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题名: 青鳉piwi和vasa基因的表达模式和功能分析
作者: 李名友
答辩日期: 2008-06-17
导师: 桂建芳 ; 洪云汉
授予单位: 中国科学院水生生物研究所
授予地点: 水生生物研究所
学位: 博士
关键词: 生殖细胞 ; opiwi,vasa ; 精子发生 ; 拟染色小体 ; 卵子发生 ; 巴尔氏小体 ; 青鳉
其他题名: Expression and Functional Analysis of piwi and vasa in Medaka
摘要: 在大多数动物里,早期的胚胎发育产生两大类细胞:体系细胞和种系细胞。种系细胞源于原始生殖细胞(PGC),PGC是从早期胚胎的体细胞中分化而来的,经过长距离的跋涉迁移到生殖腺,然后形成精子和卵子,精卵结合产生新的个体,从而保证物种的延续和进化。因此了解生殖细胞的形成、发育及其命运维持的分子机制对操作生殖细胞极为关键。作为生殖细胞的标记基因,piwi和vasa的表达模式和功能分析在物种间广为研究。本文旨在以青鳉为模式生物,研究piwi和vasa的表达模式及其在鱼类生殖细胞形成与发育过程中的作用。 Piwi基因最早是从果蝇卵巢中分离出来的,对生殖干细胞的增殖和分裂有调控作用,其功能在进化上具有保守性。目前青鳉piwi的研究还没有报道,本研究克隆到的青鳉piwi基因(opiwi)cDNA全长为2.9kb,编码857个氨基酸,在N端和C端分别具有保守的PAZ结构域和PIWI结构域。系统树显示opiwi属于piwi亚族,和斑马鱼的piwi相似性最高。Opiwi和人的piwi基因具有相似的基因结构和染色体分布。Opiwi RNA是母源性的,在整个胚胎发育过程都有表达。在早期的胚胎发育过程中,opiwi RNA和蛋白均匀分布在每个卵裂球;在体节期,opiwi除了在PGC大量表达外,我们还发现其在眼睛、脑以及尾部等体细胞也有微弱表达。oPiwi蛋白以颗粒状分布在PGC的核周。在成体组织里,opiwi RNA和蛋白虽然在性腺大量表达,在眼和脑神经系统也有表达。在性腺里,opiwi RNA和蛋白仅在生殖细胞里有表达,而在其他体细胞则没有表达。在卵子发生过程中,opiwi在卵原细胞和早期的卵母细胞大量表达,随着卵子发育,其表达逐渐下降,在成熟的卵母细胞里,其信号呈点状或薄层分布;在卵母细胞里,opiwi和线粒体共定位于卵子的生殖质结构巴尔氏小体(Balbiani body);我们还发现在卵原细胞里,oPiwi蛋白以中空的颗粒分布在其核周;此外oPiwi蛋白除了在细胞质有分布外,在细胞核也有分布,其细胞核定位和PCNA(proliferating cell nuclear antigen)及线粒体相同。在精子发生过程中,opiwi RNA和蛋白在精原细胞和精母细胞大量表达,opiwi的RNA在精子细胞和精子里没有信号,而其蛋白在精子细胞和精子里持续表达,且和线粒体共定位于生精细胞的生殖质结构拟染色体(chromatoid body);oPiwi蛋白在精原细胞的细胞质和细胞核都有分布,而在其他生精细胞则只在细胞质有分布。oPiwi还以中空的颗粒结构分布在精原细胞的核周。在干细胞系中,opiwi RNA和蛋白都有大量表达,其蛋白在细胞质和细胞核均有分布。此外,我们还发现opiwi 3’非编码区(3’untranslated region, 3’UTR)在囊胚晚期(9hpf)就能特异性地标记PGC,而目前报道最早能区分青鳉PGC的nanos3’UTR则是在原肠早期(13hpf)。我们通过减少或增加opiwi的表达来研究其功能,PGC用nanos启动子驱动的绿色荧光和vasa启动子驱动的绿色荧光双标记(NgVg),这样我们可以从原肠中期就可以追踪PGC的发育。结果表明注射opiwi的Morpholino(MOpw)消减opiwi的表达对PGC发育和早期胚胎发育以及体细胞发育具有双重作用:MOpw导致PGC数目减少以及异位PGC,同时早期的胚胎发育和晚期胚胎发育的体细胞的发育也受影响;体外细胞培养表明,注射MOpw导致PGC和体细胞的分裂受阻。而MOpw和opiwi的RNA共注射能挽救MOpw的表型。进一步的实验表明是母源性的opiwi影响生殖细胞的发育和早期的胚胎发育,而合子性的表达仅影响晚期胚胎的体细胞发育。过量表达正常或缺失去掉PAZ结构域的RNA也影响生殖细胞发育和胚胎发育。因此,opiwi除了影响青鳉的生殖细胞的形成发育这一高度保守的功能外,我们还发现其在体细胞的发育也起着重要作用。 Vasa是进化上功能保守的翻译调控因子。它最初从果蝇中分离出来,对前后轴的极化和生殖细胞的形成是必需的。vasa同时在其他物种的生殖细胞的发育也起着重要作用,但它在早期的胚胎发育和生殖细胞的发育中的具体功能还不清楚。迄今,青鳉的vasa RNA仅见于生殖细胞,vasa启动子的活性亦是生殖细胞特异的,其蛋白的表达尚无详细的报道。本研究表明vasa RNA和蛋白的表达模式和opiwi基本一致,即是母源性的,在胚胎发育的原肠前期表达很强,随后开始降低。在早期的胚胎发育过程中,vasa均匀分布在每个卵裂球;在器官发生期,vasa除了在PGC大量表达,此外还在体细胞如脑、躯干和尾部也有表达。Vasa蛋白和oPiwi蛋白以中空的颗粒分布在PGC以及性原细胞的核周。在成体组织中,vasa RNA除在性腺大量表达外,在体细胞也有微弱表达。Vasa RNA和蛋白仅在性腺的生殖细胞有表达。在卵子发生,vasa RNA和蛋白在整个卵子发生都有表达,且和oPiwi共定位于巴尔氏小体。Vasa蛋白在卵母细胞的细胞质和细胞核都有分布。在精子发生阶段,vasa RNA和蛋白在精原细胞和精母细胞大量表达,vasa RNA在精子细胞和精子里没有信号,而其蛋白在精子细胞和精子也有微弱表达。Vasa蛋白在精原细胞的细胞质和细胞核都有分布,而在其它生精细胞仅在细胞质有表达。在精子细胞和精子阶段,Vasa和oPiwi共定位于拟染色小体。与其表达模式相对应的是,vasa在青鳉鱼PGC的发育和体细胞的发育过程中具有双重作用。注射MO消减vasa的翻译表达,PGC不能迁移到目的地--生殖腺。单个细胞的体内体外实验表明消减vasa的表达不改变PGC的动性,存活能力和分裂。即使在性腺外的异位PGC也具有动性,能够存活和分裂。将注射MOvas的胚胎的细胞在中囊胚时期移植到同样时期的野生型胚胎,PGC不能迁移到生殖腺。并且,注射MOvas,还会导致早期发育阶段胚胎大量死亡,活着的胚胎也伴随大量体系细胞的死亡;将注射MOvas后的胚胎的细胞在原肠早期分离出来,在体外培养第四天,也观察到大量的体细胞死亡。因此,我们推测vasa在青鳉的胚胎发育过程中具有双重作用:在生殖细胞的发育过程中,它对生殖细胞的迁移是必要的、自主性的;在体系细胞中,它在原肠期胚胎分层和体细胞的维持方面起着重要作用。我们还证实了生殖腺外异位的PGC和体外培养的PGC有维持着它自身特性的能力,即PGC的特性是自主性的。 综上所述,opiwi和vasa主要在青鳉的生殖细胞里表达并起作用,同时我们也证实这2个基因在体细胞发育具有新功能。 关键词:生殖细胞,opiwi,vasa,精子发生,拟染色小体,卵子发生,巴尔氏小体,青鳉
