【作者】 苏颖；

【导师】 孟安明；

【作者基本信息】 清华大学 ， 生物学， 2006， 博士

【摘要】 TGFβ/Nodal信号通路是影响胚胎发育的几个重要的信号通路之一。相比于其他信号通路,Nodal信号通路对于胚胎的中内胚层的诱导起到关键的作用。本文选取了三个与TGFβ信号通路相关的基因,即小鼠Dapper2,斑马鱼hmg2l1和ppm1a,研究其时空表达谱及功能,以加深对脊椎动物胚胎发育机理的了解。我们曾报道斑马鱼Dapper2通过降解Nodal受体而抑制胚胎中胚层的发育,但哺乳动物Dapper2未见报导。鉴于Dapper2对于胚胎中胚层发育的重要性,本文首先克隆出小鼠Dapper2基因,并研究其时空表达谱,发现它在体节形成期后在体节、耳泡、神经顶板、肠等组织中特异性表达。荧光酶报告实验显示小鼠Dapper2可以抑制TGFβ信号通路活性。胚胎实验表明,在斑马鱼胚胎中过量表达小鼠Dapper2,可以抑制斑马鱼胚胎的中胚层的发育。这些实验结果都说明,小鼠Dapper2和斑马鱼dapper2在调节TGFβ信号和胚胎发育方面具有保守性。以斑马鱼Dapper2为诱饵,通过酵母双杂交筛选,鉴定出与斑马鱼Dapper2互作的Hmg2l1。在哺乳动物细胞中,转染斑马鱼Hmg2l1可以抑制β-catenin介导的Wnt信号,也可以增强Smad2/3介导的TGFβ/Nodal信号。在斑马鱼oep突变体中,过量表达hmg2l1,可以导致突变体胚胎的头部缺失。关于hmg2l1影响斑马鱼发育的作用机理还需要更进一步的研究。斑马鱼Ppm1a是镁离子依赖性的丝氨酸/苏氨酸磷酸酶,它可以使磷酸化的Smad2/3去磷酸化,从而抑制TGFβ/Nodal信号通路活性。本实验发现,斑马鱼ppm1a是全身性表达的母源基因。在斑马鱼胚胎中过量表达ppm1a,可以抑制背部中胚层组织的发育。过量表达ppm1a还可以缓解由注射Smad2的MH2结构域蛋白或smad3b mRNA而引起的背部化表型;Smad2的MH2结构域蛋白的注射也可以减轻ppm1a对于背部中胚层组织发育的抑制作用。这些结果表明Ppm1a通过影响Smad2/3来下调TGFβ/Nodal信号,从而影响胚胎背部中胚层的发育。本研究表明,Dapper2、Hmg2l1和Ppm1a都是TGFβ/Nodal的负调控因子,可能均参与控制脊椎动物胚胎的背部发育。这些数据为了解TGFβ/Nodal信号的转导调控机理和对胚胎发育的调控作用提供了新的视觉。更多还原

【Abstract】 TGFβ/Nodal signaling pathway is one of the most important signaling pathways in the development of embryos. Compared with other signals, TGFβ/Nodal plays a key role in the induction of mesendoderm. Here, my work focused on three genes, mouse Dapper2, zebrafish hmg2l1 and ppm1a, which were all related with TGFβsignaling pathway. Their expression patterns and functions were studied to further illustrate the mechanism of embryonic development.As we have reported, zebrafish Dapper2 acted as an inhibitor of mesoderm development by promoting the degradation of Nodal receptor, however, mammalian Dapper2 has never been reported. Because of the significance of zebrafish Dapper2 in mesoderm induction, the mouse dapper2 was cloned following its homolog. The study of its spatial and temporal expression pattern showed that it expressed specifically in otic vesicle, somite,gut and roof plate of mouse embryos. In the ARE-luciferase reporter assay, mouse Dapper2 can inhibit the activity of TGFβpathway. The overexpression of mouse Dapper2 in zebrafish embryos induced the reduction of mesoderm. All of the results suggested that the effects of Dapper2 in regulating TGFβsignal and zebrafish embryonic development were conserved between zebrafish and mouse.Zebrafish Hmg2l1, which was obtained by yeast two-hybridization using zebrafish Dapper2 as bait, can interact with zebrafish Dapper2. It repressed Wnt signal dependent onβ-catenin and activated TGFβ/Nodal signaling pathway transduced by Smad2/3. The overexpression of hmg2l1 led to the absence of head structure in oep mutant. More work is still needed to explain the mechanism.Zebrafish Ppm1a, a Mg2+-dependent serine/threonine phosphatase, was identified as a Smad2/3-specific phosphatase. It down-regulated TGFβ/Nodal signal by dephosphorylating the activated Smad2/3. Demonstrated by in situ hybridization, zebrafish ppm1a was a maternal gene and expressed throughout the embryos. In the zebrafish, overexpression of ppm1a can inhibit the development of dorsal mesoderm,and the abnormal phenotype can be rescued by injection of the MH2 domain of Smad2 protein. Furthermore, overexpression of ppm1a can rescued the dorsalized phenotype caused by the injection of Smad2-MH2 protein or smad3b mRNA. These results revealed that Ppm1a can down-regulate TGFβ/Nodal signal and affect the development of dorsal mesoderm by interacting with Smad2/3.My work showed that Dapper2, Hmg2l1 and Ppm1a were negative regulators of TGFβ/Nodal signal and they perhaps regulated the development of dorsal structure in vertebrate embryos. These data give some new insights on the mechanism of TGFβ/Nodal transduction and in regulation of embryonic development.更多还原