fusion Proteins %XWe describe the molecular characterization of the Drosophila gene spitz (spi), which encodes a putative 26-kD, EGF-like transmembrane protein that is structurally similar to TGF-alpha. Temporal and spatial expression patterns of spi transcripts indicate that spi is expressed throughout the embryo. Examination of mutant embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. We propose that spi may act as a ligand for cell-specific receptors, possibly rhomboid and/or the Drosophila EGF receptor homolog.
%B Genes Dev %V 6 %P 1503-17 %8 1992 Aug %G eng %N 8 %1 http://www.ncbi.nlm.nih.gov/pubmed/1644292?dopt=Abstract %0 Journal Article %J Development %D 1992 %T orthodenticle activity is required for the development of medial structures in the larval and adult epidermis of Drosophila. %A Wieschaus, E %A Perrimon, N %A Finkelstein, R %K Animals %K Drosophila %K Embryonic Induction %K Genes %K Morphogenesis %K Mosaicism %K Phenotype %XLethal alleles of orthodenticle (= otd) cause abnormalities in the embryonic head that reflect an early role in anterior pattern formation. In addition, otd activity is required for the development of the larval and adult epidermis. Clonal analysis of both viable and lethal alleles shows that the adult requirement for otd is restricted to medial regions of certain discs. When otd activity is reduced or removed, some medial precursor cells produce bristles and cuticle characteristic of more lateral structures. Similar medial defects are observed in the larval epidermis of embryos homozygous for lethal otd alleles. Antibodies to otd recognize a nuclear protein found at high levels in the medial region of the eye antennal discs, the leg discs, the genital discs and along the ventral midline of the ventral epidermis of the embryo. These results suggest that the otd gene product is required to specify medial cell fates in both the larval and adult epidermis.
%B Development %V 115 %P 801-11 %8 1992 Jul %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/1425355?dopt=Abstract %0 Journal Article %J Dev Genet %D 1992 %T Use of a yeast site-specific recombinase to generate embryonic mosaics in Drosophila. %A Dang, D T %A Perrimon, N %K Animals %K Clone Cells %K DNA Nucleotidyltransferases %K Drosophila %K Female %K Hot Temperature %K Mitosis %K Mosaicism %K Mutation %XAn efficient method for generating embryonic mosaics using a yeast site-specific recombinase (FLP), under the control of a heat shock promoter, is described. FLP-recombinase can promote mitotic exchange between homologous chromosomes that contain FRT (FLP Recombination Target) sequences. To demonstrate the efficiency of FLP-recombinase to generate embryonic mosaics, clones of the recessive and cell autonomous mutation armadillo (arm), detected by their ability to differentiate ectopic denticles in the naked cuticle of each abdominal segment, have been induced. We have analyzed the parameters of FLP-recombinase induced embryonic mitotic recombination and have demonstrated that clones can be efficiently induced during the postblastoderm mitotic divisions. We discuss applications of this technique for the analyses of the roles of various mutations during embryonic patterning.
%B Dev Genet %V 13 %P 367-75 %8 1992 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/1292893?dopt=Abstract %R 10.1002/dvg.1020130507 %0 Journal Article %J Cell %D 1992 %T wingless signaling acts through zeste-white 3, the Drosophila homolog of glycogen synthase kinase-3, to regulate engrailed and establish cell fate. %A Siegfried, E %A Chou, T B %A Perrimon, N %K Amino Acid Sequence %K Animals %K Calcium-Calmodulin-Dependent Protein Kinases %K Cell Communication %K Drosophila %K Gene Expression Regulation %K Glycogen Synthase Kinases %K Molecular Sequence Data %K Protein Kinases %K Sequence Homology, Amino Acid %K Signal Transduction %XIntrasegmental patterning in the Drosophila embryo is regulated by cell-cell communication. One of the signaling pathways that operates to specify positional information throughout the segment is mediated by the wingless (wg) protein, which is the homolog of the proto-oncogene Wnt-1. The early role of wg is to stabilize engrailed (en) expression by initiating a phase of en autoregulation in the adjacent more posterior cells. Here, we report that the segment polarity gene zeste-white 3 (zw3; also known as shaggy) acts as a repressor of en autoregulation. Genetic epistasis experiments indicate that wg signaling operates by inactivating the zw3 repression of en autoactivation. In addition, we demonstrate that zw3 encodes the Drosophila homolog of mammalian glycogen synthase kinase-3.
%B Cell %V 71 %P 1167-79 %8 1992 Dec 24 %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/1335365?dopt=Abstract %0 Journal Article %J Proc Natl Acad Sci U S A %D 1992 %T Altering the insertional specificity of a Drosophila transposable element. %A Kassis, J A %A Noll, E %A VanSickle, E P %A Odenwald, W F %A Perrimon, N %K Animals %K Base Sequence %K DNA Transposable Elements %K Drosophila melanogaster %K Enhancer Elements, Genetic %K Gene Expression Regulation %K Genes %K Molecular Sequence Data %K Oligodeoxyribonucleotides %K Promoter Regions, Genetic %XVectors derived from the Drosophila P element transposon are widely used to make transgenic Drosophila. Insertion of most P-element-derived vectors is nonrandom, but they exhibit a broad specificity of target sites. During experiments to identify cis-acting regulatory elements of the Drosophila segmentation gene engrailed, we identified a fragment of engrailed DNA that, when included within a P-element vector, strikingly alters the specificity of target sites. P-element vectors that contain this fragment of engrailed regulatory DNA insert at a high frequency near genes expressed in stripes.
%B Proc Natl Acad Sci U S A %V 89 %P 1919-23 %8 1992 Mar 1 %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/1311855?dopt=Abstract %0 Journal Article %J Genetics %D 1992 %T Genetic and developmental analysis of polytene section 17 of the X chromosome of Drosophila melanogaster. %A Eberl, D F %A Perkins, L A %A Engelstein, M %A Hilliker, A J %A Perrimon, N %K Amino Acid Sequence %K Animals %K Chromosome Banding %K Cloning, Molecular %K Crosses, Genetic %K Drosophila melanogaster %K Female %K Genetic Complementation Test %K Male %K Molecular Sequence Data %K Mutagenesis %K Phenotype %K Restriction Mapping %K Salivary Glands %K Transformation, Genetic %K X Chromosome %XPolytene section 17 of the X chromosome of Drosophila melanogaster, previously known to contain six putative lethal complementation groups important in oogenesis and embryogenesis, has here been further characterized genetically and developmentally. We constructed fcl+Y, a duplication of this region, which allowed us to conduct mutagenesis screens specific for the region and to perform complementation analyses (previously not possible). We recovered 67 new lethal mutations which defined 15 complementation groups within Df(1)N19 which deletes most of polytene section 17. The zygotic lethal phenotypes of these and preexisting mutations within polytene section 17 were examined, and their maternal requirements were analysed in homozygous germline clones using the dominant female sterile technique. We present evidence that an additional gene, which produces two developmentally regulated transcripts, is located in this region and is involved in embryogenesis, although no mutations in this gene were identified. In this interval of 37 to 43 polytene chromosome bands we have defined 17 genes, 12 (71%) of which are of significance to oogenesis or embryogenesis.
