The Effect Of HSP90 Inhibition On The Embryonic Development Of Zebrafish

HSP90(Heat shock protein 90,HSP90)is one of the most abundant chaperone of heat shock protein family,and itschaperone function has been well accepted.HSP90 consists of two isoforms,HSP90? and HSP90?.HSP90? is inducible,which expresses specially in certain tissues when faced with environment pressure.The two isoforms of HSP90,combine with different co-chaperones to maintain the stability of target proteins,playing an important role in function regulation and development of multi-celled organisms.Structurally,HSP90 is a homodimer and each protomer contains three flexibly linked regions,an N?terminal ATP?binding domain(N?domain),a middle domain(M?domain),and a C?terminal dimerization domain(C?domain).Co?chaperone regulation is a conserved feature of the eukaryotic HSP90 system.To date,more than 20 co-chaperones have been identified.They regulate the function of HSP90 in different ways such as inhibition and activation of the ATPase of Hsp90 as well as recruitment of specific client.Different co-chaperones work together to facilitate the maturation of HSP90 clients.The composition of co?chaperone complexes seems to depend to some degree on the presence of a specific client protein.HSP90 can regulate SHRs,protein kinases,and various biological processes,such as RNA processing,the progression of diseases,and the regulation of cell cycle.However,the effect of HSP90 on regulating genetic or epigenetic development of creatures remain to be studied.A hypothesis has been reported that HSP90,as a capacitor for the conditional release of stores of hidden morphogenic variation,can supply adaptive genotypes for environment survival.But it is not very clear that the buffering machine of HSP90 as a capacitor for storing morphogenic variation.Zebrafish,one of the tropical fishes,studied broadly as a model of tropical fish,has a broad scope of adaptive tempreture and can endure lower temperature to 9°C within certain time range;so studying the cold tolerance of fishes has a big biological significance.Therefore,we treated zebrafish embryos with Radicicol,releasing the buffering function of HSP90 under environment pressure and revealing the diversity of phenotype of creature,which offered a new model for the study of function of HSP90 in biological evolution and a new strategy for breeding of economic fishes adaptive to low temperatures.We examined the mRNA levels of two genes responsive to HSP90 function repression,BAG3(BCL2-associated athanogene 3)and HSPB1(heat shock protein beta-1),at 12hpf(hours post-fertilization,hpf)and 24 hpf,and observed the developmental status of zebrafish embryos at diferent developmental stages and calculated the survival rate by treating zebrafish embryos with 2,5,10?mol/L Radicicol.Tthese zebrafish were exposed to cold stress after sexual maturity,and the histone acetylation level and histone methylation level of embryos and adultswere assayed by Western blot.Results showed that: RT-qPCR showed that Radicicol treatment caused a strong increase in the mRNA levels of BAG3 and HSPB1 in zebrafish embryos at 12,24,36,48 hpf.Western blot showed the protein levels of H3K9 Ac,H3K27Ac,HDAC1,H3K4me1,H3K4me3 were upregulated in zebrafish embryos treated with 5?mol/L Radicicol at24 hpf.When both Radicicol treatment groups and control group were exposed to in cold stress(8°C 24h),we found that in the Radicicol treatment group some fishes showed enhanced cold tolerance,which could be inherited.And we also found that the F1 appeared abnormal skin patterns,which changed from dark blue stripes to spots,compared to wild type zebrafish,suggesting that HSP90 played a key role in zebrafish pigmentation regulation.Moreover,Western blot showed that the histone acetylation level of zebrafish with spot pattern was upregulated,indicatinf a close relationship between HSP90 function andepigenetic modification.It has been reported that low conductivity can comprimise the function of HSP90.So we treated zebrafish embryos in low conductivity water,the effect of which was similar to the Radicicol treatment in cold adaption.Zebrafish in low conductivity showed yellow skin color,while the wild type shows stripes and Radicicol treated zebrafish showed spots.The results showed that HSP90 regulated multiple feartures in zebrafish including temperature adaptationand skin color adaptation.In summary,inhibition of HSP90 function led to abnormal phenotypes in different aspects.The mRNA levels of histone epigenetic enzymes,DNA methylation enzymes and DNA demethylation enzymes determined changed.The protein levels of histone methylation and acetylation also changed,which all indicated that HSP90 had a close relationship with epigenetic modification.