REGULATION OF GERM CELL FATE DURING EMBRYOGENESIS

生殖细胞系对于物种的繁殖和延续是必不可少的;然而，人们对胚胎发生过程中指导其发育的分子机制知之甚少。 这项建议的长期目标是使用遗传模型系统秀丽隐杆线虫来表征这些机制。 C.秀丽线虫特别适合这项研究，因为在这种线虫中，可以连续跟踪从卵到成虫的生殖系发育。 这项建议集中在必需的生殖系因子PIE-1和它调节的两个进化上保守的机制上。第一种机制涉及胚胎生殖细胞中mRNA转录的全面抑制。 我们以前的研究表明，PIE-1通过阻断早期生殖细胞中的mRNA转录来保护生殖细胞免受体细胞转录因子的影响。 我们将提出以下问题：1）PIE-1如何定位于生殖谱系？ 2)PIE-1中的哪些结构域抑制转录？3）转录抑制对生殖细胞的命运有什么影响？ 这些问题将通过确定不同PIE-1结构域在体内的定位和功能，并通过确定这些结构域的功能因子来解决。 最近的技术进步使这些实验成为可能，这些技术进步允许在早期胚胎中表达转基因。第二种机制涉及与生殖系特异性P颗粒相关的母体RNA的调节。 我们已经确定了这样的RNA，NOS-2，并已表明，NOS-2蛋白在原始生殖细胞的表达是依赖于PIE-1。NOS-2与果蝇nanos相关，并且与另一个nanos同系物NOS-1一起对于原始生殖细胞发育是必需的。 我们将确定1）PIE-1定位和结构的哪些方面是NOS-2表达所必需的，以及2）生殖细胞命运的哪些方面是由NOS-1和NOS-2控制的。我们希望这些研究能够为基本的发育过程提供见解，包括蛋白质和mRNA的不对称分离，转录抑制和生殖细胞命运的控制。 C.线虫和脊椎动物表明，在这个简单的模型系统中收集的原则将适用于包括人类在内的其他动物。

The germ line is essential for reproduction and the perpetuation of species; yet little is known about the molecular mechanisms that guide its development during embryogenesis. The long-term goal of this proposal is to characterize these mechanisms using the genetic model system Caenorhabditis elegans. C. elegans is particularly well suited for this study, since, in this nematode, it is possible to track the development of the germline continuously from egg to adult. This proposal focuses on the essential germline factor PIE-1 and on two evolutionarily- conserved mechanisms which it regulates. The first mechanism involves the global inhibition of mRNA transcription in embryonic germ cells. Our previous studies suggest that PIE-1 protects the germline from somatic transcription factors by blocking mRNA transcription in early germ cells. We will ask the following questions: 1) how is PIE-1 localized to the germ lineage? 2) what domains in PIE-1 repress transcription? and 3) what effects does transcriptional repression have on germ cell fate? These questions will be addressed by determining the localization and function of different PIE-1 domains in vivo, and by identifying factors that function with these domains. These experiments are made possible by recent technical advances which permit the expression of transgenes in early embryos. The second mechanism involves the regulation of maternal RNAs associated with the germline-specific P granules. We have identified one such RNA, nos-2, and have shown that expression of NOS-2 protein in primordial germ cells is dependent on PIE-1. NOS-2 is related to Drosophila nanos, and together with another nanos homologue NOS-1 is essential for primordial germ cell development. We will determine 1) what aspects of PIE-1 localization and structure are required for NOS-2 expression, and 2) what aspects of germ cell fate are controlled by NOS-1 and NOS-2. We expect these studies to provide insights into basic developmental processes including the asymmetric segregation of proteins and mRNAs, transcriptional repression, and the control of germ cell fate. The many conserved characteristics between the germline of C. elegans and vertebrates suggest that principles gathered in this simple model system will be applicable to other animals, including humans.