RESUMO
Genomic imprinting-the non-equivalence of maternal and paternal genomes-is a critical process that has evolved independently in many plant and mammalian species1,2. According to kinship theory, imprinting is the inevitable consequence of conflictive selective forces acting on differentially expressed parental alleles3,4. Yet, how these epigenetic differences evolve in the first place is poorly understood3,5,6. Here we report the identification and molecular dissection of a parent-of-origin effect on gene expression that might help to clarify this fundamental question. Toxin-antidote elements (TAs) are selfish elements that spread in populations by poisoning non-carrier individuals7-9. In reciprocal crosses between two Caenorhabditis tropicalis wild isolates, we found that the slow-1/grow-1 TA is specifically inactive when paternally inherited. This parent-of-origin effect stems from transcriptional repression of the slow-1 toxin by the PIWI-interacting RNA (piRNA) host defence pathway. The repression requires PIWI Argonaute and SET-32 histone methyltransferase activities and is transgenerationally inherited via small RNAs. Remarkably, when slow-1/grow-1 is maternally inherited, slow-1 repression is halted by a translation-independent role of its maternal mRNA. That is, slow-1 transcripts loaded into eggs-but not SLOW-1 protein-are necessary and sufficient to counteract piRNA-mediated repression. Our findings show that parent-of-origin effects can evolve by co-option of the piRNA pathway and hinder the spread of selfish genes that require sex for their propagation.
Assuntos
Caenorhabditis , Impressão Genômica , RNA de Interação com Piwi , Sequências Repetitivas de Ácido Nucleico , Animais , Feminino , Masculino , Alelos , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Caenorhabditis/genética , Caenorhabditis/metabolismo , Cruzamentos Genéticos , Pai , Genoma/genética , Impressão Genômica/genética , Organismos Hermafroditas/genética , Histona Metiltransferases/genética , Histona Metiltransferases/metabolismo , Mães , Oócitos/metabolismo , RNA de Interação com Piwi/genética , Biossíntese de Proteínas , Sequências Repetitivas de Ácido Nucleico/genética , RNA Mensageiro/genética , Toxinas Biológicas/genética , Transcrição GênicaRESUMO
Nuclear Argonaute proteins, guided by their bound small RNAs to nascent target transcripts, mediate cotranscriptional silencing of transposons and repetitive genomic loci through heterochromatin formation. The molecular mechanisms involved in this process are incompletely understood. Here, we show that the SFiNX complex, a silencing mediator downstream from nuclear Piwi-piRNA complexes in Drosophila, facilitates cotranscriptional silencing as a homodimer. The dynein light chain protein Cut up/LC8 mediates SFiNX dimerization, and its function can be bypassed by a heterologous dimerization domain, arguing for a constitutive SFiNX dimer. Dimeric, but not monomeric SFiNX, is capable of forming molecular condensates in a nucleic acid-stimulated manner. Mutations that prevent SFiNX dimerization result in loss of condensate formation in vitro and the inability of Piwi to initiate heterochromatin formation and silence transposons in vivo. We propose that multivalent SFiNX-nucleic acid interactions are critical for heterochromatin establishment at piRNA target loci in a cotranscriptional manner.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Inativação Gênica/fisiologia , Complexos Multiproteicos/metabolismo , Animais , Dimerização , Proteínas de Drosophila/química , Drosophila melanogaster/metabolismo , Dineínas/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
Stem cells need to balance self-renewal and differentiation for correct tissue development and homeostasis. Defects in this balance can lead to developmental defects or tumor formation. In recent years, mRNA splicing has emerged as an important mechanism regulating cell fate decisions. Here we address the role of the evolutionarily conserved splicing co-factor Barricade (Barc)/Tat-SF1/CUS2 in Drosophila neural stem cell (neuroblast) lineage formation. We show that Barc is required for the generation of neurons during Drosophila brain development by ensuring correct neural progenitor proliferation and differentiation. Barc associates with components of the U2 small nuclear ribonucleoprotein (snRNP) complex, and its depletion causes alternative splicing in the form of intron retention in a subset of genes. Using bioinformatics analysis and a cell culture-based splicing assay, we found that Barc-dependent introns share three major traits: they are short, GC rich and have weak 3' splice sites. Our results show that Barc, together with the U2 snRNP complex, plays an important role in regulating neural stem cell lineage progression during brain development and facilitates correct splicing of a subset of introns.
