Establishing Germ Cell Fate in Xenopus

描述：(申请人提供) 在有性繁殖物种中，配子是体内唯一可以 可以产生一个基因上完整的新生物体。在这个意义上，“配子” 是物种的干细胞“(Wylie，1999)。配子来源于 早期形成的一小群称为原始生殖细胞(PGC)的细胞 正在开发中。这些独特的细胞保持完全全能，而体细胞 逐渐受到命运的限制。最终，PGC从 它们的起源地点是体细胞性腺，在那里它们分裂，进入减数分裂和 性别分化。在非洲爪哇，人们认为PGC由以下因素决定 种质的遗传，一个包含独特的专门化细胞质领域 RNA和蛋白质。事实上，对于PGC如何保存他们的 全能性或种质成分在POC发育中所起的作用。 已分离到几个种质特异的RNA，其中包括Xdazl成员 在人类中属于DAZ家族(King等人，1999年)。我们发现这是母性的 Xdazl的耗尽会导致PGC的严重减少或损失，因为它们 未能走出内胚层(Houston and King，2000)。XCAT-2， DEADSouth和Xpat是其他种质特异的RNA 特征的，但其功能尚不清楚。在这项提案中，实验 来探索生殖细胞的命运是如何决定的，并将解决 以下问题：一、是什么机制允许PGCs保持全能的命运 而植物团中的周围细胞则附着于内胚层 命运？内胚层在这一过程中扮演什么角色，如果有的话？II.采取什么措施 PGC的发育需要种质成分XCAT-2、DEADSouth、Xdazl和 XPAT？Xdazl和DEADSouth蛋白有哪些具体的分子功能 表演？翻译抑制细菌的机制是什么？ 卵子发生过程中的血浆RNAs Xdaz1、Xcat2和DEADSouth？ 一种将绿色荧光蛋白(GFP)靶向表达于胚芽的方法 提供的血浆将允许在不同的位置跟踪或分离PGC 发展中的时代。这一程序将使pGCs可用于生化。 以及第一次进行分子研究。免疫细胞化学和原位观察 杂交将被用来确定pGCs何时转录 活性以及它们是否表达内胚层诊断基因。 靶向功能获得、功能丧失和过度表达的实验 种质RNA将精确定位需要这些基因的步骤。卵母细胞 在Xcat2和Xdaz1的非编码区注射缺失突变体将 揭示翻译抑制所需的顺式作用元件。这些配置项 这些元素将被用来亲和纯化反式作用因子。结果来自 这些拟议的研究将为生殖系如何 非洲爪哇和一般作为PGC发展的关键方面的形式(全能， 迁徙)可能在整个门之间保存下来。

DESCRIPTION: (provided by applicant) In sexually reproducing species, gametes are the only cells in the body that can give rise to a genetically complete new organism. In this sense, "gametes are the stem cells of the species" (Wylie, 1999). Gametes originate from a small population of cells called primordial germ cells (PGCs) that form early in development. These unique cells retain full totipotency while somatic cells become progressively restricted in their fates. Eventually, PGCs migrate from their site of origin to the somatic gonads where they divide, enter meiosis and sexually differentiate. In Xenopus, it is thought that PGCs are determined by inheritance of germ plasm, a specialized cytoplasmic domain containing unique RNAs and proteins. Virtually nothing is known about how PGCs preserve their totipotency or what functions germ plasm components play in POC development. Several germ plasm specific RNAs have been isolated, including Xdazl a member of the DAZ family in humans (King et al., 1999). We found that maternal depletion of Xdazl results in the severe reduction or loss of PGCs because they fail to migrate out of the endoderm (Houston and King, 2000). Xcat-2, DEADSouth, and Xpat are other germ plasm specific RNAs that have been characterized, but whose functions are not known. In this proposal, experiments are outlined to explore how germ cell fate is determined and will address the following questions: I. What mechanism permits PGCs to retain a totipotent fate while surrounding cells in the vegetal mass become committed to an endoderm fate? What role, if any, does endoderm play in this process? II. What steps in PGC development require the germ plasm components Xcat-2, DEADSouth, Xdazl, and Xpat? What specific molecular functions do Xdazl and DEADSouth proteins perform? III. What is the mechanism of translational repression of the germ plasm RNAs Xdazl, Xcat2, and DEADSouth during oogenesis? A method for targeting green fluorescent protein (GFP) expression to the germ plasm is presented that will allow PGCs to be followed or isolated at different times in development. This procedure will make PGCs accessible to biochemical and molecular studies for the first time. Immunocytochemistry and in situ hybridization will be used to determine when PGCs become transcriptionally active and whether they express genes diagnostic for endoderm. Gain-of-function, loss-of-function, and over-expression experiments targeting the germ plasm RNAs will pinpoint the steps that require these genes. Oocyte injections of deletion mutants in the UTR regions of Xcat2 and Xdazl will reveal cis-acting elements required for translational repression. These cis elements will be used to affinity purify trans-acting factors. Results from these proposed studies will offer significant insights into how the germline forms in Xenopus and in general as key aspects of PGC development (totipotency, migration) are likely conserved across phyla.