%B Genetics %V 130 %P 569-83 %8 1992 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/1551578?dopt=Abstract %0 Journal Article %J Genetics %D 1992 %T Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. %A Chou, T B %A Perrimon, N %K Animals %K Chimera %K Chromosomes %K Crosses, Genetic %K DNA Nucleotidyltransferases %K Drosophila %K Female %K Genes, Lethal %K Genetic Linkage %K Germ Cells %K Hot Temperature %K Mitosis %K Mutagenesis, Site-Directed %K Recombination, Genetic %K Saccharomyces cerevisiae %K X Chromosome %XWe describe an efficient method for generating female germline mosaics by inducing site-specific homologous mitotic recombination with a yeast recombinase (FLP) which is driven by a heat shock promoter. These germline mosaics are produced in flies heterozygous for the agametic, germline-dependent, dominant female sterile (DFS) mutation ovoD1, where only flies possessing germline clones are able to lay eggs. This method, the "FLP-DFS" technique, is very efficient because more than 90% of females with germline clones can be recovered. We show that this heat-inducible, site-specific mitotic recombination system does not affect viability and that the germline clones recovered are physiologically the same as those created by X-ray induced mitotic recombination. We describe the parameters of FLP-recombinase induced germline mitotic recombination and the use of the "FLP-DFS" technique to analyze the maternal effect of X-linked zygotic lethal mutations.
%B Genetics %V 131 %P 643-53 %8 1992 Jul %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/1628809?dopt=Abstract %0 Journal Article %J Development %D 1991 %T The molecular genetics of head development in Drosophila melanogaster. %A Finkelstein, R %A Perrimon, N %K Animals %K Biological Evolution %K Drosophila melanogaster %K Genes %K Genes, Homeobox %K Head %K Morphogenesis %B Development %V 112 %P 899-912 %8 1991 Aug %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/1682131?dopt=Abstract %0 Journal Article %J In Vivo %D 1991 %T The molecular genetics of tail development in Drosophila melanogaster. %A Perkins, L A %A Perrimon, N %K Animals %K Drosophila melanogaster %K Gene Expression Regulation %K Morphogenesis %K Signal Transduction %K Transcription, Genetic %XThe formation of the telson in the Drosophila embryo, which encompasses all structures posterior to abdominal segment 7, is under the control of the "terminal class" genes. These maternally expressed genes are organized in a signal transduction pathway which implicates cell-cell interactions between the germ cell derivatives (the nurse cells and oocyte) and the surrounding follicle cell epithelium. Activation of this localized signal transduction pathway at the termini of the embryo is believed to specify the domains of activation and repression of a set of zygotic genes whose interactions specify the various cell states required for the proper formation of tail structures.
%B In Vivo %V 5 %P 521-31 %8 1991 Sep-Oct %G eng %N 5 %1 http://www.ncbi.nlm.nih.gov/pubmed/1768804?dopt=Abstract %0 Journal Article %J Genes Dev %D 1991 %T The crooked neck gene of Drosophila contains a motif found in a family of yeast cell cycle genes. %A Zhang, K %A Smouse, D %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Cell Cycle %K Cell Division %K Cloning, Molecular %K DNA %K Drosophila %K Drosophila Proteins %K Genes, Fungal %K Insect Hormones %K Molecular Sequence Data %K Multigene Family %K Phenotype %K Repetitive Sequences, Nucleic Acid %K Restriction Mapping %K Sequence Alignment %K Transcription, Genetic %XThe crooked neck (crn) gene of Drosophila encodes a protein of 702 amino acids and contains 16 tandemly arranged copies of a 34-amino-acid repeat that is similar to the tetratrico peptide repeat (TPR). Multiple copies of the TPR motif have also been found in a family of yeast genes, including several members that are necessary for cell division. TPR-containing proteins encoded by the yeast genes CDC16, CDC23, and nuc2+ are required for progression through the G2/M transition of the cell cycle. Loss of zygotic expression of crn causes defects in the proliferation of brain neuroblasts and results in the absence of identified neuronal lineages in the central and peripheral nervous systems. The sequence similarity and mutant phenotypes are consistent with a cell cycle requirement for the crn gene product.
%B Genes Dev %V 5 %P 1080-91 %8 1991 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/2044955?dopt=Abstract %0 Journal Article %J Dev Genet %D 1991 %T Generating lineage-specific markers to study Drosophila development. %A Perrimon, N %A Noll, E %A McCall, K %A Brand, A %K Animals %K beta-Galactosidase %K Cloning, Molecular %K DNA Transposable Elements %K Drosophila %K Female %K Gene Expression %K Genetic Markers %K Immunoenzyme Techniques %K Male %K Mutation %K Organ Specificity %K Promoter Regions, Genetic %XTo generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of beta-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.
%B Dev Genet %V 12 %P 238-52 %8 1991 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/1651183?dopt=Abstract %R 10.1002/dvg.1020120309 %0 Book Section %B Cell Activation: Genetic Approaches %D 1991 %T Segment polarity genes and intercellular communication in Drosophila %A Klingensmith, J %A Perrimon, Norbert %E Mond, J %E Weiss,A. %E Cambier,J. %B Cell Activation: Genetic Approaches %I Raven Press %C New York, NY %P 251-274 %G eng %0 Journal Article %J Trends Genet %D 1990 %T Serine/threonine protein kinases in Drosophila. %A Siegfried, E %A Ambrosio, L %A Perrimon, N %K Animals %K Cell Differentiation %K Drosophila %K Protein Kinases %K Protein-Serine-Threonine Kinases %K Signal Transduction %XThe study of serine/threonine kinases in Drosophila is coming of age. Recently several kinases have been identified and their role in cell determination has been established. This review discusses these recent findings and describes the potential for genetic analyses of kinase activity and signal transduction.
%B Trends Genet %V 6 %P 357-62 %8 1990 Nov %G eng %N 11 %1 http://www.ncbi.nlm.nih.gov/pubmed/2150900?dopt=Abstract %0 Journal Article %J Dev Biol %D 1990 %T Genetic dissection of a complex neurological mutant, polyhomeotic, in Drosophila. %A Smouse, David %A Perrimon, Norbert %K Animals %K Axons %K Brain %K Cell Division %K Cell Survival %K Drosophila %K Epidermis %K Gene Expression Regulation %K Genes, Regulator %K Mosaicism %K Mutation %K Neurons %K Phenotype %K Ring Chromosomes %XNull mutations at the polyhomeotic locus of Drosophila produce a complex phenotype during embryogenesis, which includes death of the ventral epidermis, misregulation of homeotic and segmentation gene expression, and global misrouting of CNS axons. It is shown here, through the use of mosaic analyses, double mutant combinations, and in vitro culture experiments, that all aspects of the phenotype with the exception of the axonal phenotype are cell autonomous. The changes in homeotic and segmentation gene expression in the CNS are not caused by death of the ventral epidermis, but are cell autonomous effects which most likely cause changes in neuronal cell identity. The axonal phenotype associated with ph mutations is also independent of epidermal cell death, but may be due to the nonautonomous effects of altered neuronal identities or to death or transformation of some as yet unidentified cell type. Despite the apparent autonomy of the ph mutation, mutant neurons can influence the development of adjacent wild-type neurons, presumably by depriving them of their normal fasciculation partners.
%B Dev Biol %V 139 %P 169-85 %8 1990 May %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/2328834?dopt=Abstract %0 Journal Article %J Genes Dev %D 1990 %T The orthodenticle gene encodes a novel homeo domain protein involved in the development of the Drosophila nervous system and ocellar visual structures. %A Finkelstein, R %A Smouse, D %A Capaci, T M %A Spradling, A C %A Perrimon, N %K Alleles %K Amino Acid Sequence %K Animals %K Base Sequence %K Central Nervous System %K Cloning, Molecular %K Drosophila %K Gene Expression Regulation %K Genes, Homeobox %K Molecular Sequence Data %K Mutation %K Phenotype %K Photoreceptor Cells %K Sequence Homology, Nucleic Acid %K X Chromosome %XThe orthodenticle (otd) locus of Drosophila is required for embryonic development, and null mutations of otd cause defects in head development and segmental patterning. We show here that otd is necessary for the formation of the embryonic central nervous system (CNS). otd mutations result in the formation of an abnormal neuropil and in the disappearance of identified neurons associated with the midline of the CNS. In addition, otd is allelic to ocelliless (oc), a mutation that causes the deletion of the ocelli of the adult fly. We have identified a transcription unit corresponding to the otd locus and find that it is expressed early in a stripe near the anterior pole of the cellular blastoderm and later in the region of the CNS from which these neurons normally arise. The predicted otd protein contains a well-conserved homeo domain and is therefore likely to be a transcriptional regulator involved in specifying cell fate both in the embryonic CNS and in the ocelli.
