Tipping M, Perrimon N. Drosophila as a model for context-dependent tumorigenesis. J Cell Physiol. 2014;229 (1) :27-33. Abstract

Drosophila 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.

2014_J Cell Phys_Tipping.pdf
Zhou R, Mohr S, Hannon GJ, Perrimon N. Inducing RNAi in Drosophila cells by soaking with dsRNA. Cold Spring Harb Protoc. 2014;2014 (5). Abstract

RNA 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.

2014_CSH Perspect_Zhou.pdf
Piccirillo R, Demontis F, Perrimon N, Goldberg AL. Mechanisms of muscle growth and atrophy in mammals and Drosophila. Dev Dyn. 2014;243 (2) :201-15. Abstract

BACKGROUND: 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.

2014_Dev Dyn_Piccirillo.pdf
Owusu-Ansah E, Perrimon N. Modeling metabolic homeostasis and nutrient sensing in Drosophila: implications for aging and metabolic diseases. Dis Model Mech. 2014;7 (3) :343-50. Abstract

Over 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.

Mohr SE, Hu Y, Kim K, Housden BE, Perrimon N. Resources for functional genomics studies in Drosophila melanogaster. Genetics. 2014;197 (1) :1-18. Abstract

Drosophila 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.

Mohr SE, Smith JA, Shamu CE, Neumüller RA, Perrimon N. RNAi screening comes of age: improved techniques and complementary approaches. Nat Rev Mol Cell Biol. 2014;15 (9) :591-600. Abstract

Gene 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.

2014_Nat Rev MCB_Mohr.pdf
Droujinine IA, Yan D, Perrimon N. A sharp end to sugary Wingless travels. J Cell Biol. 2014;206 (7) :819-21. Abstract

Drosophila 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. 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.

Housden BE, Perrimon N. Spatial and temporal organization of signaling pathways. Trends Biochem Sci. 2014;39 (10) :457-64. Abstract

The 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.

2014_Trends Bio Sci_Housden.pdf
Doupé DP, Perrimon N. Visualizing and manipulating temporal signaling dynamics with fluorescence-based tools. Sci Signal. 2014;7 (319) :re1. Abstract

The 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.

2014_Sci Sig_Doupe.pdf
Perrimon N. Drosophila developmental biology methods. Methods. 2014;68 (1) :1. 2014_Methods_Perrimon.pdf
Sopko R, Foos M, Vinayagam A, Zhai B, Binari R, Hu Y, et al. Combining genetic perturbations and proteomics to examine kinase-phosphatase networks in Drosophila embryos. Dev Cell. 2014;31 (1) :114-27. Abstract

Connecting 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.

2014_Dev Cell_Sopko.pdf Supplement.pdf
Gerstein MB, Rozowsky J, Yan K-K, Wang D, Cheng C, Brown JB, et al. Comparative analysis of the transcriptome across distant species. Nature. 2014;512 (7515) :445-8. Abstract

The 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.

2014_Nature_Gerstein.pdf Supplement.pdf
Song W, Veenstra JA, Perrimon N. Control of lipid metabolism by tachykinin in Drosophila. Cell Rep. 2014;9 (1) :40-7. Abstract

The 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.

2014_Cell Rep_Song.pdf Supplement.pdf Erratum.pdf
Brown JB, Boley N, Eisman R, May GE, Stoiber MH, Duff MO, et al. Diversity and dynamics of the Drosophila transcriptome. Nature. 2014;512 (7515) :393-9. Abstract

Animal 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.

2014_Nature_Brown.pdf Supplemental
Amcheslavsky A, Song W, Li Q, Nie Y, Bragatto I, Ferrandon D, et al. Enteroendocrine cells support intestinal stem-cell-mediated homeostasis in Drosophila. Cell Rep. 2014;9 (1) :32-9. Abstract

Intestinal 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.

2014_Cell Rep_Amcheslavsky.pdf Supplement.pdf
Shulman JM, Imboywa S, Giagtzoglou N, Powers MP, Hu Y, Devenport D, et al. Functional screening in Drosophila identifies Alzheimer's disease susceptibility genes and implicates Tau-mediated mechanisms. Hum Mol Genet. 2014;23 (4) :870-7. Abstract

Using 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.

2014_Human Mol Gene_Shulman.pdf Supplemental
Griffin R, Binari R, Perrimon N. Genetic odyssey to generate marked clones in Drosophila mosaics. Proc Natl Acad Sci U S A. 2014;111 (13) :4756-63. Abstract

Chimaeras, 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.

Vinayagam A, Zirin J, Roesel C, Hu Y, Yilmazel B, Samsonova AA, et al. Integrating protein-protein interaction networks with phenotypes reveals signs of interactions. Nat Methods. 2014;11 (1) :94-9. Abstract

A 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.

2014_Nat Methods_Vinayagam.pdf Supplemental
Demontis F, Patel VK, Swindell WR, Perrimon N. Intertissue control of the nucleolus via a myokine-dependent longevity pathway. Cell Rep. 2014;7 (5) :1481-94. Abstract

Recent 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.

2014_Cell Rep_Demontis.pdf Supp. Info 1.pdf Supp. Info 2.pdf
Yilmazel B, Hu Y, Sigoillot F, Smith JA, Shamu CE, Perrimon N, et al. Online GESS: prediction of miRNA-like off-target effects in large-scale RNAi screen data by seed region analysis. BMC Bioinformatics. 2014;15 :192. Abstract

BACKGROUND: 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.

2014_BMC Bio_Yilmazel.pdf