Manifestation of appropriate behavior in adult animals requires developmental mechanisms that help in the formation of correctly wired neural circuits. Flight circuit development in Drosophila requires store-operated calcium entry (SOCE) through the STIM/Orai pathway. SOCE-associated flight deficits in adult Drosophila derive extensively from regulation of gene expression in pupal neurons, and one such SOCE-regulated gene encodes the small GTPase Ral. The cellular mechanism by which Ral helps in maturation of the flight circuit was not understood. Here, we show that knockdown of components of a Ral effector, the exocyst complex, in pupal neurons also leads to reduced flight bout durations, and this phenotype derives primarily from dopaminergic neurons. Importantly, synaptic release from pupal dopaminergic neurons is abrogated upon knockdown of dSTIM, Ral, or exocyst components. Ral overexpression restores the diminished synaptic release of dStim knockdown neurons as well as flight deficits associated with dSTIM knockdown in dopaminergic neurons. These results identify Ral-mediated vesicular release as an effector mechanism of neuronal SOCE in pupal dopaminergic neurons with functional consequences on flight behavior.

No abstract

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.

Anaplastic lymphoma kinase (Alk) is a receptor tyrosine kinase of the insulin receptor super-family that functions as oncogenic driver in a range of human cancers such as neuroblastoma. In order to investigate mechanisms underlying Alk oncogenic signaling, we conducted a genetic suppressor screen in Drosophila melanogaster. Our screen identified multiple loci important for Alk signaling, including members of Ras/Raf/ERK-, Pi3K-, and STAT-pathways as well as tailless (tll) and foxo whose orthologues NR2E1/TLX and FOXO3 are transcription factors implicated in human neuroblastoma. Many of the identified suppressors were also able to modulate signaling output from activated oncogenic variants of human ALK, suggesting that our screen identified targets likely relevant in a wide range of contexts. Interestingly, two misexpression alleles of wallenda (wnd, encoding a leucine zipper bearing kinase similar to human DLK and LZK) were among the strongest suppressors. We show that Alk expression leads to a growth advantage and induces cell death in surrounding cells. Our results suggest that Alk activity conveys a competitive advantage to cells, which can be reversed by over-expression of the JNK kinase kinase Wnd.

Numerous roles for the Alk receptor tyrosine kinase have been described in Drosophila, including functions in the 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 analysed 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/scRNA-seq and protein 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 Spar mutant flies exhibit defects in sleep and circadian rhythm control. In summary, we report a novel activity regulating neuropeptide precursor gene that is regulated by Alk signaling in the Drosophila CNS.