%B Genes Dev %V 4 %P 1516-27 %8 1990 Sep %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/1979296?dopt=Abstract %0 Journal Article %J Nature %D 1990 %T The orthodenticle gene is regulated by bicoid and torso and specifies Drosophila head development. %A Finkelstein, R %A Perrimon, N %K Animals %K Blastoderm %K Drosophila %K Gene Expression Regulation %K Genes, Homeobox %K Head %K Mutation %K Phenotype %K Transcription, Genetic %XIn the Drosophila embryo, cell fate along the anterior-posterior axis is determined by maternally expressed genes. The activity of the bicoid (bcd) gene is required for the development of larval head and thoracic structures, and that of maternal torso (tor) for the development of the unsegmented region of the head (acron). In contrast to the case of thoracic and abdominal segmentation, the hierarchy of zygotically expressed genes controlling head development has not been clearly defined. The bcd protein, which is expressed in a gradient, activates zygotic expression of the gap gene hunchback (hb), but hb alone is not sufficient to specify head development. Driever et al. proposed that at least one other bcd-activated gene controls the development of head regions anterior to the hb domain. We report here that the homeobox gene orthodenticle (otd), which is involved in head development, could be such a gene. We also show that otd expression responds to the activity of the maternal tor gene at the anterior pole of the embryo.
%B Nature %V 346 %P 485-8 %8 1990 Aug 2 %G eng %N 6283 %1 http://www.ncbi.nlm.nih.gov/pubmed/1974036?dopt=Abstract %R 10.1038/346485a0 %0 Journal Article %J Nature %D 1990 %T Putative protein kinase product of the Drosophila segment-polarity gene zeste-white3. %A Siegfried, E %A Perkins, L A %A Capaci, T M %A Perrimon, N %K Amino Acid Sequence %K Animals %K Base Sequence %K Chromosome Mapping %K Cloning, Molecular %K DNA %K Drosophila melanogaster %K Genes %K Molecular Sequence Data %K Morphogenesis %K Protein Kinases %K Restriction Mapping %K RNA, Messenger %XThe metameric pattern of the Drosophila embryo is regulated by a combination of maternal and zygotic genes. The segment-polarity class of genes are required for the correct patterning within each segmental unit. Mutations in any one of these genes results in deletions and duplications of parts of each segment. The segment-polarity genes act coordinately by means of local cellular interactions to assign and maintain an identity for each cell in the segment, and to establish segment boundaries. Here we describe the molecular characterization of a novel segment-polarity gene, zeste-white3 (zw3). Embryos derived from germ lines that are homozygous for zw3 mutations (zw3 embryos) have phenotypes similar to embryos that are mutant for the segment-polarity gene naked (nkd). These embryos lack most of the ventral denticles, which are differentiated structures derived from the most anterior region of each segment. We have isolated the zw3 gene and compared the structure of one maternal and one zygotic transcript encoded by the gene. The zw3 gene is unique among the segment-polarity genes so far characterized, in that it encodes proteins that have homology to serine-threonine protein kinases. This indicates that zw3 may play a part in a signal transduction pathway involved in the establishment of cell identity within each embryonic segment.
%B Nature %V 345 %P 825-9 %8 1990 Jun 28 %G eng %N 6278 %1 http://www.ncbi.nlm.nih.gov/pubmed/2113617?dopt=Abstract %R 10.1038/345825a0 %0 Journal Article %J Proc Natl Acad Sci U S A %D 1990 %T Drosophila homolog of the mammalian jun oncogene is expressed during embryonic development and activates transcription in mammalian cells. %A Zhang, Kang %A Chaillet, J Richard %A Perkins, Lizabeth A %A Halazonetis, Thanos D %A Perrimon, Norbert %K Amino Acid Sequence %K Animals %K Base Sequence %K Blotting, Northern %K DNA-Binding Proteins %K Drosophila melanogaster %K Embryo, Nonmammalian %K Humans %K Molecular Sequence Data %K Multigene Family %K Plasmids %K Protein-Tyrosine Kinases %K Proto-Oncogene Proteins c-jun %K Proto-Oncogenes %K Restriction Mapping %K RNA %K Sequence Homology, Nucleic Acid %K Transcription Factors %XBy means of low-stringency cross-species hybridization to Southern DNA blots, human c-jun sequences were used to identify a unique Drosophila melanogaster locus (Djun). The predicted DJun protein is highly homologous to members of the mammalian Jun family in both the DNA binding and leucine zipper regions. Djun was mapped by in situ hybridization to position 46E of the second chromosome. It encodes a 1.7-kilobase transcript constitutively expressed at all developmental stages. Functionally, Djun in cooperation with mouse c-fos can trans-activate activator protein 1 DNA binding site when introduced into mammalian cells. Taken together, these data suggest that Djun, much like its mammalian homolog, may activate transcription of genes involved in regulation of cell growth, differentiation, and development. Furthermore, the identification of Djun allows one to exploit the genetics of Drosophila to identify genes in signal transduction pathways involving Djun and thus c-jun.
%B Proc Natl Acad Sci U S A %V 87 %P 6281-5 %8 1990 Aug %G eng %N 16 %1 http://www.ncbi.nlm.nih.gov/pubmed/1696724?dopt=Abstract %0 Journal Article %J Mol Cell Biol %D 1990 %T Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster. %A Perkins, Lizabeth A %A Doctor, John S %A Zhang, Kang %A Stinson, Leslie %A Perrimon, Norbert %A Craig, Elizabeth A %K Amino Acid Sequence %K Animals %K Base Sequence %K Drosophila melanogaster %K Embryo, Nonmammalian %K Exons %K Genes %K Heat-Shock Proteins %K Humans %K Molecular Sequence Data %K Multigene Family %K Restriction Mapping %K Sequence Homology, Nucleic Acid %K Transcription, Genetic %XThe Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.
%B Mol Cell Biol %V 10 %P 3232-8 %8 1990 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/2111451?dopt=Abstract %0 Journal Article %J Genetics %D 1989 %T Developmental genetics of loci at the base of the X chromosome of Drosophila melanogaster. %A Perrimon, Norbert %A Smouse, David %A Miklos, George L Gabor %K Animals %K Chromosome Mapping %K Drosophila melanogaster %K Female %K Gastrula %K Genes, Lethal %K Genetic Complementation Test %K Germ Cells %K Nervous System %K Phenotype %K Suppression, Genetic %K X Chromosome %XWe have conducted a genetic and developmental analysis of the 26 contiguous genetic complementation groups within the 19D3-20F2 interval of the base of the X chromosome, a region of 34 polytene bands delimited by the maroon-like and suppressor of forked loci. Within this region there are four loci which cause visible phenotypes but which have little or no effect on zygotic viability (maroon-like, little fly, small optic lobes and sluggish). There are 22 loci which, when mutated, are zygotic lethals and three of these, legless/runt, folded gastrulation and 13E3, have severe effects on embryonic development. In addition, three visible phenotypes have been defined only by overlapping deficiencies (melanized-like, tumorous head, and varied outspread). We have analyzed the lethal phases and maternal requirement of 58 mutations at 22 of the zygotic lethal loci by means of germline clone analysis using the dominant female sterile technique. Additionally, all lethal complementation groups, as well as a specific subset of deficiencies, have been studied histologically for defects in the development of the central and peripheral embryonic nervous systems.
