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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.