新基因hacp在斑马鱼原始生殖细胞增殖中的作用及分子机制研究

The study of molecular mechanism of primordial germ cells (PGCs) proliferation is one of the important issue in animal embryology,developmental biology and medicine fields.On basis of our previous study about hacp mRNA expression during the period of PGCs proliferation and the increased numbers of PGCs and vasa mRNA after overexpression of Hacp in zebrafish, the endogenous temporal and special distribution pattern of hacp mRNA and protein during zebrafish embryonic development and in the adult tissues will be analyzed using in situ hybridization and immunohistochemistry in this project. RNA interference , overexpression and rescue experiments will be performed to study the function of hacp gene .The roles of hacp will be illuminated in the process of the PGCs proliferation.Chromatin Immunoprecipitation and EMSA will be used to certify the target gene binding sequence of Hacp protein and the element in Hacp in the promoter of the target gene. The expression of the Hacp target gene in developing embryos and the abnormity of the number of PGCs, in which the expression of hacp was inhibited or over-expressed will also be investigated by the qRT-PCR and in situ hybridization. The molecular mechanisms of hacp regulation during the PGCs proliferation will also be illuminated in vitro and in vivo. This project will unravel the essential regulation factors about PGCs proliferation and its molecular mechanism. More importantly, it will obviously broaden our horizon on the development of vertabrate animals and provide critical contribution on the studies about the mechanism of germ stem cell deferentiation and human generative cells tumorigenesis.

原始生殖细胞（PGCs）增殖分子机制的研究是动物胚胎学、发育生物学和医学关注的重点之一。本项目是在发现了hacp mRNA在斑马鱼PGCs增殖期大量表达，在过表达Hacp的胚胎中,PGCs标记物vasa mRNA和PGCs数量增加的基础上，采用原位杂交和免疫组化等方法，研究hacp mRNA和蛋白在斑马鱼早期胚胎发育过程中的时空表达谱，分析其与PGCs增殖的关系；通过RNA干涉、过表达和拯救实验，研究hacp基因的功能，明确其在PGCs增殖过程中的作用；采用CHIP和EMSA等技术, 获得Hacp蛋白结合的靶基因，确定其在靶基因启动子上的特异结合元件；通过干涉和过表达实验，确定Hacp调控下游靶基因的表达与PGCs数目异常变化的关系，明确Hacp表达调控在PGCs增殖中的作用，阐明hacp调控PGCs增殖的分子机理，为生殖干细胞分化和人类生殖细胞肿瘤的发生机理提供重要的基础理论资料。

原始生殖细胞（PGCs）增殖的分子机制研究是动物发育生物学和医学关注的重点之一。Nanog在维持哺乳动物胚胎干细胞多能性和自我更新方面起着关键作用。通过本项目研究， 我们从斑马鱼中克隆了一段编码同源异型盒结构域的cDNA序列（提交时命名为hacp，后改名为znanog)；证明了znanog mRNA和蛋白在斑马鱼的精原细胞、早期卵母细胞和胚胎发育过程中的PGCs中表达。通过基因功能的研究，我们发现在敲除zNanog的胚胎中，PGCs 的标记基因vasa 的表达和vasa mRNA 阳性细胞数目增加, 其定位异常,分析表明zNanog在斑马鱼早期胚胎发育过程的 PGCs增殖中发挥了重要作用。通过ChIP-Seq和荧光素酶等方法研究，获得了zNanog结合的靶基因znlk1，确定了其在znlk1启动子上结合的特异序列；在体外细胞和斑马鱼胚胎发育中，证明了zNanog上调znlk1 mRNA和蛋白的表达；通过MO-zNano,过表达和挽救试验研究，发现了在注射了zNanog morpholino (MO-zNanog）的胚胎中，vasa mRNA、蛋白和Vasa mRNA阳性细胞数目增加；在MO-zNanog 胚胎中过表达znlk1 mRNA，与MO-zNanog 胚胎比较，在共注射MO-zNanog和znlk1 mRNA的胚胎中，vasa mRNA阳性细胞数目减少，表明znlk1 mRNA能挽救zNanog缺失，以上结果说明zNanog直接结合到znlk1启动子上调控PGCs的增殖。 结果为研究原始生殖细胞增殖和人类生殖细胞肿瘤的发生分子机理提供了基础理论资料。