【作者】 龙娟；

【导师】 王德寿；

【作者基本信息】 西南大学 ， 动物学， 2019， 硕士

【摘要】 转化生长因子-β(Transforming growth factorβ,TGF-β)信号通路是一个包含众多成员的大家族,主要由膜外的配体、膜上的受体以及胞内的SMADs蛋白组成。该信号参与介导了多种生物学过程,尤其在胚胎、组织和器官的发育与形成等方面发挥着重要作用。目前的研究报道都局限于单个TGF-β信号通路成员或者单个亚家族成员在某些组织或者某个发育时期的表达或功能研究,且研究对象大多为人类、小鼠和斑马鱼等模式动物,而大部分TGF-β信号通路成员在真骨鱼类中的表达模式和功能仍不清楚。此外,TGF-β信号通路被认为是动物基因组进化过程的良好标记,但TGF-β信号通路成员在多数动物类群中没有被全面鉴定过,其演化还不清晰。近年来,研究表明TGF-β信号通路在真骨鱼类性别决定、分化和性腺发育中也具有非常重要的作用,有多个成员被证实为鱼类的性别决定基因,还有很多成员也参与了鱼类的性别分化。本研究从全基因组水平鉴定了包括尼罗罗非鱼在内的24种代表性动物的TGF-β信号通路成员,分析了该信号通路在无脊椎动物进化过程中以及脊椎动物4次基因组复制过程中的系统演化。基于尼罗罗非鱼成鱼的8个不同组织转录组及不同发育时期雌雄性腺的16个转录组,分析了TGF-β信号通路成员的表达模式,并通过qPCR、原位杂交和免疫组化进行了实验验证。主要结果如下:本研究分离鉴定了海绵、水母、丝盘虫、绦虫、线虫、黑腹果蝇、水蛭、牡蛎、海胆、玻璃海鞘、文昌鱼、七鳃鳗、象鲨、斑点雀鳝、河鲀、尼罗罗非鱼、青鳉、斑马鱼、鲤鱼、腔棘鱼、非洲爪蟾、巨蟒、鸡和人24种代表性动物的TGF-β信号通路成员,分析表明TGF-β信号最早出现于海绵动物,是后生动物的关键特征之一。在海绵动物中已经出现多个配体、I型受体和SMADs,而仅有一个II型受体ACVR2,表明ACVR2可能是II型受体的祖先。在无脊椎动物和无颌类脊椎动物中,TGF-β配体、受体和SMADs的总数较少,且各物种间变化不大。而经过2R,有颌类脊椎动物中该信号通路成员发生了显著的扩张,四足动物已经拥有了30多个配体、7个I型受体、5个II型受体和8个SMADs,配体的扩张尤为明显。经过3R和4R,在真骨鱼类中有43-82个配体、9-22个I型受体、7-14个II型受体和13-31个SMADs,其成员明显多于四足动物。TGF-β信号通路成员在2R和3R后显著扩增,为脊椎动物组织和器官的复杂化奠定了物质基础。另外,脊椎动物中配体的数量远远超过了受体和SMADs,这表明多个配体可以共享相同的受体和SMADs。我们首次在软骨鱼类中分离出BMP16,也首次在四足动物中分离出了TGFB2、TGFBR2、ACVR1、SMAD4和SMAD6的复制基因。通过对TGF-β信号通路成员共线性分析,发现该信号通路成员间形成了5个紧密排列的基因簇,且这些基因簇在脊椎动物高度保守,表明这些基因间可能有协同作用。对尼罗罗非鱼成鱼8个组织的转录组数据分析表明,TGF-β多数配体呈现组织特异性表达,主要表达于心脏、性腺、肝脏和脑中:其中有6个配体(bmp16、bmp5、inhbba、bmp10a、bmp10b和bmp4)主要表达在心脏,有5个配体(admp2、bmp7a、gdf9、bmp15和gdf3)主要表达在卵巢,有4个配体(inha、gsdf、amh和gdf6b)主要表达在精巢,有3个配体(gdf2、inhbe和inhbab)主要表达在肝脏,有3个配体(gdf8a、gdf11和bmp7b)主要表达在脑;而大多数受体和SMADs在多个组织中都有表达,呈现泛表达模式,这可能是因为配体比较多样,且在不同的组织起作用的配体,共享同一个受体和SMADs。对尼罗罗非鱼不同发育阶段雌雄性腺转录组数据分析发现,TGF-β信号通路中有13个基因在性腺高表达并有明显性差,其中包括9个在卵巢高表达的基因(admp2、bmp7a、gdf9、bmp15、gdf3、acvr2ba、smad1、smad5和smad8)和4个在精巢高表达的基因(amh、gsdf、inha和amhr2)。为了更进一步了解TGF-β信号通路成员在性腺中的细胞定位,我们挑选16个基因(bmp15、gdf9、gdf3、ALK2a、ALK3、ALK6b、bmpr2a、bmpr2b、smad1、smad2a、smad2b、smad3a、smad3b、smad4ab、smad5和smad8)进行了原位杂交,结果表明这些基因都表达于卵巢的卵母细胞,其中大多数受体和SMADs还表达于成鱼精巢的精母细胞,表明TGF-β信号广泛地参与到尼罗罗非鱼的性腺发育中,尤其是对于卵母细胞的发育具有重要作用。利用实验室前期制备的特异性抗体进行免疫组化分析,结果表明Gsdf高表达于精巢精原细胞周围的支持细胞,也少量表达于不同发育阶段卵巢卵原细胞周围的体细胞,Amh在5 dah(Days after hatching,dah)高表达于精巢生殖细胞周围的体细胞,在后期主要表达于精巢的叶边细胞和支持细胞,也少量表达于卵巢初级生长阶段卵泡的颗粒细胞中。结合本实验室已有的敲除实验结果进一步表明gsdf和amh在性别决定和分化中有重要作用。综上,本研究从全基因组水平分析了TGF-β信号通路成员在动物界的起源和演化,并以尼罗罗非鱼为研究对象分析了TGF-β信号通路成员的表达模式,为全面理解TGF-β信号通路成员的进化及功能提供了新视角,为进一步研究TGF-β信号通路在不同组织特别是在性别决定、分化和性腺发育中的作用奠定了基础。更多还原

【Abstract】 Transforming growth factor-β(TGF-β)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-β 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-β signaling pathway members in teleosts are still unclear.In addition,the TGF-β signaling is considered to be a good marker for the evolutionary process of animal genome,but the members of the TGF-β signaling pathway have not been fully identified in most animal groups,and its evolution is still unclear.Recent studies showed that TGF-β 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-β 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-β 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-β 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-β 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-β 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-β 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-β signaling in teleosts are obviously more than those of tetrapods.The significant expansion of TGF-β 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-β 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-β 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-β 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-β 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-β signaling pathway in animal kingdom at genome level,and analyzed the expression patterns of TGF-β signaling pathway members in tilapia.This study provided a new perspective for comprehensively understanding the evolution and function of TGF-β signaling pathway members,and laied the foundation for further study the role of TGF-β signaling pathway in different tissues,especially in sex determination,differention and gonadal development.更多还原