Assuntos
Ciclo Celular , Linhagem da Célula , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Fatores de Transcrição/metabolismo , Processamento Alternativo/genética , Animais , Composição de Bases/genética , Sequência de Bases , Padronização Corporal/genética , Encéfalo/anatomia & histologia , Contagem de Células , Proliferação de Células , Células Clonais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Íntrons/genética , Camundongos , Modelos Biológicos , Mutação/genética , Neurônios/citologia , Neurônios/metabolismo , Fenótipo , Ligação Proteica , Interferência de RNA , Sítios de Splice de RNA/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Fatores de TempoRESUMO
The HOIP ubiquitin E3 ligase generates linear ubiquitin chains by forming a complex with HOIL-1L and SHARPIN in mammals. Here, we provide the first evidence of linear ubiquitination induced by a HOIP orthologue in Drosophila We identify Drosophila CG11321, which we named Linear Ubiquitin E3 ligase (LUBEL), and find that it catalyzes linear ubiquitination in vitro We detect endogenous linear ubiquitin chain-derived peptides by mass spectrometry in Drosophila Schneider 2 cells and adult flies. Furthermore, using CRISPR/Cas9 technology, we establish linear ubiquitination-defective flies by mutating residues essential for the catalytic activity of LUBEL Linear ubiquitination signals accumulate upon heat shock in flies. Interestingly, flies with LUBEL mutations display reduced survival and climbing defects upon heat shock, which is also observed upon specific LUBEL depletion in muscle. Thus, LUBEL is involved in the heat response by controlling linear ubiquitination in flies.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/fisiologia , Resposta ao Choque Térmico/fisiologia , Proteínas de Ligação a RNA/genética , Animais , Catálise , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Proteínas de Drosophila/metabolismo , Mutação , NF-kappa B/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Members of the diverse heterochromatin protein 1 (HP1) family play crucial roles in heterochromatin formation and maintenance. Despite the similar affinities of their chromodomains for di- and tri-methylated histone H3 lysine 9 (H3K9me2/3), different HP1 proteins exhibit distinct chromatin-binding patterns, likely due to interactions with various specificity factors. Previously, we showed that the chromatin-binding pattern of the HP1 protein Rhino, a crucial factor of the Drosophila PIWI-interacting RNA (piRNA) pathway, is largely defined by a DNA sequence-specific C2H2 zinc finger protein named Kipferl (Baumgartner et al., 2022). Here, we elucidate the molecular basis of the interaction between Rhino and its guidance factor Kipferl. Through phylogenetic analyses, structure prediction, and in vivo genetics, we identify a single amino acid change within Rhino's chromodomain, G31D, that does not affect H3K9me2/3 binding but disrupts the interaction between Rhino and Kipferl. Flies carrying the rhinoG31D mutation phenocopy kipferl mutant flies, with Rhino redistributing from piRNA clusters to satellite repeats, causing pronounced changes in the ovarian piRNA profile of rhinoG31D flies. Thus, Rhino's chromodomain functions as a dual-specificity module, facilitating interactions with both a histone mark and a DNA-binding protein.
Assuntos
Cromatina , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona , Proteínas de Drosophila , Drosophila melanogaster , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Cromatina/metabolismo , Cromatina/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolução Molecular , Filogenia , Ligação Proteica , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/genética , Histonas/metabolismo , Histonas/genética , DNA/metabolismo , DNA/genéticaRESUMO
RNA interference systems depend on the synthesis of small RNA precursors whose sequences define the target spectrum of these silencing pathways. The Drosophila Heterochromatin Protein 1 (HP1) variant Rhino permits transcription of PIWI-interacting RNA (piRNA) precursors within transposon-rich heterochromatic loci in germline cells. Current models propose that Rhino's specific chromatin occupancy at piRNA source loci is determined by histone marks and maternally inherited piRNAs, but also imply the existence of other, undiscovered specificity cues. Here, we identify a member of the diverse family of zinc finger associated domain (ZAD)-C2H2 zinc finger proteins, Kipferl, as critical Rhino cofactor in ovaries. By binding to guanosine-rich DNA motifs and interacting with the Rhino chromodomain, Kipferl recruits Rhino to specific loci and stabilizes it on chromatin. In kipferl mutant flies, Rhino is lost from most of its target chromatin loci and instead accumulates on pericentromeric Satellite arrays, resulting in decreased levels of transposon targeting piRNAs and impaired fertility. Our findings reveal that DNA sequence, in addition to the H3K9me3 mark, determines the identity of piRNA source loci and provide insight into how Rhino might be caught in the crossfire of genetic conflicts.
