Establishing Germ Cell Fate in Xenopus

描述：（申请人提供） 在有性繁殖的物种中，配子是体内唯一的细胞， 可以产生一个基因完整的新生物体。在这个意义上，"配子 是物种的干细胞"（Wylie，1999）。配子起源于 一小群细胞称为原始生殖细胞（PGCs）， 参与发展.这些独特的细胞保留了完全的全能性， 他们的命运逐渐受到限制。最终，PGCs从 它们的起源地到体细胞性腺，在那里它们分裂，进入减数分裂， 性别差异。在非洲爪蟾中，人们认为PGCs是由 遗传的种质，一个专门的细胞质领域，含有独特的 RNA和蛋白质。几乎没有人知道PGCs如何保存它们的 全能性或种质成分在POC发育中起什么作用。 已经分离出几种种质特异性RNA，包括Xdazl， DAZ家族在人类中的作用（King等，1999年）。我们发现母亲 Xdazl的耗尽导致PGCs的严重减少或损失， 未能迁移出内胚层（Houston和King，2000年）。Xcat-2， DEADSouth和Xpat是已经被发现的其他种质特异性RNA。 其特征是，但其功能尚不清楚。在这项研究中，实验 概述了如何生殖细胞的命运是确定的，并将解决 以下问题：一。什么机制允许PGCs保留全能命运 而植物块中的周围细胞则定向于内胚层 命运？内胚层在这个过程中起什么作用？二.什么步骤 PGC发育需要种质组分Xcat-2、DEADSouth、Xdazl和 Xpat？Xdazl和DEADSouth蛋白质有哪些特定的分子功能 表演？三.翻译抑制的机制是什么 卵子发生过程中的血浆RNA Xdaz1、Xcat2和DEADSouth？ 一种将绿色荧光蛋白（GFP）表达靶向至胚芽的方法， 血浆中的蛋白质，这将使PGC被跟踪或分离在不同的 时代发展。该程序将使PGCs可用于生物化学 和分子学研究。免疫细胞化学和原位 杂交将用于确定PGC何时转录地 活性以及它们是否表达内胚层诊断基因。 功能获得、功能丧失和过表达实验靶向 生殖质RNA将精确定位需要这些基因的步骤。卵母 在Xcat2和Xdazl的UTR区域中注射缺失突变体将 揭示了翻译抑制所需的顺式作用元件。这些顺 元件将用于亲和纯化反式作用因子。结果 这些拟议的研究将提供重要的见解， 在非洲爪蟾中和一般作为PGC发育的关键方面（全能性， 迁移）可能是跨门保存。

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.