%B Genetics %V 121 %P 313-31 %8 1989 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/2499511?dopt=Abstract %0 Journal Article %J Development %D 1989 %T A Drosophila gene expressed in the embryonic CNS shares one conserved domain with the mammalian GAP-43. %A Ng, Shi-Chung %A Perkins, Lizabeth A %A Conboy, Gary %A Perrimon, Norbert %A Fishman, Mark C %K Amino Acid Sequence %K Animals %K Central Nervous System %K Chromosome Mapping %K Drosophila %K GAP-43 Protein %K Genes %K Genes, Overlapping %K Membrane Proteins %K Molecular Probe Techniques %K Nerve Tissue Proteins %XBy cross hybridization with the mammalian growth-related protein, GAP-43, we have isolated several Drosophila cDNAs and genomic sequences. These sequences correspond to a single copy gene that encodes two developmentally regulated transcripts 2.4 and 2.0 kb in length. The predicted protein sequence from the cDNAs contains a stretch of 20 amino acids closely related to the mammalian GAP-43 protein. These residues are also highly conserved in a cDNA isolated from the nematode C. elegans. Prior to dorsal closure, expression of the Drosophila gene is observed in non-neuronal tissues, especially in the mesectoderm and presumptive epidermis, both in a metameric pattern. After dorsal closure, expression becomes restricted to sets of cells that are segmentally reiterated along the periphery of the nervous system. These cells appear to include at least one specific set of glia that may establish scaffolding for the development of the longitudinal neuropile.
%B Development %V 105 %P 629-38 %8 1989 Mar %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/2693037?dopt=Abstract %0 Journal Article %J Development %D 1989 %T l(1)pole hole is required maternally for pattern formation in the terminal regions of the embryo. %A Ambrosio, Linda %A Mahowald, Anthony P %A Perrimon, Norbert %K Animals %K Blastoderm %K Cell Differentiation %K Drosophila melanogaster %K Gastrula %K Gene Expression %K Microscopy, Electron, Scanning %K Mitosis %K Morphogenesis %K Mutation %K Nervous System %K Zygote %XMaternal expression of the l(1)pole hole (l(1)ph) gene product is required for the development of the Drosophila embryo. When maternal l(1)ph+ activity is absent, alterations in the embryonic fate map occur as visualized by the expression of segmentation genes fushitarazu and engrailed. If both maternal and zygotic activity is absent, embryos degenerate around 7 h of development. If only maternal activity is missing, embryos complete embryogenesis and show deletions of both anterior and posterior structures. Anteriorly, structures originating from labral and acron head regions are missing. Posteriorly, abdominal segments A8, 9 and 10, the telson and the proctodeum are missing. Similar pattern deletions are observed in embryos derived from the terminal class of female sterile mutations. Thus, the maternal l(1)ph+ gene product is required for the establishment of cell identities at the anterior and posterior poles of the Drosophila embryo.
%B Development %V 106 %P 145-58 %8 1989 May %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/2516795?dopt=Abstract %0 Journal Article %J Dev Biol %D 1989 %T Multiple functions of a Drosophila homeotic gene, zeste-white 3, during segmentation and neurogenesis. %A Perrimon, Norbert %A Smouse, David %K Animals %K Central Nervous System %K Drosophila melanogaster %K Embryo, Nonmammalian %K Genes, Homeobox %K Genes, Lethal %K Morphogenesis %K Phenotype %XLack of both maternal and zygotic gene activity at the zeste-white 3 (zw3) locus causes severe developmental transformations. Embryos derived from germ cells that lack zw3+ gene activity die during embryogenesis and have a phenotype that is similar to that of embryos mutant for the segment polarity gene naked (nkd). In both nkd and germ line clone-derived zw3 embryos the pattern elements derived from the anterior-most part of each segment, the denticle belts, are deleted. Similar abnormal patterns of the zygotically expressed genes engrailed and Ultrabithorax are detected in both mutants, suggesting that the two genes are involved in the same developmental process. Additionally, the induction of clones of zw3 mutant cells in imaginal discs causes homeotic transformations of noninnervated hair cells into innervated sensory bristles. The multiple roles of zw3 during development and its possible interactions with the zygotic gene nkd are discussed.
%B Dev Biol %V 135 %P 287-305 %8 1989 Oct %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/2570722?dopt=Abstract %0 Journal Article %J Nature %D 1989 %T Requirement of the Drosophila raf homologue for torso function. %A Ambrosio, Linda %A Mahowald, Anthony P %A Perrimon, Norbert %K Animals %K Blotting, Northern %K DNA Transposable Elements %K Drosophila melanogaster %K Morphogenesis %K Nucleic Acid Hybridization %K Oogenesis %K Phosphoproteins %K Protein Kinases %K Proto-Oncogene Proteins %K Proto-Oncogene Proteins c-raf %K Restriction Mapping %K RNA, Messenger %K Suppression, Genetic %K Transcription, Genetic %XIn Drosophila the correct formation of the most anterior and posterior regions of the larva, acron and telson is dependent on the maternally expressed terminal class of genes. In their absence, the anterior head skeleton is truncated and all the structures posterior to the abdominal segment seven are not formed. The protein predicted to be encoded by one of these genes, torso (tor), seems to be a transmembrane protein with an extracytoplasmic domain acting as a receptor and a cytoplasmic domain containing tyrosine kinase activity. Here we report that another member of the terminal-genes class, l(1)polehole (l(1)ph), which is also zygotically expressed, is the Drosophila homologue of the v-raf oncogene and encodes a potential serine-and-threonine kinase. We also show that functional l(1)ph gene product is required for the expression of a gain-of-function tor mutant phenotype, indicating that l(1)ph acts downstream of tor. Together, these results support the idea that the induction of terminal development occurs through a signal transduction system, involving the local activation of the tor-encoded tyrosine kinase at the anterior and posterior egg poles, resulting in the phosphorylation of the l(1)ph gene product. In turn, downstream target proteins may be phosphorylated, ultimately leading to the regionalized expression of zygotic target genes. Such a process is in agreement with the finding that both tor and l(1)ph messenger RNAs are evenly distributed.
%B Nature %V 342 %P 288-91 %8 1989 Nov 16 %G eng %N 6247 %1 http://www.ncbi.nlm.nih.gov/pubmed/2554148?dopt=Abstract %R 10.1038/342288a0 %0 Journal Article %J Dev Biol %D 1989 %T The segment polarity phenotype of Drosophila involves differential tendencies toward transformation and cell death. %A Klingensmith, John %A Noll, Elizabeth %A Perrimon, Norbert %K Alleles %K Animals %K Cell Differentiation %K Cell Survival %K Drosophila %K Epidermis %K Female %K Gene Expression Regulation %K Homozygote %K Male %K Mutation %K Phenotype %K Temperature %XThe segment polarity genes of Drosophila are required for intrasegmental organization, as revealed by their abnormal cuticular morphology in mutant embryos. Lesions in most of these loci result in a similar cuticular phenotype, in which the normally naked, posterior region of the segment is covered to varying degrees by ectopic denticles. A temperature-sensitive allele of armadillo, which allows us to vary the level of arm+ activity, generates this entire range of phenotypes, suggesting that these genes affect a common pathway. Previous work with a strong allele of arm revealed the locus to be cell-autonomous, in that small homozygous epidermal clones secreted denticles. We have conducted a similar clonal analysis at all levels of arm+ activity. This shows a differential tendency toward cell transformation and cell death within the segment. Antibodies to segmentation gene-fusion products show that the cell death is primarily in the most posterior region of the segment. We suggest that differential cell respecification, resulting in transformation or death, is involved in generating the segment polarity phenotype.