The genes within our DNA encode the essentials of our body plan and how each task in the body is achieved. However, our genome also contains many repetitive regions of DNA that do not encode functional genes. Some of these regions are genetic parasites known as transposons that try to multiply and spread around the DNA of their host. To prevent transposon DNA from interfering with the way the body operates, humans and other animals have evolved elaborate defense mechanisms to identify transposons and prevent them from multiplying. In one such mechanism, known as the piRNA pathway, the host makes small molecules known as piRNAs that have sequences complementary to those of transposons, and act as guides to silence the transposons. The instructions to make these piRNAs are stored in the form of transposon fragments in dedicated regions of host DNA called piRNA clusters. These clusters thereby act as genetic memory, allowing the host to recognize and silence specific transposons in other locations within the host's genome. In fruit flies, a protein called Rhino binds to piRNA clusters that are densely packed to allow piRNAs to be made. However, it remained unclear how Rhino is able to identify and bind to piRNA clusters, but not to other similarly densely packed regions of DNA. Baumgartner et al. used a combination of genetic, genomic, and imaging approaches to study how Rhino finds its way in the fruit fly genome. They found that another protein called Kipferl interacts with Rhino and is required for Rhino to bind to nearly all piRNA clusters. Since Kipferl can by itself bind to the sequences that Rhino needs to find, the results suggest that Kipferl acts to recruit and initiate Rhino binding within densely packed piRNA clusters. Further experiments found that, in flies lacking Kipferl, Rhino binds to regions of DNA called Satellite repeats, hinting that these selfish sequences may compete for Rhino for their own benefit. The finding that Kipferl and Rhino work together to define the memory system of the piRNA pathway strongly advances our understanding of how a sequence-specific defense system based on small RNAs can be established.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Elementos de DNA Transponíveis/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Guanosina/metabolismo , Precursores de RNA/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Dedos de ZincoRESUMO
Nuclear Argonaute proteins, guided by small RNAs, mediate sequence-specific heterochromatin formation. The molecular principles that link Argonaute-small RNA complexes to cellular heterochromatin effectors on binding to nascent target RNAs are poorly understood. Here, we explain the mechanism by which the PIWI-interacting RNA (piRNA) pathway connects to the heterochromatin machinery in Drosophila. We find that Panoramix, a corepressor required for piRNA-guided heterochromatin formation, is SUMOylated on chromatin in a Piwi-dependent manner. SUMOylation, together with an amphipathic LxxLL motif in Panoramix's intrinsically disordered repressor domain, are necessary and sufficient to recruit Small ovary (Sov), a multi-zinc-finger protein essential for general heterochromatin formation and viability. Structure-guided mutations that eliminate the Panoramix-Sov interaction or that prevent SUMOylation of Panoramix uncouple Sov from the piRNA pathway, resulting in viable but sterile flies in which Piwi-targeted transposons are derepressed. Thus, Piwi engages the heterochromatin machinery specifically at transposon loci by coupling recruitment of a corepressor to nascent transcripts with its SUMOylation.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Sítios de Ligação/genética , Cromatina/genética , Cromatina/metabolismo , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Inativação Gênica , Genes de Insetos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Moleculares , Mutação , Proteínas Nucleares/química , Células-Tronco de Oogônios/metabolismo , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Sumoilação/genética , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismoRESUMO
The Drosophila PDGF/VEGF receptor (PVR) has known functions in the guidance of cell migration. We now demonstrate that during embryonic hematopoiesis, PVR has a role in the control of antiapoptotic cell survival. In Pvr mutants, a large fraction of the embryonic hemocyte population undergoes apoptosis, and the remaining blood cells cannibalistically phagocytose their dying peers. Consequently, total hemocyte numbers drop dramatically during embryogenesis, and large aggregates of engorged macrophages carrying multiple apoptotic corpses form. Hemocyte-specific expression of the pan-caspase inhibitor p35 in Pvr mutants eliminates hemocyte aggregates and restores blood cell counts and morphology. Additional rescue experiments suggest involvement of the Ras pathway in PVR-mediated blood cell survival. In cell culture, we demonstrate that PVR directly controls survival of a hemocyte cell line. This function of PVR shows striking conservation with mammalian hematopoiesis and establishes Drosophila as a model to study hematopoietic cell survival in development and disease.
