Roles Of Bmal1a In Zebrafish Circadian Regulation

As a positive factor in the regulation of the circadian clock,Bmall forms a heterodimer with Clock to initiate transcription by binding to the E-box of the promoter regions of per and cry.There are two bmall genes in zebrafish(Danio rerio),namely bmalla and bmallb,which are co-orthologs of the fruitfly(Drosophila melanogaster)cycle and mouse(Mus musculus)Bmall gene.Previous studies showed that loss of Bmall/cycle led to arrhythmic behaviors in mice or flies,and that the role of Bmallb in zebrafish is differed from BMAL1/CYCLE in mice and flies.However,the role of zebrafish Bmal1 a in circadian regulation is still unclear.Here,qRT-PCR results showed the mRNA level of bmall a exhibited significant oscillations in zebrafish.Sequence analysis revealed that the zebrafish bmalla promoter region harbors two RORE elements.Dual-luciferase reporter assays and ChIP assays showed that the circadian clock directly regulates the expression of bmall a gene through these two RORE elements.In order to investigate the function of Bmal1a in zebrafish circadian clocks,we examined locomotor activities and expression of key circadian genes in bmalla-/-and bmall a-/-;bmallb-/-mutants.Behavioral assays showed that bmalla-/-and bmalla-/-;bmallb-/-larvae displayed a prolonged period and a delayed phase under DD(constant darkness)and LL(constant light)conditions,compared with WT;under LD(Light-and-Dark)condition,the locomotor activities of bmalla-/-;bmallb-/-larvae are significantly decreased.qRT-PCR results showed that core circadian gene bmall a is up-regulated,bmallb,perla,per 3 and cry1bb are down-regulated in bmalla-/-under DD condition,while under LD condition,core circadian genes bmalla,bmallb,bmal2,perla,per3 and cry1bb are down-regulated;In bmalla-/-;bmal1b-/-,core circadian genes bmalla and bmal2 are up-regulated,bmallb,per la,per3 and crylbb are down-regulated under DD condition;bmall a and perla are up-regulated,bmallb,bmal2,per3 and crylbb are down-regulated under LD condition.To further investigate the role of Bmall a in the circadian clock,we generated heat shock-inducible bmalla overexpressing transgenic fish lines Tg(hsp70l:bmalla;CG2).The behavioral assays showed that the locomotor activities of zebrafish are increased significantly and the behavioral rhythmicity is altered following the heat shock treatments.qRT-PCR results showed that the core circadian genes bmalla,bmal2,perla,per3,cry1bb are significantly altered in the bmalla overexpressing transgenic zebrafish.In order to further study its tissue-specific role,we generated pineal gland-specific knockout bmalla transgenic zebrafish Tg(U6x:sgRNA(bmalla):LC);Tg(exorh:Cas9:CG2)and pineal gland-specific overexpression bmalla transgenic zebrafish Tg(exorh:bmalla;CG2).Further examination of these two transgenic zebrafish lines should help gain an understanding of Bmal1a's function in the pineal gland.In summary,our findings indicate that Bmal1a plays an important role in the zebrafish circadian clock.