%B Dev Biol %V 134 %P 130-45 %8 1989 Jul %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/2731645?dopt=Abstract %0 Journal Article %J Genetics %D 1989 %T Zygotic lethals with specific maternal effect phenotypes in Drosophila melanogaster. I. Loci on the X chromosome. %A Perrimon, Norbert %A Engstrom, Lee %A Mahowald, Anthony P %K Animals %K Clone Cells %K Drosophila melanogaster %K Female %K Genes, Lethal %K Genetic Linkage %K Homozygote %K Mutation %K Oogenesis %K Phenotype %K X Chromosome %K Zygote %XIn order to identify all X-linked zygotic lethal loci that exhibit a specific maternal effect on embryonic development, germline clonal analyses of X-linked zygotic lethal mutations have been performed. Two strategies were employed. In Screen A germline clonal analysis of 441 mutations at 211 previously mapped X-linked loci within defined regions was performed. In Screen B germline clonal analysis of 581 larval and pupal mutations distributed throughout the entire length of the X chromosome was performed. These approaches provide an 86% level of saturation for X-linked late zygotic lethals (larval and pupal) with specific maternal effect embryonic lethal phenotypes. The maternal effect phenotypes of these mutations are described.
%B Genetics %V 121 %P 333-52 %8 1989 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/2499512?dopt=Abstract %0 Journal Article %J Genes Dev %D 1988 %T polyhomeotic: a gene required for the embryonic development of axon pathways in the central nervous system of Drosophila. %A Smouse, D %A Goodman, C %A Mahowald, A %A Perrimon, N %K Animals %K Axons %K Drosophila %K Embryo, Nonmammalian %K Genes, Homeobox %K Mutation %K Nervous System %XHypomorphic alleles of the locus polyhomeotic (ph) produce multiple, homeotic-like transformations in adult flies that mimic dominant mutations in the Antennapedia and Bithorax complexes. Analysis of null alleles of ph has revealed a complex, embryonically lethal phenotype that includes cell death of the ventral epidermis and abnormalities in the patterns of expression of homeotic and segmentation genes. There is also a dramatic alteration in the pattern of axon pathways in the central nervous system, such that the wild-type array of segmentally repeated commissures and connectives is replaced by bundles of axons confined to the hemiganglia of origin. It is possible that this axonal phenotype is the result of loss of neuronal identity caused by abnormal homeotic and segmentation gene expression.
%B Genes Dev %V 2 %P 830-42 %8 1988 Jul %G eng %N 7 %1 http://www.ncbi.nlm.nih.gov/pubmed/2905316?dopt=Abstract %0 Journal Article %J Genetics %D 1988 %T Genetic evidence that the sans fille locus is involved in Drosophila sex determination. %A Oliver, Brian %A Perrimon, Norbert %A Mahowald, Anthony P %K Animals %K Chromosome Mapping %K Crosses, Genetic %K Drosophila %K Female %K Genes, Lethal %K Genes, Regulator %K Genetic Complementation Test %K Genotype %K Heterozygote %K Homozygote %K Male %K Mutation %K Phenotype %K Sex Determination Analysis %K Temperature %XFemales homozygous for sans fille1621 (= fs(1)1621) have an abnormal germ line. Instead of producing eggs, the germ-line cells proliferate forming ovarian tumors or excessive numbers of nurse cells. The Sex-lethal gene product(s) regulate the branch point of the dosage compensation and sex determination pathways in the soma. The role of Sex-lethal in the germ line is not clear but the germ line of females homozygous for female sterile Sex-lethal alleles or germ-line clones of loss-of-function alleles are characterized by ovarian tumors. Females heterozygous for sans fille1621 or Sex-lethal are phenotypically wild type with respect to viability and fertility but females trans-heterozygous for sans fille1621 and Sex-lethal show ovarian tumors, somatic sexual transformations, and greatly reduced viability.
%B Genetics %V 120 %P 159-71 %8 1988 Sep %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/3220249?dopt=Abstract %0 Journal Article %J Dev Biol %D 1988 %T The maternal effect of lethal(1)discs-large-1: a recessive oncogene of Drosophila melanogaster. %A Perrimon, Norbert %K Age Factors %K Animals %K Drosophila melanogaster %K Female %K Gene Expression Regulation %K Genes, Lethal %K Genes, Recessive %K Genetic Complementation Test %K Infertility, Female %K Microscopy, Electron, Scanning %K Morphogenesis %K Mutation %K Neoplasms, Experimental %K Oncogenes %K Phenotype %K Temperature %XThe maternal effect phenotypes of recessive mutations at the Drosophila zygotic lethal gene l(1)discs-large-1 (l(1)dlg-1) are described. L(1)dlg-1 is located in 10B7-8 on the salivary gland chromosome map. A complex complementation pattern is observed among the nine characterized alleles. Larvae missing zygotic l(1)dlg-1+ gene activity die due to aberrant growth of imaginal cells at the larval-pupal transition. Embryos lacking both maternal and zygotic activity of l(1)dlg-1+, i.e., embryos derived from homozygous l(1)dlg-1 germ line clones for null alleles, show neurogenesis and morphogenesis defects that result in very abnormal embryos. Although differentiated, most tissues are morphologically misshapen. This maternal effect is rescuable to some extent. One allele, l(1)dlg-1HF321, is a temperature-sensitive mutation for the zygotic lethality. Embryos derived from homozygous l(1)dlg-1HF321 females at 18 degrees C exhibit defects associated with dorsal closure and head involution. More extreme phenotypes are observed when females are shifted to higher temperatures and include defective dorsal closure, collapse of the somatic musculature, and an oversized central nervous system. The possible involvement of the recessive oncogene l(1)dlg-1 in cell adhesion is discussed.
%B Dev Biol %V 127 %P 392-407 %8 1988 Jun %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/3132409?dopt=Abstract %0 Book Section %B Primers in Developmental Biology %D 1987 %T Maternal contributions to early development in Drosophila %A Perrimon, Norbert %A Mahowald, Anthony P %E Malacinski, George M %B Primers in Developmental Biology %P 305-328 %G eng %0 Journal Article %J Mol Cell Biol %D 1987 %T Drosophila melanogaster homologs of the raf oncogene. %A Mark, George E %A MacIntyre, Ross J %A Digan, Mary E %A Ambrosio, Linda %A Perrimon, Norbert %K Amino Acid Sequence %K Animals %K Base Sequence %K Chromosome Mapping %K Cloning, Molecular %K DNA Restriction Enzymes %K Drosophila melanogaster %K Genes %K Mice %K Nucleic Acid Hybridization %K Oncogenes %K Plasmids %K Protein Kinases %K Sequence Homology, Nucleic Acid %XA murine v-raf probe, representing the kinase domain, was used to identify two unique loci in Drosophila melanogaster DNA. The most closely related to v-raf was mapped by in situ hybridization to position 2F5-6 (Draf-1) on the X chromosome, whereas the other raf-related gene (Draf-2) was found at position 43A2-5 on chromosome 2. The nucleotide and amino acid homologies of Draf-1 to the kinase domain of v-raf are 61 and 65%, respectively. The large amount of a 3.2-kilobase Draf-1 transcript detected in eggs as a maternal message decreases during embryonic development, and significant steady-state levels are observed throughout the remainder of morphogenesis. We speculate that the Draf-1 locus plays an important role in early embryogenesis.