Assuntos
Células Sanguíneas/metabolismo , Sobrevivência Celular/genética , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/embriologia , Embrião não Mamífero/embriologia , Hematopoese/genética , Receptores Proteína Tirosina Quinases/fisiologia , Proteínas Virais , Animais , Apoptose/genética , Células Sanguíneas/citologia , Agregação Celular/genética , Células Cultivadas , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Macrófagos/fisiologia , Modelos Animais , Mutação/genética , Fagocitose/genética , Proteínas/genética , Proteínas/metabolismo , Receptores Proteína Tirosina Quinases/genética , Transdução de Sinais/genética , Proteínas ras/genética , Proteínas ras/metabolismoRESUMO
Proper synaptic development is critical for establishing all aspects of neural function including learning, memory, and locomotion. Here, we describe the phenotypic consequences of mutations in the wishful thinking (wit) gene, the Drosophila homolog of the vertebrate BMP type II receptor. Mutations in wit result in pharate lethality that can be rescued by expression of a wit transgene in motor neurons but not in muscles. Mutant larvae exhibit small synapses, severe defects in evoked junctional potentials, a lower frequency of spontaneous vesicle release, and an alteration in the ultrastructure of synaptic active zones. These results reveal a novel role for BMP signaling in regulating Drosophila neuromuscular junction synapse assembly and activity and may indicate that similar pathways could govern vertebrate synapse development.
Assuntos
Sistema Nervoso Central/anormalidades , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Mutação/fisiologia , Junção Neuromuscular/anormalidades , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Fatores de Transcrição , Animais , Padronização Corporal/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/genética , Sistema Nervoso Central/crescimento & desenvolvimento , Sistema Nervoso Central/ultraestrutura , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/ultraestrutura , Feminino , Genes Letais/genética , Imuno-Histoquímica , Masculino , Dados de Sequência Molecular , Neurônios Motores/metabolismo , Neurônios Motores/ultraestrutura , Junção Neuromuscular/crescimento & desenvolvimento , Junção Neuromuscular/ultraestrutura , Plasticidade Neuronal/genética , Neurotransmissores/genética , Neurotransmissores/metabolismo , Fenótipo , Proteínas Serina-Treonina Quinases/isolamento & purificação , Receptores de Superfície Celular/isolamento & purificação , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais/genéticaRESUMO
Traditional loss-of-function studies in Drosophila suffer from a number of shortcomings, including off-target effects in the case of RNA interference (RNAi) or the stochastic nature of mosaic clonal analysis. Here, we describe minimal in vivo GFP interference (miGFPi) as a versatile strategy to characterize gene function and to conduct highly stringent, cell type-specific loss-of-function experiments in Drosophila miGFPi combines CRISPR/Cas9-mediated tagging of genes at their endogenous locus with an immunotag and an exogenous 21 nucleotide RNAi effector sequence with the use of a single reagent, highly validated RNAi line targeting this sequence. We demonstrate the utility and time effectiveness of this method by characterizing the function of the Polymerase I (Pol I)-associated transcription factor Tif-1a, and the previously uncharacterized gene MESR4, in the Drosophila female germline stem cell lineage. In addition, we show that miGFPi serves as a powerful technique to functionally characterize individual isoforms of a gene. We exemplify this aspect of miGFPi by studying isoform-specific loss-of-function phenotypes of the longitudinals lacking (lola) gene in neural stem cells. Altogether, the miGFPi strategy constitutes a generalized loss-of-function approach that is amenable to the study of the function of all genes in the genome in a stringent and highly time effective manner.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Drosophila/genética , Drosophila/genética , Interferência de RNA , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Animais , Animais Geneticamente Modificados , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Feminino , Células Germinativas , Proteínas de Fluorescência Verde/genética , Mutação , Isoformas de Proteínas/genética , RNA Guia de Cinetoplastídeos , Proteínas Repressoras/metabolismo , Células-TroncoRESUMO
The CRISPR-associated RNA-guided nuclease Cas9 has emerged as a powerful tool for genome engineering in a variety of organisms. To achieve efficient gene targeting rates in Drosophila, current approaches require either injection of in vitro transcribed RNAs or injection into transgenic Cas9-expressing embryos. We report a simple and versatile alternative method for CRISPR-mediated genome editing in Drosophila using bicistronic Cas9/sgRNA expression vectors. Gene targeting with this single-plasmid injection approach is as efficient as in transgenic nanos-Cas9 embryos and allows the isolation of targeted knock-out and knock-in alleles by molecular screening within 2 months. Our strategy is independent of genetic background and does not require prior establishment of transgenic flies.
