Roles Of Clock1a In The Zebrafish Circadian Clock

Approximately 24-hour rhythms of activities in all animals from bacteria to human are controlled by the circadian clock. The circadian clock functions at cell and tissue. Circadian misalignment can cause a variety of diseases, such as sleep disorders, metabolic disorders, premature aging and different mental disorders.The mammalian circadian clock controlled by a series of key circadian genes, including Clock and Bmal1 as well as Cryptochromes(Crys) and Period(Pers). CLOCK:BMALl heterodimer bind to E box elements in the promoters of Per and Cry genes and thereby activate their transcriptions. After translation, dimerization and translocation to the nucleus, the PER and CRY proteins physically interact with CLOCK:BMALl complex and thereby inhibit their own transcriptional.In zebrafish, there are three clock genes named clock1 a, clock1 b and clock2. They are rhythmically expressed in different tissues/organs. Zebrafish have evolved different mechanisms controlling the expression of clock genes, and the functions of these three clock genes have diverged. However, how these three clock genes function in zebrafish is still unclear.In this study, we generated a zebrafish clock1 a mutant through CRISPR-Cas9. This clock1 a mutant zebrafish harbors a CRISPR-Cas9 induced 7-bp deletion, which is a frame shift resulting in a truncated Clock1 a with only 101 amino acids. Luciferase reporter assays show that the mutate form of clock1 a lose its activating effect on e4bp4-2b gene. Quantitative Real-Time PCR showed altered expression of key circadian clock genes per1 a, per1 b, per2, per3, cry1 aa, cry1 ab, cry1 ba and cry1 bb in homozygous clock1 a mutant zebrafish larvae under both light/dark(LD) and constant dark(DD) conditions. Behavioral assays show that clock1 a mutant fish display hyperactivity under LD condition and completely lose circadian rhythms under DD condition. Altered expression of key circadian clock genes and disrupted locomotor rhythms in clock1 a mutants suggest that Clock1 a is essential for zebrafish circadian regulation and maintaining zebrafish locomotor rhythms. Hyperactivity phenotype was also observed in clock1 a mutant adult fish, and the mirror-attack test shows that clock1 a mutant adult fish are more aggressive/hyperactive, suggesting zebrafish clock1 a mutant also display mania-like syndrome as well as mice Clock mutant. Lithium is a mood stabilizer that is particularly effective in treating mania. We found Li+ treatment can effectively rescue mania-like behavior.In addition, clock1 a mutants female show increased reproductive capacity. Compared with WT female, clock1 a mutants female produce more embryos. And the expression of lh,lhr,fsh,fshr,vtg are all up-regulated. But clock1 a knockout have no effect on male fertility.We also employed deep sequencing to conduct transcriptome analysis of clock1 a at ZT2(11:00, 98hpf) and ZT14(23:00, 110hpf), and revealed that 244 up-regulated genes and 406 down-regulated genes at ZT2, 184 up-regulated genes and 249 down-regulated genes at ZT14, and 159 differentially expressed genes at both ZT2 and ZT14. Apip is an APAF1 interacting protein, the expression of apip is reduced by 7-fold, which means maybe apip is a target of Clock1 a, and Clock1 a regulates apoptosis.We successfully generated a clock1 a knockout zebrafish. It explored the functions of clock1 a in core circadian clock gene expression, the behavior rhythm and reproduction. It improves the existing feedback loop, also provides a basis for further research.