%B Mol Cell Biol %V 7 %P 2134-40 %8 1987 Jun %G eng %N 6 %1 http://www.ncbi.nlm.nih.gov/pubmed/3037346?dopt=Abstract %0 Journal Article %J Dev Biol %D 1987 %T Multiple functions of segment polarity genes in Drosophila. %A Perrimon, Norbert %A Mahowald, Anthony P %K Alleles %K Animals %K Crosses, Genetic %K Drosophila %K Female %K Genes %K Genes, Lethal %K Homozygote %K Male %K Microscopy, Electron, Scanning %K Mutation %K Phenotype %Xl(1)dishevelled (l(1)dsh) is a late zygotic lethal mutation that exhibits a rescuable maternal effect lethal phenotype. l(1)dsh/Y embryos, derived from females possessing a homozygous l(1)dsh germline clone, exhibit a segment polarity embryonic phenotype. Analysis of the development of these embryos indicates: (1) that segmental boundaries do not form although the correct number of tracheal pits is formed; (2) that pockets of cell death occur between the tracheal pits; and (3) that engrailed expression becomes abnormal during germ band shortening. We propose that, in the absence of both maternal and zygotic expression of l(1)dsh+, cells from each posterior compartment die. Subsequently, cells from the anterior compartment must rearrange their positional values to generate the segment polarity phenotype. We have compared the phenotype of five other segment polarity loci: four embryonic lethals [l(1)armadillo, l(2)gooseberry, l(2)wingless, and l(3)hedgehog]; and the late zygotic lethal, l(1)fused. Only l(2)wingless embryos exhibit early segmentation defects similar to those found in l(1)dsh/Y embryos derived from homozygous germline clones. In contrast, segmentation is essentially normal in l(1)armadillo, l(2)gooseberry, l(3)hedgehog, and l(1)fused embryos. The respective maternal and zygotic contribution and the roles of the segment polarity loci for the patterning of the embryo and the adult are discussed.
%B Dev Biol %V 119 %P 587-600 %8 1987 Feb %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/3803719?dopt=Abstract %0 Journal Article %J Genes Dev %D 1987 %T The ovo locus is required for sex-specific germ line maintenance in Drosophila. %A Oliver, Brian %A Perrimon, Norbert %A Mahowald, Anthony P %K Alleles %K Animals %K Chromosome Deletion %K Chromosome Mapping %K Drosophila %K Female %K Genes %K Homozygote %K Mutation %K Phenotype %K Sex Determination Analysis %K X Chromosome %XMutations at the ovo locus result in a defective female germ line. The male germ line is not affected. Adult females homozygous for loss-of-function alleles have no germ line stem cells. The sex-specific phenotype is evident at late blastoderm and early gastrula stages when the pole cells of embryos homozygous for a loss-of-function allele begin to die. This is the only zygotically acting gene known that is required specifically for embryonic germ line survival. Females heterozygous for dominant alleles or homozygous for alleles reducing gene activity exhibit a range of defects in oogenesis. We have mapped the ovo locus to position 4E1-2 of the salivary gland X chromosome by using a set of cytologically visible deletions.
%B Genes Dev %V 1 %P 913-23 %8 1987 Nov %G eng %N 9 %1 http://www.ncbi.nlm.nih.gov/pubmed/3428601?dopt=Abstract %0 Journal Article %J Dev Biol %D 1987 %T Region-specific defects in l(1)giant embryos of Drosophila melanogaster. %A Petschek, Jane P %A Perrimon, Norbert %A Mahowald, Anthony P %K Alleles %K Animals %K Drosophila melanogaster %K Embryo, Nonmammalian %K Embryonic and Fetal Development %K Genes, Lethal %K Microscopy, Electron, Scanning %XLack of zygotic expression of the l(1)giant locus (l(1)gt;3A1), produces embryos with defects in abdominal A5, 6, and 7 and within the head. Scanning electron microscopy at the time of segment formation reveals two regions of defects in the segmentation pattern: anteriorly the labial lobe and thoracic segments T1 and T2 are fused; posteriorly, abdominal segments A5-7 are disrupted. The mature embryo shows incomplete head involution and defects within A5-7; fusion of T1 and T2 is no longer observed. Localized cell death within neural and mesodermal tissues is observed at 7 hr of development; later ventral ganglia, A5-7, are missing. Double-mutant analyses of l(1)gt with maternal effect lethal mutations and mutations that generate homeotic, segment number, gap, or segment polarity phenotypes indicate that normal activity of l(1)gt is required for differentiation of two embryonic domains: one corresponding to labial, T1 and T2 segments, and the second corresponding to abdominal segments 5, 6, and 7.
%B Dev Biol %V 119 %P 175-89 %8 1987 Jan %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/3098602?dopt=Abstract %0 Book Section %B Gametogenesis and the Early Embryo %D 1986 %T The maternal role of zygotic lethals during early embryogenesis in Drosophila %A Perrimon, Norbert %A Mahowald, Anthony P %E Gall, Joseph G %B Gametogenesis and the Early Embryo %I Alan R. Liss, Inc %C New York, NY %P 221-237 %G eng %0 Journal Article %J Dev Biol %D 1986 %T Developmental analysis of the torso-like phenotype in Drosophila produced by a maternal-effect locus. %A Degelmann, A %A Hardy, P A %A Perrimon, N %A Mahowald, A P %K Alleles %K Animals %K Blastoderm %K Drosophila %K Embryo, Nonmammalian %K Microscopy, Electron %K Microscopy, Electron, Scanning %K Phenotype %XThe segmental plan of the Drosophila embryo is already established at the blastoderm stage through the action of maternal effect genes which determine the polarity of the embryo and zygotically active genes involved in segmentation. We have analyzed the first example of a group of maternally acting genes which are necessary for establishing the developmental potential of the posterior 25% of the blastoderm. Females, homozygous for the X-linked maternal-effect mutation female sterile(1)Nasrat211 [fs(1)N211], produce embryos, characterized as torso-like, which lack all posterior endodermal derivatives as well as structures characteristic of abdominal segments 8 to 10. In addition, anterior endodermal derivatives are deficient and the absence of pharyngeal musculature causes a collapse of the cephalopharyngeal apparatus. The columnar blastoderm cell layer is defective at the posterior tip below the pole cells in these embryos. This defect, however, is presumably secondary to some abnormal feature of pole cell formation since in double mutants of fs(1)Nasrat211; tudor3 the blastoderm is normal but the embryos still show the torso-like phenotype. In situ hybridization with RNA probes derived from the fushi tarazu gene establishes that the cellular determination of the posterior blastoderm of embryos produced by fs(1)N211 is changed. This represents the first direct demonstration that a maternal-effect mutation alters the spatial distribution of a zygotic gene product involved in the segmental patterning of the embryo.
%B Dev Biol %V 115 %P 479-89 %8 1986 Jun %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/3709971?dopt=Abstract %0 Journal Article %J Dev Biol %D 1986 %T l(1)hopscotch, A larval-pupal zygotic lethal with a specific maternal effect on segmentation in Drosophila. %A Perrimon, Norbert %A Mahowald, Anthony P %K Abdomen %K Animals %K Chromosome Mapping %K Dosage Compensation, Genetic %K Drosophila melanogaster %K Genes, Lethal %K Genetic Complementation Test %K Infertility %K Larva %K Morphogenesis %K Mutation %K Phenotype %K Pupa %K Thorax %XThe maternal and zygotic effect phenotypes of mutations at the l(1)hopscotch (l(1)hop) locus are described. l(1)hop is located in 10B6-8 on the salivary gland chromosome map and 17 alleles have been characterized. A complex complementation pattern is observed among the 17 alleles. The lethal phase of null alleles of l(1)hop occurs at the larval-pupal interface associated with a small disc phenotype. Embryos produced from homozygous l(1)hop germline clones show segment specific defects. The extent of these defects depends upon both the strength of the allele and the paternal contribution. In the most extreme case embryos exhibit defects associated with five segments T2, T3, A4, A5, and A8. In the less extreme phenotype defects are only associated with A5. Thus, activity of l(1)hop+ is required both for the maintenance and continued cell division of diploid imaginal precursors and for the establishment of the full array of segments.