Assuntos
Sistemas CRISPR-Cas , Drosophila/genética , Marcação de Genes/métodos , Genoma de Inseto , Edição de RNA , Animais , Plasmídeos/genéticaRESUMO
Pain is a significant medical concern and represents a major unmet clinical need. The ability to perceive and react to tissue-damaging stimuli is essential in order to maintain bodily integrity in the face of environmental danger. To prevent damage the systems that detect noxious stimuli are therefore under strict evolutionary pressure. We developed a high-throughput behavioral method to identify genes contributing to thermal nociception in the fruit fly and have reported a large-scale screen that identified the Ca²âº channel straightjacket (stj) as a conserved regulator of thermal nociception. Here we present the minimal anatomical and neuronal requirements for Drosophila to avoid noxious heat in our novel behavioral paradigm. Bioinformatics analysis of our whole genome data set revealed 23 genes implicated in Ca²âº signaling that are required for noxious heat avoidance. One of these genes, the conserved thermoreceptor TrpA1, was confirmed as a bona fide "pain" gene in both adult and larval fly nociception paradigms. The nociceptive function of TrpA1 required expression within the Drosophila nervous system, specifically within nociceptive multi-dendritic (MD) sensory neurons. Therefore, our analysis identifies the channel TRPA1 as a conserved regulator of nociception.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Nociceptividade/fisiologia , Canais de Cátion TRPC/metabolismo , Temperatura , Animais , Antenas de Artrópodes/metabolismo , Aprendizagem da Esquiva , Sinalização do Cálcio/genética , Dendritos/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/genética , Ensaios de Triagem em Larga Escala , Canais Iônicos/metabolismo , Larva/metabolismo , Canal de Cátion TRPA1RESUMO
BACKGROUND: In multicellular animals, cell size is controlled by a limited set of conserved intracellular signaling pathways, which when deregulated contribute to tumorigenesis by enabling cells to grow outside their usual niche. To delineate the pathways controlling this process, we screened a genome-scale, image-based Drosophila RNA interference dataset for double-stranded RNAs that reduce the average size of adherent S2R+ cells. RESULTS: Automated analysis of images from this RNA interference screen identified the receptor tyrosine kinase Pvr, Ras pathway components and several novel genes as regulators of cell size. Significantly, Pvr/Ras signaling also affected the size of other Drosophila cell lines and of larval hemocytes. A detailed genetic analysis of this growth signaling pathway revealed a role for redundant secreted ligands, Pvf2 and Pvf3, in the establishment of an autocrine growth signaling loop. Downstream of Ras1, growth signaling was found to depend on parallel mitogen-activated protein kinase (MAPK) and phospho-inositide-3-kinase (PI3K) signaling modules, as well as the Tor pathway. CONCLUSIONS: This automated genome-wide screen identifies autocrine Pvf/Pvr signaling, upstream of Ras, MAPK and PI3K, as rate-limiting for the growth of immortalized fly cells in culture. Since, Pvf2/3 and Pvr show mutually exclusive in vivo patterns of gene expression, these data suggest that co-expression of this receptor-ligand pair plays a key role in driving cell autonomous growth during the establishment of Drosophila cell lines, as has been suggested to occur during tumor development.