| The terrestrial ancestors of cetaceans took over the world's waters through rapid radiation adaptation about 53-56 million years ago from the land ocean and became fully aquatic.In order to adapt to aquatic habitats,whales have evolved a unique onehemispheric slow-wave sleep pattern.This is when one hemisphere enters slow-wave sleep(SWS),while the other hemisphere remains awake,with the near-total disappearance of rapid-eye-movement(REM)sleep.However,the molecular mechanisms underlying the unique USWS pattern in cetaceans remain unclear.As an important negative regulator in the circadian clock regulation loop,PER3 gene plays an important role in the sleep regulation of mammals.It has been reported that PER3 gene knockout can lead to abnormal circadian rhythm sleep homeostasis in mice.In view of this,PER3 gene was selected as a candidate gene in this study.First,compared with other mammals,we identified whether there is a specific amino acid mutation in PER3 gene of whales.If there is a specific mutation,the following scientific questions will be explored through in vivo and in vitro experiments: 1)In vitro experiments were conducted to determine whether PER3 gene and PER3-Mut carrying its specific amino acid mutation in whales still have the ability to inhibit CLOCK and BMAL1 genes and whether they affect the stability of PER3 protein? 2)In vivo experiment,since zebrafish has a biological rhythm similar to that of mammals,zebrafish was used as the model animal in this study.(1)Transient overexpression of PER3 and PER3-Mut gene was conducted in vivo.Through transcriptome sequencing and comparative analysis,whether the expression of genes or pathways regulating sleep has been changed?(2)Stable overexpression of PER3 gene and PER3-Mut in zebrafish can cause zebrafish to produce similar whale USWS sleep reduction behavior through behavior monitoring?Based on the above studies,we hope to preliminarily reveal whether the core circadian clock gene PER3 has an adaptive function change and clarify the relationship between its functional change and the unique sleep pattern of USWS in whales.Compared with other land mammals,four specific amino acid mutations(E53K,E54 D,P334L,S636R)were identified in PER3 gene of cetaceans.In order to further explore whether PER3 gene mutations in whales affect their function,this study conducted site-directed mutagenesis experiments in vivo and in vitro with zebrafish as the model animal.First of all,the luciferase reporting system was used to detect that under the action of the zebrafish per1 b promoter,PER3 in cetacean could still inhibit the expression of clock1 a and bmal1 b,and compared with the zebrafish per3,PER3-Mut,which carries a cetacean-specific mutation,has a slightly increased inhibition of upstream clock1 a and bmal1 b genes.CLOCK and BMAL1 are strong positive regulators in the circadian clock regulation loop.Mutation in Clock leads to increased arousal and decreased sleep phenotypes in mice,while Bmal1 knockout mice show circadian rhythm disorder and sleep fragmentation phenotypes.Therefore,cetacean-specific amino acid mutations may induce reduced sleep in cetaceans by inhibiting upstream genes CLOCK and BMAL1,which is consistent with the phenotype of increased wakefulness and sleep fragmentation caused by USWS sleep.Secondly,to further explore whether PER3 gene and its specific amino acid mutations affect protein stability,the protein synthesis inhibitor actinomycetes(CHX)was used to treat cells to detect the protein expression levels at different time points.The results showed that relative protein levels of whale PER3(Flag-PER3)and zebrafish Per3(Flag-PER3-Mut)with cetacean-specific mutations were significantly lower than those of wild-type zebrafish Per3(Flag-Per3),and their half-life was shortened,suggesting that the protein stability might be affected.Finally,the PER3 gene of dolphin(PER3),the PER3 gene of zebrafish carrying a cetacean-specific mutation(PER3-Mut)and the per3 gene of zebrafish themselves(per3)were overexpressed in zebrafish,and the behavior of the transgenic embryos was observed for five consecutive days using the zebrafish embryo behavior monitor.The results showed that compared with transgenic zebrafish overexpressing per3 group,the activity of zebrafish overexpressing PER3 and PER3-MUT group was significantly reduced,suggesting that the rhythm may have been changed.In addition,transcriptome sequencing was performed on transgenic zebrafish,and the main results were as follows:(1)Compared with the transient overexpression of per3 gene,a total of 294 differentially expressed genes(DEGs)were identified in the PER3 overexpression group.Gene enrichment analysis showed that differentially expressed genes were mainly enriched several neurotransmitter receptors closely related to sleep regulation,including glutamate receptor,acetylcholine receptor and ?-aminobutyric acid receptor.It has been proved that glutamate is an excitatory neurotransmitter.Compared with per3 of zebrafish itself,glutamate receptor grin1 b,gria2b and gria3 a were significantly upregulated in the PER3 overexpression group,which will lead to a large amount of calcium ions entering the neurons and a large amount of excitatory neurotransmitter glutamate release,leading to increased arousal of the body.This suggests that PER3 gene contributes to the maintenance of arousal in whales,which is consistent with the unique USWS sleep pattern in whales where one hemisphere is always awake.Similarly,overexpression of the PER3 gene in whale results in a significant down-regulation of the arousal inducing acetylcholine receptor chrne gene,which has been shown to cause sleep disorders and circadian rhythm disturbances.Therefore,down-regulation of the PER3 gene may help whales maintain arousal.In addition,overexpression of whale PER3 gene leads to significant upregulation of ?-aminobutyric acid(GABA)receptors gabbr1 b and gabrb4.Previous studies have confirmed that sufficient amount of ?-aminobutyric acid makes the body easier to enter deep sleep,so the significant upregulation of gabbr1 b and gabrb4 promotes increased synthesis of GABA and contributes to the short sleep of fragmented sleep in whales.In addition,sufficient amount of GABA will also cause REM reduction,which is also consistent with the REM sleep loss phenotype in the sleep pattern of USWS in cetaceans.(2)Compared with the transient overexpression of per3 gene in zebrafish,179 DEGs were identified in the Per3-Mut-overexpression group,which were mainly concentrated in biological processes such as visual system development(GO: 0150063)and drug metabolism(GO:0017144).A total of 8 genes were found during the development of the visual system,including significantly down-regulated genes crygm2d11,crygm2d14 and crygm2d15.These genes are involved in the regulation of lens transparency and indirectly affect the photoreceptor afferent pathway that regulates circadian rhythm.Thus,a specific PER3 mutation in cetaceans may regulate sleep by affecting lens transparency and photoreceptor afferent pathways.In addition,compared with the control group,overexpression of the whale PER3 and Per3-Mut-overexpression group resulted in significantly reduced expression of the clock1 b,clock2,bmal1 a and cry3 genes in zebrafish.In conclusion,PER3 gene and its specific amino acid mutations in whales enhance the transcriptional inhibitory activity of upstream clock genes clock1 a and bmal1 b at the cellular level,while the stability of their expressed proteins is significantly decreased.At the living level of zebrafish,compared with transgenic zebrafish with overexpression of per3,the motor activity of zebrafish with overexpression of PER3 and PER3-Mut was significantly reduced,and the expression of neurotransmitter genes closely related to sleep regulation and core circadian clock genes were significantly altered.The results of this study provide preliminary molecular evidence for USWS sleep in cetaceans and lay a foundation for further studies on the regulatory mechanism of USWS sleep in cetaceans. |
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