RESUMEN
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.
Asunto(s)
Quinasa de Linfoma Anaplásico , Proteínas de Unión al ADN , Proteínas de Drosophila , Desarrollo Embrionario , Proteínas del Tejido Nervioso , Proteínas Proto-Oncogénicas , Factores de Transcripción , Animales , Quinasa de Linfoma Anaplásico/genética , Diferenciación Celular/genética , Proteínas de Unión al ADN/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Proteínas de Drosophila/genética , Desarrollo Embrionario/genética , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Mesodermo/crecimiento & desarrollo , Mesodermo/metabolismo , Desarrollo de Músculos/genética , Músculos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , RNA-Seq , Transducción de Señal/genética , Análisis de la Célula Individual , Factores de Transcripción/genéticaRESUMEN
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.
