Roles Of Clock2 In The Zebrafish Circadian Clock

In order to adapt to periodic changes of environment factors such as light,temperature and humidity caused by the earth's rotation,almost all living things have a nearly 24-hour circadian rhythms.Circadian clocks regulate the activities of the organisms at the molecular,biochemical,cellular,physiological and behavioral levels.The molecular mechanisms of the circadian clock depend on a transcription/translation feedback loop.In mammals,this loop consists of positive factors and negative factors: CLOCK and BMAL form heterodimer,binding to in E/E'-box in the Per and Cry promoter regions to activate their transcription.Subsequently,PER and CRY proteins form another heterodimer in the cytoplasm and enter into nucleus,and inhibit their own transcription by interacting with the CLOCK: BMAL heterodimer.There are three clock genes in the zebrafish,clock1 a,clock1b and clock2.clock2 also named Npas2.Mammalian Npas2 is necessary to support the body's normal circadian rhythms,and play an important role in many aspects such as regulation of the circadian clock,cell growth,differentiation,apoptosis,and inhibit tumor growth.However,the function and regulatory mechanisms of clock2 in zebrafish are not yet determinded to date.In order to study the regulatory mechanisms of zebrafish clock2,we isolated the clock2 promoter.Luciferase report assays showed the Clock1a:Bmal1b heterodimer can't activate clock2 expression;but Tefa activates its expression and Rev-erb? inhibist clock2 expression.Ch IP assays showed that Tefa binds to the D-box region in the clock2 promoter.To explore the role of Clock2 in zebrafish circadian clock,we also used a zebrafish clock2 mutant generated previously with CRISPR-Cas9 Behavioral assays show that clock2 mutant fish display reduced locomotor activities under light/dark(LD)condition after 5 hpf and altered circadian rhythms under constant dark(DD)condition,with 0.6-hour shortened period,3.5-hour delayed phase,invariably of their locomotor activities under constant dark(DD)condition;1.4-hour lengthened period,0.5-hour delayed phase,invariably of their locomotor activities under constant light(LL)condition.Quantitative Real-Time PCR showed altered expression of key circadian clock2 genes per1 a,per1b,per2,per3,cry1 aa,cry1ab,cry1 ba and cry1 bb in homozygous clock2 mutant zebrafish larvae under dark/dark(DD)condition and light/light(LL)condition.Both clock1 a and clock1 b are up-regulated in the homozygous clock2 mutant fish under light/dark(LD)condition but down-regulated in homozygous clock2 mutant fish larvae under constant dark(DD)and constant light(LL)conditions.Our studies demonstrate that Clock2 has distinct roles in the zebrafish circadian clock.In addition,clock2 homozygotes showed growth retardation and lower male to female ratio compared to wild types.We also conducted RNA-seq-based transcriptome analysis of clock2 mutant embryos at ZT0(120 hpf)and found 277 genes were up-regulated,144 genes were down-regulated.Among them,melanogenesis-related genes tyr ? prkacbb-2 ? ednaba and cell cycle/progesterone-mediated oocyte maturation-related genes gadd45 a ? anapc5 ?cadm1b are all altered,which is reconfirmed by qRT-PCR analyses.These results indicate that Clock2 plays an important role in other biological processes such as melanogenesis,growth,development and cell cycle.Our research indicates that zebrafish clock2 is a direct target of the circadian clock and also play important roles in circadian regulation.We also found that core circadian genes were altered in the clock2 mutant.These results showed that Clock2 has a vital function in the circadian clocks,development and behavior.