RESUMO
Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the central nervous system (CNS), however the molecular details are poorly understood. To gain mechanistic insight, we employed Targeted DamID (TaDa) transcriptional profiling to identify targets of Alk signaling in the larval CNS. TaDa was employed in larval CNS tissues, while genetically manipulating Alk signaling output. The resulting TaDa data were analyzed together with larval CNS scRNA-seq datasets performed under similar conditions, identifying a role for Alk in the transcriptional regulation of neuroendocrine gene expression. Further integration with bulk and scRNA-seq datasets from larval brains in which Alk signaling was manipulated identified a previously uncharacterized Drosophila neuropeptide precursor encoded by CG4577 as an Alk signaling transcriptional target. CG4577, which we named Sparkly (Spar), is expressed in a subset of Alk-positive neuroendocrine cells in the developing larval CNS, including circadian clock neurons. In agreement with our TaDa analysis, overexpression of the Drosophila Alk ligand Jeb resulted in increased levels of Spar protein in the larval CNS. We show that Spar protein is expressed in circadian (clock) neurons, and flies lacking Spar exhibit defects in sleep and circadian activity control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.
Assuntos
Quinase do Linfoma Anaplásico , Sistema Nervoso Central , Proteínas de Drosophila , Animais , Sistema Nervoso Central/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Quinase do Linfoma Anaplásico/metabolismo , Quinase do Linfoma Anaplásico/genética , Larva/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Neuropeptídeos/metabolismo , Neuropeptídeos/genética , Transdução de Sinais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/genética , Drosophila/genética , Drosophila/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão GênicaRESUMO
Development of the Drosophila visceral muscle depends on Anaplastic Lymphoma Kinase (Alk) receptor tyrosine kinase (RTK) signaling, which specifies founder cells (FCs) in the circular visceral mesoderm (VM). Although Alk activation by its ligand Jelly Belly (Jeb) is well characterized, few target molecules have been identified. Here, we used targeted DamID (TaDa) to identify Alk targets in embryos overexpressing Jeb versus embryos with abrogated Alk activity, revealing differentially expressed genes, including the Snail/Scratch family transcription factor Kahuli (Kah). We confirmed Kah mRNA and protein expression in the VM, and identified midgut constriction defects in Kah mutants similar to those of pointed (pnt). ChIP and RNA-Seq data analysis defined a Kah target-binding site similar to that of Snail, and identified a set of common target genes putatively regulated by Kah and Pnt during midgut constriction. Taken together, we report a rich dataset of Alk-responsive loci in the embryonic VM and functionally characterize the role of Kah in the regulation of embryonic midgut morphogenesis.
Assuntos
Quinase do Linfoma Anaplásico , Proteínas de Ligação a DNA , Proteínas de Drosophila , Desenvolvimento Embrionário , Proteínas do Tecido Nervoso , Proteínas Proto-Oncogênicas , Fatores de Transcrição , Animais , Quinase do Linfoma Anaplásico/genética , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Desenvolvimento Embrionário/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/genética , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Desenvolvimento Muscular/genética , Músculos/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas Proto-Oncogênicas/genética , RNA Mensageiro/genética , RNA-Seq , Transdução de Sinais/genética , Análise de Célula Única , Fatores de Transcrição/genéticaRESUMO
Anaplastic lymphoma kinase (Alk) is an evolutionary conserved receptor tyrosine kinase belonging to the insulin receptor superfamily. In addition to its well-studied role in cancer, numerous studies have revealed that Alk signaling is associated with a variety of complex traits such as: regulation of growth and metabolism, hibernation, regulation of neurotransmitters, synaptic coupling, axon targeting, decision making, memory formation and learning, alcohol use disorder, as well as steroid hormone metabolism. In this study, we used BioID-based in vivo proximity labeling to identify molecules that interact with Alk in the Drosophila central nervous system (CNS). To do this, we used CRISPR/Cas9 induced homology-directed repair (HDR) to modify the endogenous Alk locus to produce first and next generation Alk::BioID chimeras. This approach allowed identification of Alk proximitomes under physiological conditions and without overexpression. Our results show that the next generation of BioID proteins (TurboID and miniTurbo) outperform the first generation BirA* fusion in terms of labeling speed and efficiency. LC-MS3-based BioID screening of AlkTurboID and AlkminiTurbo larval brains revealed an extensive neuronal Alk proximitome identifying numerous potential components of Alk signaling complexes. Validation of Alk proximitome candidates further revealed co-expression of Stardust (Sdt), Discs large 1 (Dlg1), Syntaxin (Syx) and Rugose (Rg) with Alk in the CNS and identified the protein-tyrosine-phosphatase Corkscrew (Csw) as a modulator of Alk signaling.
