Publications

2021
Larkin A, Marygold SJ, Antonazzo G, Attrill H, Dos Santos G, Garapati PV, et al. FlyBase: updates to the Drosophila melanogaster knowledge base. Nucleic Acids Res. 2021;49 (D1) :D899-D907. Abstract
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.
2021_NucAcidsRes_Larkin.pdf
Bosch JA, Birchak G, Perrimon N. Precise genome engineering in using prime editing. Proc Natl Acad Sci U S A. 2021;118 (1). Abstract
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.
2021_PNAS_Bosch.pdf 2021_PNAS_Bosch_Supp.pdf
Hu Y, Comjean A, Rodiger J, Liu Y, Gao Y, Chung V, et al. FlyRNAi.org-the database of the Drosophila RNAi screening center and transgenic RNAi project: 2021 update. Nucleic Acids Res. 2021;49 (D1) :D908-D915. Abstract
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.
2020_NucAcidsRes_Hu.pdf
Bosch JA, Chen C-L, Perrimon N. Proximity-dependent labeling methods for proteomic profiling in living cells: An update. Wiley Interdiscip Rev Dev Biol. 2021;10 (1) :e392. Abstract
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.
2021_WileyDevBio_Bosch.pdf
2020
Hu Y, Chung V, Comjean A, Rodiger J, Nipun F, Perrimon N, et al. BioLitMine: Advanced Mining of Biomedical and Biological Literature About Human Genes and Genes from Major Model Organisms. G3 (Bethesda). 2020;10 (12) :4531-4539. Abstract
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.
2020_G3_Hu.pdf
Parkhitko AA, Ramesh D, Wang L, Leshchiner D, Filine E, Binari R, et al. Downregulation of the tyrosine degradation pathway extends lifespan. Elife. 2020;9. Abstract
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.
2020_eLife_Parkhitko.pdf
Ingaramo MC, Sánchez JA, Perrimon N, Dekanty A. Fat Body p53 Regulates Systemic Insulin Signaling and Autophagy under Nutrient Stress via Drosophila Upd2 Repression. Cell Rep. 2020;33 (4) :108321. Abstract
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.
2020_Cell Rep_Ingaramo.pdf
Ghosh AC, Tattikota SG, Liu Y, Comjean A, Hu Y, Barrera V, et al. PDGF/VEGF signaling from muscles to hepatocyte-like cells protects against obesity. Elife. 2020;9. Abstract
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.
2020_eLife_Ghosh.pdf
Parkhitko AA, Filine E, Mohr SE, Moskalev A, Perrimon N. Targeting metabolic pathways for extension of lifespan and healthspan across multiple species. Ageing Res Rev. 2020;64 :101188. Abstract
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.
2020_AgeResRev_Parkhitko.pdf
Xia B, Amador G, Viswanatha R, Zirin J, Mohr SE, Perrimon N. CRISPR-based engineering of gene knockout cells by homology-directed insertion in polyploid Drosophila S2R+ cells. Nat Protoc. 2020;15 (10) :3478-3498. Abstract
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.
2020_Nature Prot_Xia.pdf Supplement.pdf
Ewen-Campen B, Perrimon N. Expanding the horizons of genome editing in the fruit fly with Cas12a. Proc Natl Acad Sci U S A. 2020;117 (39) :24019-24021. 2020_PNAS_Ewen-Campen.pdf
Ewen-Campen B, Comyn T, Vogt E, Perrimon N. No Evidence that Wnt Ligands Are Required for Planar Cell Polarity in Drosophila. Cell Rep. 2020;32 (10) :108121. Abstract
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.
2020_Cell Rep_Ewen-Campen.pdf Supplement.pdf
Cho B, Yoon S-H, Lee D, Koranteng F, Tattikota SG, Cha N, et al. Single-cell transcriptome maps of myeloid blood cell lineages in Drosophila. Nat Commun. 2020;11 (1) :4483. Abstract
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.
2020_Nature Comm_Cho.pdf Supplement.pdf
Gu L, Wang L, Chen H, Hong J, Shen Z, Dhall A, et al. CG14906 (mettl4) mediates m6A methylation of U2 snRNA in Drosophila. Cell Discov. 2020;6 :44. 2020_Cell Discov_Gu.pdf
Bajpai A, Ahmad QT, Tang H-W, Manzar N, Singh V, Thakur A, et al. A model of oral peptide therapeutics for adult Intestinal Stem Cell tumors. Dis Model Mech. 2020;Abstract
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/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) protein, specifically its TONDU domain, competitively inhibits YAP-TEAD interaction, resulting in arrest of tumor growth. Here, we show that either overexpression of the TONDU peptide or its oral uptake leads to suppression of Yorkie (Yki)-driven intestinal stem cell (ISC) tumors in the adult midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with ChIP 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.
2020_DMM_Bajpai.pdf
Tattikota SG, Cho B, Liu Y, Hu Y, Barrera V, Steinbaugh MJ, et al. A single-cell survey of Drosophila blood. Elife. 2020;9. Abstract
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.
2020_eLife_Tattikota.pdf
Kim K, Lane EA, Saftien A, Wang H, Xu Y, Wirtz-Peitz F, et al. Drosophila as a model for studying cystic fibrosis pathophysiology of the gastrointestinal system. Proc Natl Acad Sci U S A. 2020;Abstract
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.
2020_PNAS_Kim.pdf Supplement.pdf
Vásquez-Procopio J, Osorio B, Cortés-Martínez L, Hernández-Hernández F, Medina-Contreras O, Ríos-Castro E, et al. Intestinal response to dietary manganese depletion in Drosophila. Metallomics. 2020;12 (2) :218-240. Abstract
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.
2020_RSC_Vasquez-Procopio.pdf Supp. Figures.pdf Supp. Tables.zip
Hung R-J, Hu Y, Kirchner R, Liu Y, Xu C, Comjean A, et al. A cell atlas of the adult midgut. Proc Natl Acad Sci U S A. 2020;117 (3) :1514-1523. Abstract
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.
2020_PNAS_Hung.pdf Supplement.pdf
Xu C, Franklin B, Tang H-W, Regimbald-Dumas Y, Hu Y, Ramos J, et al. An in vivo RNAi screen uncovers the role of AdoR signaling and adenosine deaminase in controlling intestinal stem cell activity. Proc Natl Acad Sci U S A. 2020;117 (1) :464-471. Abstract
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.
2020_PNAS_Xu.pdf Supplement.pdf

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