英文摘要: In most animals, primordial germ cells(PGCs)are set aside from somatic cells in early stage of embryo development and migrate to the gonads where they differentiate into gametes for transmitting genetic information from one generation to another. Understanding the mechanism of germline development is very important for genetic manipulation of germ cells. Piwi and vasa were studied among different organisms. This work was aimed at making use of the medaka as a model to study the expression pattern and function of piwi and vasa, two highly conserved germ cell markers in the germline development. Piwi was first isolated from Drosophila which is important for germ line stem cell self renewal. Piwi encodes an RNA binding protein and has been shown to be highly conserved in structure and germ cell specific. At present, medaka piwi has not been reported. Here we show that medaka piwi (opiwi) is 2.9kb for a putative protein of 857 aa with conserved PAZ domain at its N-terminus and PIWI domain at its C-terminus respectively. A phylogenetic tree shows that opiwi belongs to the piwi sub-family and has a closest identity with that of zebrafish piwi. Opiwi has the same genome structure and chromosome location as the human piwi gene. During embryogenesis, opiwi RNA is maternally provided and its expression persists throughout embryogenesis. During early stages of embryo development, opiwi RNA and protein evenly distribute in every blastomere. During the somitegenesis stage, opiwi RNA expression is high in PGC and weak in somatic cells such as the eye, brain and tail. In PGCs oPiwi protein distributes in peri-muclear particles. In spermatogonia and oogonia, oPiwi protein localizes to the same structure. In adult tissues, except for their preferential expression in gonads, opiwi RNA and protein are also weakly expressed in the eye and brain. In the gonads, both opiwi RNA and protein expression are restricted to germ cells. In the ovary, opiwi RNA and protein are high in oogonia and early stages of oocytes, declines in growing oocytes, in the late and mature stage oocytes, opiwi either distributes into dot-like structure in the yolk or forms a thin layer around the cortex of oocytes. In addition, opiwi RNA and protein localize to Balbiani body with mitochondria in early stage of oocytes. In the testis, opiwi is high in spermatogonia and spermertocyte, opiwi RNA is absent from spermatids and sperm, in contrast, oPiwi is persisting in spermatids where it localizes to chromatoid body also with mitochondria. Furthermore, we find that gfp fused to opiwi3’UTR (untranslated region, 3’UTR) can identify first PGCs as early as later blastula stage (9hpf), compared to the early gastrula stage (13hpf) when PGCs become distinguished by nanos3’UTR... Opiwi function was investigated by increasing or decreasing its expression, PGCs are distinguished at the mid gastrula by gfp expression in double transgenic medaka driven by nanos promoter and vasa promoter (NgVg). Injection of opiwi morpholino (MOpw) gives rise to decreased PGC number and somatic development defect during organgenesis. Opiwi depleted embryos in cell culture shows that opiwi depletion affected PGCs and somatic cells proliferation and survival. By injection of splicing MO and antibody, we find that its maternal expression affects germ cells development while