Vipr2/Vpac2 Contributes To Circadian Clock Regulation In Zebrafish

VIPR2.also called VPAC2.is a G protein-coupled receptor for VIP(neuropeptide vasoactive intestinal peptide)and PACAP(pituitary adenylate cyclase-activating polypeptide).Zebrafish have three orthologs of mammalian Vpac genes,vpac1a,vpac1b and vpac2.Mammalian VIP and PACAP play critical roles in the suprachiasmatic nucleus(SCN),and VPAC2-activated signal transduction pathways contribute to the regulation of circadian synchronization in the SCN.However,the mechanisms underlying how vipr2/vpac2 gene contributes to zebrafish circadian regulation is still largely unknown.Here we generated two zebrafish vipr2 mutant lines with CRISPR-Cas9.Loss of vipr2 results in 1.1 h phase delay and 1 h shortened period under DD condition;then causes 1.4 h phase delay and 0.8 h lengthed period under LL condition.Heat-shock incubated over-expressed the vipr2 lengthed the locomotor period under DD condition,which is opposited to the mutant.Key circadian clock genes,per1a.per1b,cry1aa and cry1bb,themRNA level was downregulated in the vipr2 zebrafish mutants,the peak of per3.cry1baare advanced.Especialy.the circadain rhythmicity of per3-Luc was disrupted in vipr2 mutant,accompany with phase advanced and period lengthed.In situ hybridization result shows that vipr2 is expressed highly in the brain,suggesting that it has a role in neuronal communication.We will generate knock-in fish for examining expression of vipr2 in specific neurons in the brain.Generates the Tg(vipr2:ntr,CG2)transgenic fish to study the circadian function of vipr2 neurons.Our results reveal the important roles of the vipr2/vpac2 gene in zebrafish circadian clock.