Establishing Germ Cell Fate in Xenopus

描述(由申请人提供)：了解在细胞中保持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，是终极干细胞，因为它们保持着完全的发育潜力，而其他胚胎细胞则逐渐受到命运的限制。我们的长期目标是建立在脊椎动物模式生物非洲爪哇中PGC的规范和分化过程中起作用的关键调控通路。在果蝇和线虫的遗传学研究以及非洲爪哇的分子胚胎学研究中，已经确定了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。已鉴定的基因产物是如何“联网”的，更重要的是，这些基因产物保持全能性的真正机制在很大程度上仍不清楚。本申请将解决我们知识中的这一差距。非洲爪哇是这些研究的首选系统，因为它提供了生化和胚胎学水平上的完全可及性的独特组合。根据我们以前的数据，我们假设在胚胎中建立胚层期间，通过靶向翻译抑制特定母体mRNAs和Xcat2控制的mRNA转录的全局抑制相结合的方式，在PGCs中保持全能性。为了验证我们的中心假设，我们将完成以下具体目标：1)通过结合实时RT-PCR、注射标记的Vegt转录本和免疫定位来评估Vegt在分离的PGCs中的稳定性、翻译和核定位，确定母体Vegt功能在PGCs中是如何受到限制的。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作为翻译抑制物在保持PGC全能性方面所起的作用。这些RNA在PGC中的错误表达将被测试它们对PGC命运的影响。3)通过体内功能分析筛选抑制转录的Xcat2相互作用因子，确定在原始生殖细胞中瞬时转录抑制的机制。这项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类型的细胞，可用于治疗包括糖尿病、帕金森和心血管疾病在内的退行性疾病。我们对原生殖细胞的研究，将探索不同的机制如何合作，以保存细胞中的遗传幼稚或基态。我们的发现将与成人躯体干细胞如何维持多潜能状态有关，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.