the zygotic expression affects somatic development. Injection of opiwi RNA and dominant negative RNA without PAZ domain also decreases PGC number and defects in early stages of embryo development. Vasa was identified in a genetic screening as one of the several maternal-effect genes for the embryonic polarity and germ cell formation in Drosophila. It is best known for its expression and role in the germline in different organisms. So far, Medaka vasa RNA is restricted to germ cells and its promoter activity is germ cells specific. However, Vasa protein expression and function still remains unclear. Here we report that vasa RNA and protein have the same expression pattern of that of opiwi , Vasa RNA is maternally provided with high expression till early gastrula stage, its expression persists throughout the embryogenesis. During early stages of embryo development, vasa is evenly distributed in every blastomere. During organgenesis stage, we find that vasa RNA is weak in somatic cells such as brain, trunk and tail except for its preferential expression in PGCs. In adult tissues, vasa RNA also is weak in somatic tissues although it is high in the gonads. In the gonads, vasa RNA and protein are restricted to germ cells. In the ovary, vasa expression is throughout the whole stage of oogenesis and co-localized with opiwi in Balbiani body. In the testis, vasa is co-localized with oPiwi in chromatoid body in spermatids. In spermatogonia , Vasa distributes in peri-nuclear particles with oPiwi. Vasa and oPiwi have the same expression pattern in oogonia and PGCs. In madaka, vasa has dual roles in PGC migration and somatic development. Loss of function by injection of MOvas led to many ectopic PGCs. MOvas embryos subjected to cell culture show that vasa depletion does not affect PGC motility, proliferation and survival. Surprisingly, even PGCs at ectopic sites are able to maintain the PGC identity and capable of proliferation, mobility and survival. By chimeric analysis we show that vasa depleted PGCs can move but can not migrate properly in the normal host environment. Surprisingly, upon vasa knockdown, embryonic lethality is compromised during organogenesis, and massive somatic cell death takes place in living embryos and their cell cultures. Therefore, medaka vasa has dual roles. In germline development vasa specifically controls PGC migration cell-autonomously. In somatic development vasa controls embryonic viability and cell survival. Accordingly, the medaka vasa has the conserved role in the germline and previously unidentified roles in somatic development, suggesting that vasa has a broader role than previously thought. In conclusion, both opiwi and vasa expression and function are mainly in germ cells; however we find that opiwi and vasa have a new role in the maintenance of somatic cells development. Key words: germ cells, opiwi, vasa, spermatogenesis, chromatoid body, oognesis,balbiani body, medaka
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/11952
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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青鳉piwi和vasa基因的表达模式和功能分析.李名友[d].中国科学院水生生物研究所,2008.20-25
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