A pupal transcriptomic screen identifies Ral as a target of store-operated calcium entry in Drosophila neurons

Shlesha Richhariya,Gaiti Hasan,Katharine Abruzzi,Siddharth Jayakumar,Michael Rosbash

doi:10.1038/srep42586

Abstract

Transcriptional regulation by Store-operated Calcium Entry (SOCE) is well studied in non-excitable cells. However, the role of SOCE has been poorly documented in neuronal cells with more complicated calcium dynamics. Previous reports demonstrated a requirement for SOCE in neurons that regulate Drosophila flight bouts. We refine this requirement temporally to the early pupal stage and use RNA-sequencing to identify SOCE mediated gene expression changes in the developing Drosophila pupal nervous system. Down regulation of dStim, the endoplasmic reticular calcium sensor and a principal component of SOCE in the nervous system, altered the expression of 131 genes including Ral, a small GTPase. Disruption of Ral function in neurons impaired flight, whereas ectopic expression of Ral in SOCE-compromised neurons restored flight. Through live imaging of calcium transients from cultured pupal neurons, we confirmed that Ral does not participate in SOCE, but acts downstream of it. These results identify neuronal SOCE as a mechanism that regulates expression of specific genes during development of the pupal nervous system and emphasizes the relevance of SOCE-regulated gene expression to flight circuit maturation.

Highlights

Transcriptional regulation by Store-operated Calcium Entry (SOCE) is well studied in non-excitable cells
Knockdown of dStim was chosen over dOrai, primarily because the dStim RNAi strain is both specific and more effective compared to the dOrai RNAi strains[11,19]
Control flies without the GAL4 but only the UAS-dStimIR transgene when subjected to the elevated temperature throughout, exhibited near normal flight (Fig. 1b), confirming that the flight phenotype is due to neuronal knockdown of dStim

Summary

Drosophila neurons

Shlesha Richhariya[1], Siddharth Jayakumar[1,2], Katharine Abruzzi[3], Michael Rosbash3 & Gaiti Hasan[1]. Similar manipulations solely in the larval stage or for 4 days in the adult stage did not reduce the duration of flight bouts (Fig. 4d) These data confirm a requirement for Ral in pupal development of the Drosophila flight circuit. We further tested if a similar effect on Ral levels was obtained upon knockdown of the inositol trisphosphate receptor (IP3R), the calcium channel on the ER membrane encoded by itpr that regulates SOCE in Drosophila neurons[11]. Larval levels of Ral were reduced in heterozygous dOrai[3] mutant animals (Fig. 5b), suggesting that the regulation of Ral expression by SOCE was not restricted to neurons or to the pupal stage. Expression of RalWT failed to rescue SOCE in dStim knockdown neurons, further supporting Ral function downstream of SOCE in pupal neurons

Discussion

Methods

Additional Information