Publications

2020
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_corrected.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 mA methylation of U2 snRNA in . Cell Discov. 2020;6 :44. 2020_Cell Discov_Gu.pdf
Bajpai A, Quazi TA, 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;13 (7). 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/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.
2020_DMM_Bajpai.pdf
Tattikota SG, Cho B, Liu Y, Hu Y, Barrera V, Steinbaugh MJ, et al. A single-cell survey of 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. as a model for studying cystic fibrosis pathophysiology of the gastrointestinal system. Proc Natl Acad Sci U S A. 2020;117 (19) :10357-10367. 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
Zirin J, Hu Y, Liu L, Yang-Zhou D, Colbeth R, Yan D, et al. Large-Scale Transgenic Resource Collections for Loss- and Gain-of-Function Studies. Genetics. 2020;214 (4) :755-767. Abstract
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.
2020_Genetics_Zirin.pdf Supplement.zip
Bosch JA, Knight S, Kanca O, Zirin J, Yang-Zhou D, Hu Y, et al. Use of the CRISPR-Cas9 System in Drosophila Cultured Cells to Introduce Fluorescent Tags into Endogenous Genes. Curr Protoc Mol Biol. 2020;130 (1) :e112. Abstract
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.
2020_CurrProt_Bosch.pdf
Bosch JA, Colbeth R, Zirin J, Perrimon N. Gene Knock-Ins in Using Homology-Independent Insertion of Universal Donor Plasmids. Genetics. 2020;214 (1) :75-89. Abstract
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.
2020_Genetics_Bosch.pdf
Alliance-of-Genome-Resources-Consortium. Alliance of Genome Resources Portal: unified model organism research platform. Nucleic Acids Res. 2020;Abstract
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.
2020_NucAcidsRes_AGRC.pdf
2019
Amamoto R, Garcia MD, West ER, Choi J, Lapan SW, Lane EA, et al. Probe-Seq enables transcriptional profiling of specific cell types from heterogeneous tissue by RNA-based isolation. Elife. 2019;8. Abstract
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.
2019_eLife_Amamoto.pdf Supp. File 1.xlsx Source Code.zip
Kwon YV, Zhao B, Xu C, Lee J, Chen C-L, Vinayagam A, et al. The role of translationally controlled tumor protein in proliferation of intestinal stem cells. Proc Natl Acad Sci U S A. 2019;Abstract
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.
2019_PNAS_Kwon.pdf Supplement.pdf
AGRC. The Alliance of Genome Resources: Building a Modern Data Ecosystem for Model Organism Databases. Genetics. 2019;213 (4) :1189-1196. Abstract
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.
2019_Genetics_AGRC.pdf

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