%B Dev Biol %V 118 %P 28-41 %8 1986 Nov %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/3095163?dopt=Abstract %0 Journal Article %J Genetics %D 1986 %T X-linked female-sterile loci in Drosophila melanogaster. %A Perrimon, Norbert %A Mohler, Dawson %A Engstrom, Lee %A Mahowald, Anthony P %K Alleles %K Animals %K Chromosome Mapping %K Drosophila melanogaster %K Embryo, Nonmammalian %K Female %K Genetic Complementation Test %K Infertility, Female %K Oogenesis %K Phenotype %K X Chromosome %XWe have examined the number of X-linked loci specifically required only during oogenesis. Complementation analyses among female-sterile (fs) mutations obtained in two mutagenesis screens--GANS' and MOHLER's--indicate that any fs locus represented by two or more mutant alleles in GANS' collection are usually present in MOHLER's collection. However, when a locus is represented by a single allele in one collection, it is generally not present in the other collection. We propose that this discrepancy is due to the fact that most "fs loci" represented by less than two mutant alleles are, in fact, vital (zygotic lethal) genes, and that the fs alleles are hypomorphic mutations of such genes. In support of this hypothesis we have identified lethal alleles at 12 of these "fs loci." The present analysis has possibly identified all maternal-effect lethal loci detectable by mutations on the X chromosome and has allowed us to reevaluate the number of "ovary-specific fs" loci in the Drosophila genome. Finally, germline clone analysis of a large number of fs mutations was performed in order to estimate the relative contribution of germline and somatic cell derivatives to oogenesis and to embryonic development. All the maternal-effect lethal loci tested are germline-dependent.
%B Genetics %V 113 %P 695-712 %8 1986 Jul %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/3089870?dopt=Abstract %0 Book Section %B Developmental Biology: A Comprehensive Synthesis %D 1985 %T Genetic analysis of oogenesis and the role of maternal gene expression in early development %A Konrad, K D %A Engstrom, Lee %A Perrimon, Norbert %A Mahowald, Anthony P %E Brower, L %B Developmental Biology: A Comprehensive Synthesis %I Oogenesis %V 1 %P 577-617 %G eng %0 Journal Article %J Mol Gen Genet %D 1985 %T Clonal analysis of two mutations in the large subunit of RNA polymerase II of Drosophila. %A Mortin, M A %A Perrimon, N %A Bonner, J J %K Animals %K Cell Differentiation %K Cell Division %K Cloning, Molecular %K Drosophila melanogaster %K Genes %K Genotype %K Macromolecular Substances %K Microscopy, Electron, Scanning %K Mutation %K RNA Polymerase II %XTwo mutations in the gene, RpII215, were analyzed to determine their effects on cell differentiation and proliferation. The mutations differ in that one, RpII215ts (ts), only displays a conditional recessive lethality, while the other, RpII215Ubl (Ubl), is a recessive lethal mutation that also displays a dominant mutant phenotype similar to that caused by the mutation Ultrabithorax (Ubx). Ubl causes a partial transformation of the haltere into a wing; however, this transformation is more complete in flies carrying both Ubl and Ubx. The present study shows that patches of Ubl/-tissue in gynandromorphs are morphologically normal. cuticle that has lost the wild-type copy of the RpII215 locus fails to show a haltere to wing transformation, nor does it show the synergistic enhancement of Ubx by Ubl. We conclude that an interaction between the two RpII215 alleles, Ubl and RpII215+, is responsible for the mutant phenotype. Gynandromorphs carrying the ts allele, when raised at permissive temperature, display larger patches of ts/-cuticle than expected, possibly indicating that the proliferation of ts/+ cells is reduced. This might result from an antagonistic interaction between different RpII215 alleles. Classical negative complementation does not appear to be the cause of the antagonistic interactions described above, as only one RpII215 subunit is thought to be present in an active multimeric polymerase enzyme. We have therefore coined the term 'negative heterosis' to describe the aforementioned interactions. We also observed that the effects of mutationally altered RNA polymerase II on somatic cells are different from its effects on germ cells.(ABSTRACT TRUNCATED AT 250 WORDS)
%B Mol Gen Genet %V 199 %P 421-6 %8 1985 %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/3929014?dopt=Abstract %0 Journal Article %J Genetics %D 1985 %T Developmental genetics of the 2C-D region of the Drosophila X chromosome. %A Perrimon, Norbert %A Engstrom, Lee %A Mahowald, Anthony P %K Animals %K Chromosome Mapping %K Drosophila melanogaster %K Female %K Genes, Lethal %K Genetic Linkage %K Mutation %K Phenotype %K X Chromosome %XWe have conducted a genetic and developmental analysis of genes within the 2C-D area of the X chromosome. Phenotypes of 33 mutations representing nine adjacent complementation groups including eight recessive lethals and one visible homeotic mutation (polyhomeotic) are described. Germline clonal analysis of the eight zygotic lethals has revealed three types of gene requirements: normal activity at two pupal lethal loci (corkscrew and C204) and one larval lethal locus (ultraspiracle) is required for normal embryogenesis; normal activity at three larval lethal loci (DF967, VE651 and Pgd) is required for normal oogenesis; and activity at only one locus (EA82), a larval lethal, appears to have no maternal requirement. Ambiguous results were obtained for the GF316 lethal complementation group. Analysis of mitotic figures of the pupal lethals indicates that C204 disrupts an essential mitotic function. This result correlates with the preblastoderm arrest observed among embryos derived from germline clones of C204. Embryos derived from germline clones of corkscrew (csw) exhibit a "twisted" phenotype. The recessive lethal ultraspiracle (usp) disrupts the organization of the posterior tip of the larval both zygotically and maternally: second instar usp/Y larvae derived from heterozygous usp/+ mothers possess an extra set of spiracles, whereas usp/Y embryos derived from females possessing a germline clone (usp/usp) exhibit a localized ventral defect in the ninth or posterior eighth abdominal segment. Analysis of the phenotypes of deficiency-hemizygous embryos indicates the presence of an embryonic zygotic lethal locus, as yet unidentified, which produces central nervous system and ventral hypoderm degeneration. Additional information on the genetic organization of loci within the adjacent 2E area are also described.(ABSTRACT TRUNCATED AT 250 WORDS)
%B Genetics %V 111 %P 23-41 %8 1985 Sep %G eng %N 1 %1 http://www.ncbi.nlm.nih.gov/pubmed/3928431?dopt=Abstract %0 Journal Article %J Dev Biol %D 1985 %T A pupal lethal mutation with a paternally influenced maternal effect on embryonic development in Drosophila melanogaster. %A Perrimon, Norbert %A Engstrom, Lee %A Mahowald, Anthony P %K Alleles %K Animals %K Animals, Wild %K Blastoderm %K Drosophila melanogaster %K Female %K Genes %K Humans %K Interphase %K Larva %K Male %K Mutation %K Phenotype %K Pupa %K Spermatozoa %K X Chromosome %K Y Chromosome %K Zygote %XThe maternal effect and zygotic phenotype of l(1)pole hole (l(1)ph) is described. l(1)ph is a zygotic lethal mutation which affects cell division of adult precursor cells in Drosophila larvae. The locus is located in 2F6 on the salivary gland chromosome map and four alleles have been characterized. Germ-line clonal analysis of amorphic alleles indicates that l(1)ph has a maternal effect lethal phenotype. Two lethal phenotypes are observed among embryos derived from female germ-line clones homozygous for amorphic alleles dependent upon the zygotic activity of l(1)ph+ introduced via the sperm. Class 1: If no wild-type dose of the gene is introduced, embryos form abnormal blastoderms in which nuclear migration and cell formation is disrupted leading to an ill-defined cuticular pattern. Class 2: If a wild-type copy of the gene is introduced, blastoderm cells do not form beneath the pole cells (the pole hole phenotype); subsequently such embryos are missing cuticular structures posterior to the seventh abdominal segment (the torso phenotype). When the zygotic activity l(1)ph+ is modulated using position effect variegation a new phenotype is observed among class 2 embryos in which torso embryos are twisted along their longitudinal axis.
