Establishing Germ Cell Fate in Xenopus

描述（由申请人提供）：了解保持细胞全能性或多能性的细胞机制是干细胞和发育生物学的中心目标。原始生殖细胞（PGCs）是配子的前体，是最终的干细胞，因为它们保留了完整的发育潜力，而其他胚胎细胞的命运逐渐受到限制。我们的长期目标是建立在脊椎动物模式生物爪蟾中PGCs的规范和分化过程中运作的关键调控途径。在果蝇和秀丽隐杆线虫的遗传学研究以及爪蟾的分子胚胎学研究中发现，PGCs中的基因产物似乎可以保护它们免受不适当的体细胞分化。鉴定出的基因产物是如何“联网”的，重要的是，它们保持全能性的真正机制在很大程度上仍然未知。本应用程序将解决我们知识上的这一空白。非洲爪蟾是这些研究的首选系统，因为它在生化和胚胎学水平上都提供了一个独特的可及性组合。基于我们之前的数据，我们假设在胚胎中建立胚层期间，PGCs的全能性是通过靶向翻译抑制特定母体mRNA和Xcat2控制的mRNA转录的整体抑制相结合而保持的。为了验证我们的中心假设，我们将完成以下具体目标：1)利用Real Time RT-PCR、注射标记的VegT转录物和免疫定位相结合，通过评估VegT的稳定性、翻译和核定位，确定母体VegT功能如何在PGCs中受到限制。2)通过使用下拉、RT-PCR和克隆方法鉴定与Xcat2共纯化的rna，确定Xcat2作为翻译抑制因子在保持PGC全能性中的作用。这些rna在PGCs中的错误表达将被测试其对PGC命运的影响。3)通过体内功能实验选择抑制转录的Xcat2相互作用因子，确定原始生殖细胞中瞬时抑制转录的机制。本研究与公共卫生的相关性：干细胞具有很高的治疗价值，因为它们能够发育成各种各样的细胞类型，可用于治疗退行性疾病，包括糖尿病、帕金森病和心血管疾病。我们对PGCs（全能性的主要范例）的研究将探索不同的机制如何合作来保存细胞中的遗传幼稚态或基态。我们的发现将与成体干细胞如何维持多能状态有关，这是干细胞生物学中的重要问题。

DESCRIPTION (provided by applicant): Understanding cellular mechanisms that preserve totipotency or pluripotency in cells are central goals of both stem cell and developmental biology. Primordial germ cells (PGCs), precursors to the gametes, are the ultimate stem cell as they retain full developmental potential while other embryonic cells become progressively restricted in their fates. Our long term goal is to establish the key regulatory pathways that operate during the specification and differentiation of PGCs in the vertebrate model organism Xenopus. Genetic studies in Drosophila and C. elegans and molecular embryological studies in Xenopus have identified gene products in PGCs that appear to protect them from inappropriate somatic differentiation. How the identified gene products are "networked" and importantly, the real mechanism through which these preserve totipotency, remain largely unknown. The present application will address this gap in our knowledge. Xenopus is the system of choice for these studies because it offers a unique combination of total accessibility at both the biochemical and embryological level. Based on our previous data, we hypothesize that during the time that germ layers are established in the embryo, totipotency is preserved in PGCs by a combination of targeted translational repression of specific maternal mRNAs and global repression of mRNA transcription controlled by Xcat2. To test our central hypothesis we will complete the following specific aims: 1) Determine how maternal VegT function is restricted in PGCs by assessing VegT stability, translation, and nuclear localization in isolated PGCs using a combination of Real Time RT-PCR, injected tagged VegT transcripts and immunolocalization. 2) Determine the role Xcat2 plays as a translational repressor in preserving PGC totipotency by identifying the RNAs that co-purify with Xcat2 using a pull-down, RT-PCR, and cloning approach. Mis-expression of these RNAs in PGCs will be tested for their effects on PGC fate. 3) Determine what mechanism is responsible for the transient repression of transcription in primordial germ cells by selecting for Xcat2 interacting factors that repress transcription in vivo functional assays. Relevance of this research to public health: Stem cells are of high therapeutic value because of their ability to develop into a wide variety of cell types that could be used in the treatment of degenerative diseases including diabetes, Parkinson's and cardiovascular disease. Our studies on PGCs, prime exemplars of totipotency, will explore how different mechanisms cooperate to preserve genetically naive or ground states in cells. Our findings will be relevant to how adult somatic stem cells maintain a pluripotent condition, important issues in stem cell biology.