Bioinformatical Analysis Of ATF/CREB Family And The Role Of Creb1 On Gametogenesis In Nile Tilapia

The ATF/CREB(Activating transcription factor/cAMP response element binding protein)family of transcription factors represent a large group of basic region-leucine zipper(bZIP)proteins that regulate diverse cellular responses,including cell survival,proliferation,differentiation and apoptosis.Gene duplication is thought to increase genome complexity,and the whole genome duplication provides a large amount of raw materials for evolutionary adaptation and innovation.Previous analysis showed that early diversification of bZIPs in multiple eukaryotic lineages was a prerequisite for the evolution of complex multicellular organisms.In recent years,the genome-wide identification of the ATF/CREB family has been,to some extent,performed in plants and mammals(E.g.human and mouse).However,there is a lack of genome-wide research on the family in most animal groups,particularly in teleosts.The origin and evolution of this family in the animal kingdom are unclear,and the relevance between its expansion of ATF/CREB family and four whole-genome duplications in vertebrates is yet to be studied.The expression pattern of the entire ATF/CREB family has not been reported.Cyclic AMP response element-binding protein 1(CREB1),a member of the ATF/CREB family,is known to be involved in oogenesis and spermatogenesis.There is only one Creb1 in tetrapods,while two copies are reserved in teleosts,creb1 a and creb1 b.Studies showed that creb1 a and creb1 b were highly expressed in the ovary and testis of Nile tilapia(Oreochromis niloticus),respectively.As there is no evidence of direct knockout of Creb1,its specific roles in gameteogenesis are still unclear.In this study,comprehensive bioinformatics-based analyses of ATF/CREB family were performed,including genome-wide search in 22 representative animal species,chromosomal location,phylogeny and synteny.Based on the transcriptome of 8 different tissues of Nile tilapia adult fish and transcriptome of female and male gonads at four critical developmental stages,the spatial and temporal expression profiles of ATF/CREB family were analyzed.Polyclonal antibody to Nile tilapia Creb1 b was successfully prepared.Mutations of creb1 a and creb1 b were performed by CRISPR/Cas9 to analyze their effects on oogenesis and spermatogenesis in the Nile tilapia.The main results are as follows:1)The origin and evolution of the ATF/CREB family.We identified ATF/CREB family members in 22 representative animals.In the primitive multicellular animals,such as sponge,jellyfish,trichoplax and nematode,1-3 ATF/CREBs were isolated.In annelid,mollusca,arthropoda,protochordate and jawless vertebrates,4-6 ATF/CREBs were isolated,while in jawed vertebrates,elephant shark,coelacanth,spotted gar and tetrapods which experienced 2R event,the number of ATF/CREBs has increased to 12-14.Significant expansion of ATF/CREBs(18-21 members)was observed from teleosts which experienced 3R event,and even 30 ATF/CREBs members were observed in 4R-derivative species,common carp.These results indicated that the family probably originated from the early diverging metazoans and significantly expanded in vertebrates with a large number of copies reserved in the teleosts due to multiple whole genome duplications.Our results also indicated that duplicates of atf6 were derived from 2R,and duplicates of creb1,crem,jdp2,creb5,atf4,atf5 and atf7 were products of 3R.Synteny analysis showed that gene clusters jdp2-batf and atf7-atf1 were highly conserved in vertebrates and actinopterygians,respectively.2)Expression patterns of ATF/CREB family.Expression analyses based on transcriptome data of eight adult tissues showed that most ATF/CREBs expressed in at least one tissues,with 12 in testis,11 in head kidney,9 in brain,9 in heart,7 in liver,5 in ovary,5 in kidney and 4 in muscle.Analyses of the gonadal transcriptomic data from four critical development stages(5,30,90 and 180 dah)revealed that 19 ATF/CREBs were expressed in Nile tilapia gonads.At 5 dah,creb1 a,creb1b,jdp2 b and atf5 a displayed sexual dimorphic expression.At 90 and 180 dah,creb1 a,jdp2b,atf4 b and atf5 b were highly expressed in the XX gonad,while creb1 b,creb5a,crema,cremb,atf1,atf4 a and atf7 a were highly expressed in the XY gonad,which indicated that ATF/CREBs might play important roles in the development of gonads.The expression patterns of 4 pair of duplications(creb1a and creb1 b,crema and cremb,atf4 a and atf4 b,jdp2a and jdp2b)derived from 3R were verified by in situ hybridization.The results were consistent with transcriptomic data.creb1 a and atf4 a were found to be expressed mainly in phase I and II oocytes of the ovary,while creb1 b and atf4 b mainly in spermatogenic cells of the testis,indicating expression divergence of duplicated genes from 3R.In addition,immunohistochemical analysis showed that Creb1 b was specifically expressed in the primary and secondary spermatocytes,which was consistent with the results of in situ hybridization.Therefore,Creb1 b could be used as a marker of spermatocytes.3)Roles of creb1 a and creb1 b on gametogenesis.creb1 a and creb1 b were mutated using CRISPR/Cas9 in Nile tilapia,and cerb1 a F0 mutant fish and creb1 b homozygous mutant fish were obtained,respectively.The target sites adjacent to PAM(protospacer adjacent motif)were designed on the third exon of creb1a(BsajI)and the second exon of cerb1b(NciI).Two restriction enzymes mentioned above were used to screen the creb1 a and creb1 b deficient F0 fish.creb1 b F1 offspring were obtained by F0 male fish mated with wild type female fish,enzyme digestion screening,and subclone sequencing.XX and XY heterozygous fish with a 5 bp deletion were selected to breed creb1 b homozygous mutants.Histological analysis of gonads revealed degenerated oocytes and increased somatic cells in creb1 a deficient XX fish,but there was no obvious phenotype for creb1 a deficient XY fish.Large empty space and fewer spermatocytes were observed in the testis of creb1 b homozygous mutant XY fish at 90 dah,but spermatogenesis was normal at 150 dah.Degenerated oocytes,increased somatic cells and down-regulated cyp19a1 a expression were observed in creb1 b homozygous mutant XX fish.The results showed that creb1 a was involved in the development of oocytes,while creb1 b played roles in the development of oocytes and the early stage of spermatogenesis.However,other similar proteins might compensate for the function of creb1 b in spermatogenesis.In conclusion,the ATF/CREB family probably originated from the primitive multicellular animals and significantly expanded due to multiple whole genome duplications.Analysis of the expression pattern of the entire family showed that most members of the ATF/CREB family were expressed in at least one tissues,and some members were differentially expressed in the gonads.We obtained creb1 a and creb1 b mutants in Nile tilapia and analyzed their phenotype.The results indicated that creb1 a only played roles in oogenesis,while creb1 b was involved in both oogenesis and spermatogenesis.This study enriched the understanding of the evolution and expression of the ATF/CREB family and provided a new perspective for understanding its potential functions,particularly the role in gonad development and gameteogenesis.In addtion,functional studies of creb1 revealed its important role in gameteogenesis in teleosts.