%B Dev Biol %V 110 %P 480-91 %8 1985 Aug %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/3926563?dopt=Abstract %0 Book Section %B Molecular Biology of Development %D 1984 %T Genetic approach to early development %A Mahowald, Anthony P %A Konrad, K D %A Engstrom, Lee %A Perrimon, Norbert %B Molecular Biology of Development %I Alan R. Liss, Inc %C New York, NY %P 185-197 %G eng %0 Journal Article %J Genetics %D 1984 %T Clonal Analysis of Dominant Female-Sterile, Germline-Dependent Mutations in Drosophila Melanogaster. %A Perrimon, Nobert %XThree allelic, dominant and germline-dependent female-sterile mutations (ovo(D) mutations) can be classified according to the severity of the ovarian abnormalities that they produce. The size and frequency of +/+ germline clones, induced in ovo(D)/+ females, were compared with K10/K10 germline clones induced in K10/+ control females. The frequency of germline clones induced by irradiation of first instar larvae is similar for the three dominant alleles and K10 ; however, the clone size increased with the strength of the allele tested, compared with K10 clones. When clones were induced later in development, the clone frequencies decreased with the strength of the alleles. These results are discussed in the context of the antimorphic nature of these mutations and the characteristics of germline development. The use of these alleles as tools in the genetic analysis of development is discussed.
%B Genetics %V 108 %P 927-39 %8 1984 Dec %G eng %N 4 %1 http://www.ncbi.nlm.nih.gov/pubmed/17246244?dopt=Abstract %0 Journal Article %J Genetics %D 1984 %T Developmental genetics of the 2E-F region of the Drosophila X chromosome: a region rich in "developmentally important" genes. %A Perrimon, Norbert %A Engstrom, Lee %A Mahowald, Anthony P %K Alleles %K Animals %K Chromosome Mapping %K Drosophila melanogaster %K Female %K Fertility %K Genes, Lethal %K Genes, Recessive %K Phenotype %K X Chromosome %XWe have analyzed the 2E1-3A1 area of the X chromosome with special attention to loci related to embryogenesis. Published maps indicate that this chromosomal segment contains ten bands. Our genetic analysis has identified 11 complementation groups: one recessive visible (prune), two female steriles and eight lethals. One of the female sterile loci is fs(1)k10 for which homozygous females produce both egg chambers and embryos with a dorsalized morphology. The second female sterile is the paternally rescuable fs(1)pecanex in which unrescued embryos have a hypertrophic nervous system. Of the eight lethal complementation groups two are recessive embryonic lethals: hemizygous giant (gt) embryos possess segmental defects, and hemizygous crooked neck (crn) embryos exhibit a twisted phenotype. Analysis of these mutations in the female germ line indicates that gt does not show a maternal effect, whereas normal activity of crn is required for germ cell viability. Analysis of the maternal effect in germ line clones of the remaining six recessive lethal complementation groups indicates that four are required for germ cell viability and one produces ambiguous results for survival of the germ cells. The remaining, l(1)pole hole, is a recessive early pupal lethal in which embryos derived from germ line clones and lacking wild-type gene activity exhibit the "torso" or "pole hole" phenotype.
%B Genetics %V 108 %P 559-72 %8 1984 Nov %G eng %N 3 %1 http://www.ncbi.nlm.nih.gov/pubmed/6437900?dopt=Abstract %0 Journal Article %J J Embryol Exp Morphol %D 1984 %T Differentiation markers in the Drosophila ovary. %A White, Robert AH %A Perrimon, Norbert %A Gehring, Walter J %K Animals %K Antibodies, Monoclonal %K Antigens %K Cell Differentiation %K Drosophila melanogaster %K Female %K Fluorescent Antibody Technique %K Lectins %K Oogenesis %K Ovary %K Wheat Germ Agglutinins %XA library of monoclonal antibodies, raised against imaginal discs of Drosophila melanogaster, was screened for binding to differentiation antigens in the adult ovary by immunofluorescence. Several lectins were similarly assayed. Two antibodies, DOV 1 and DOV 2, and wheat germ agglutinin exhibited binding which was restricted to particular stages of ovarian cell differentiation. DOV 2 also showed a marked preferential binding to the cell surface of germ line cells in the ovary. A differentiation of the portion of the tunica propria covering the anterior part of the germarium was revealed by the monoclonal antibody DOV 3. Another monoclonal antibody, DOV 4, identified a molecular specialization of the chorion at the tip of the micropyle. These markers should provide tools for the molecular analysis of oogenesis.
%B J Embryol Exp Morphol %V 84 %P 275-86 %8 1984 Dec %G eng %1 http://www.ncbi.nlm.nih.gov/pubmed/6442733?dopt=Abstract %0 Journal Article %J Dev Biol %D 1984 %T The effects of zygotic lethal mutations on female germ-line functions in Drosophila. %A Perrimon, Norbert %A Engstrom, Lee %A Mahowald, Anthony P %K Alleles %K Animals %K Clone Cells %K Crosses, Genetic %K Drosophila %K Embryo, Nonmammalian %K Female %K Genes, Lethal %K Heterozygote %K Larva %K Male %K Mutation %K Oocytes %K Oogenesis %K Phenotype %XMany genetic loci that result in lethality when mutated may also have an essential role in oogenesis. The maternal effects of EMS-induced zygotic lethal mutations at 48 loci were examined using the dominant female-sterile technique. Three categories of effects were found. In the first group (13 out of 48), no maternal effect was detected. The second set (20 out of 48) exhibited maternal effects on oogenesis, embryogenesis, or both. In 13 of this last group, only a few eggs were produced before a progressive deterioration of development occurred. It is suggested that perdurance of the wild-type gene product could produce this result. The third group (15 out of 48) produced cell lethality in germ-line clones, an effect that may be related to their role in indispensable cell functions. Three loci were found which, in germ-line clones, produced embryonic phenotypes that resemble maternal effect mutations. The implications of this study for the genetic analysis of early development are discussed.
%B Dev Biol %V 105 %P 404-14 %8 1984 Oct %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/6479445?dopt=Abstract %0 Journal Article %J Dev Biol %D 1983 %T Clonal analysis of the tissue specificity of recessive female-sterile mutations of Drosophila melanogaster using a dominant female-sterile mutation Fs(1)K1237. %A Perrimon, Norbert %A Gans, Madeleine %K Animals %K Chromosome Mapping %K Clone Cells %K Drosophila melanogaster %K Female %K Genes, Recessive %K Genetic Linkage %K Genitalia %K Infertility, Female %K Mutation %K Oogenesis %K X Chromosome %XUsing the newly isolated, germ line-dependent dominant female-sterile mutation Fs(1)K1237, we have characterized the germ line or somatic line dependence of 25 X-linked recessive female-sterile mutations. Since Fs(1)K1237/+ females fail to lay eggs, only germ line cells which lose Fs(1)K1237 as a result of X-ray-induced mitotic recombination are capable of producing eggs. Such recombination events will render genes on the homologous chromosome homozygous. If this chromosome carries a recessive female-sterile mutation, the fertility will be restored only if the altered function is not required in the germ line. Using this test, we have classified 25 recessive female-sterile mutations: 12 affect germ line function, 12 affect somatic line function, and one gave an ambiguous result for which an explanation is proposed. For a few of the somatic line-dependent mutants, we found that some eggs derived from germ line clones showed the same phenotype as eggs laid by females homozygous for the recessive female-sterile mutation. These results are discussed in terms of a coincident production of clones in the follicle cells.
%B Dev Biol %V 100 %P 365-73 %8 1983 Dec %G eng %N 2 %1 http://www.ncbi.nlm.nih.gov/pubmed/6418585?dopt=Abstract