Bioinformatic Analysis Of TGF-? Signaling Pathway Members And Their Expression In Nile Tilapia

Transforming growth factor-?(TGF-?)signaling pathway is a large family which contains many members including extracellular ligands,membrane receptors and intracellular SMADs.This signaling is involved in many biological processes,especially in the development and formation of embryos,tissues and organs.Current research are limited to the expression or function of a single TGF-? signaling member or a single subfamily member in some tissues or at certain developmental stages.Most of the research subjects are model animals such as human,mouse and zebrafish.However,the expression patterns and function of most of the TGF-? signaling pathway members in teleosts are still unclear.In addition,the TGF-? signaling is considered to be a good marker for the evolutionary process of animal genome,but the members of the TGF-? signaling pathway have not been fully identified in most animal groups,and its evolution is still unclear.Recent studies showed that TGF-? signaling pathway plays an important role in sex determination,differentiation and gonadal development of teleosts,some of them were identified as sex-determining genes or participants in such process at least.Many members have been identified as sex-determining genes in teleosts,and many members have also participated in sex differentiation.In this study,we identified TGF-? signaling pathway members in 24 representative animals at genome level,including tilapia,analyzed the phylogenetic evolution of this signaling pathway in the evolutionary process of invertebrates and in the four round of genome duplication processes of vertebrates.Based on the transcriptomes of 8 different tissues of tilapia adult fish and 16 transcriptomes of female and male gonads at different developmental stages,the expression patterns of TGF-? signaling pathway members were analyzed and verified by qPCR,in situ hybridization and immunohistochemistry.The main results were shown as follows:We identified the TGF-? signaling pathway members of sponges,jellyfish,trichoplax,tapeworm,nematode,fruit fly,leech,oyster,common urchin,vase tunicate,lancelet,lamprey,elephant shark,spotted gar,fugu,nile tilapia,medaka,zebrafish,grass carp,coelacanth,xenopus,burmese python,chicken and human.The results showed that the TGF-? signaling first appeared in the sponges of metazoans,which is one of the key characteristics of metazoan.Multiple ligands,type I receptors and SMADs have appeared in sponges,while only one type II receptor ACVR2 was found in early metazoans,suggesting that ACVR2 may be the ancestor of type II receptor.The total numbers of TGF-? ligands,receptors and SMADs in all the invertebrates and jawless vertebrates were relatively small and there was little change among species.In contrast,expansion of the pathway members,especially ligands,was observed in jawed vertebrates due to 2R event.TGF-? signaling contains more than 30 ligands,7 type I receptors,5 type II receptors and 8 SMADs in tetrapods.There are 43-82 ligands,9-22 type I receptors,7-14 type II receptors and 13-31 SMADs in teleosts.Because of 3R and 4R,the members of TGF-? signaling in teleosts are obviously more than those of tetrapods.The significant expansion of TGF-? signaling pathway members in animal evolution lays a material foundation for the need of more genes to meet the complexity of tissues and organs in animal evolution.In addition,the number of ligands in jawed vertebrates far exceeds the number of receptors and SMADs,suggesting that multiple ligands can share the same receptors and SMADs.We isolated BMP16 from whale shark for the first time,and the replication genes of TGFB2,TGFBR2,ACVR1,SMAD4 and SMAD6 from tetrapods for the first time.By syntenic analysis of TGF-? signaling pathway members,it was found that five closely arranged gene clusters formed among the members of the signaling pathway,and these gene clusters were highly conserved in vertebrates,indicating that there might be synergistic effects among these genes.The transcriptome data of eight tilapia adult tissues showed that most of the ligands of TGF-? exhibited a tissue-specific expression patterns,especially expressed in heart,gonad,liver and brain.Six ligands(bmp16,bmp5,inhbba,bmp10 a,bmp10b and bmp4)were mainly expressed in heart,five ligands(admp2,bmp7 a,gdf9,bmp15 and gdf3)were mainly expressed in ovary,four ligands(inha,gsdf,amh and gdf6b)were mainly expressed in testis,three ligands(gdf2,inhbe and inhbab)were mainly expressed in liver,and three ligands(gdf8a,gdf11 and bmp7b)were mainly expressed in brain.However,most receptors and SMADs were expressed in multiple tissues indicating they were shared by different ligands.The transcriptome data of male and female gonads of tilapia at different developmental stages showed that 13 genes of TGF-? signaling pathway members were highly expressed in gonads,including 9 genes(admp2,bmp7 a,gdf9,bmp15,gdf3,acvr2 ba,smad1,smad5 and smad8)in ovary and 4 genes(amh,gsdf,inha and amhr2)in testis.To further understand the cellular localization of TGF-? signaling pathway members in gonads,16 genes(bmp15,gdf9,gdf3,ALK2 a,ALK3,ALK6 b,bmpr2a,bmpr2 b,smad1,smad2 a,smad2b,smad3 a,smad3b,smad4 ab,smad5 and smad8)were selected for in situ hybridization in gonads of tilapia.The results showed that these genes were expressed in oocytes of ovary.Most of receptors and SMADs were also expressed in spermatocytes of adult testis,indicating that this signaling is widely involved in gonadal development of tilapia,especially in oocyte development.Immunohistochemical analysis with specific antibodies prepared in the early stage of our lab showed that Gsdf was highly expressed in Sertoli cells neighboring spermatogonia of the XY gonads and little expressed in somatic cells neighboring oogonia of the XX gonads at various stages,Amh was highly expressed in the somatic cells surrounding germ cells in the testis at 5 dah(Days after hatching,dah),and later stages in myoid cells and Sertoli cells,and little expressed in the granulosa cells of the follicles in the primary growth stage in the ovary.It is further demonstrated that amh and gsdf play an important role in sex determination and differentiation.In conclusion,this study analyzed the phylogeny and evolution of TGF-? signaling pathway in animal kingdom at genome level,and analyzed the expression patterns of TGF-? signaling pathway members in tilapia.This study provided a new perspective for comprehensively understanding the evolution and function of TGF-? signaling pathway members,and laied the foundation for further study the role of TGF-? signaling pathway in different tissues,especially in sex determination,differention and gonadal development.