REGULATION OF GERM CELL FATE DURING EMBRYOGENESIS

胚胎发生过程中生殖细胞命运的调节

• 批准号: 6388016
• 负责人: GERALDINE Catherine Joelle SEYDOUX
• 金额: $ 27.01万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6388016
• 关键词: Caenorhabditis elegans; DNA directed RNA polymerase; chimeric proteins; embryogenesis; enzyme inhibitors; gene expression; gene induction /repression; genetic transcription; germ cells; immunofluorescence technique; in situ hybridization; invertebrate embryology; messenger RNA; nucleic acid sequence; phenotype; protein structure function; yeast two hybrid system

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.

生殖系对于物种的繁殖和永续是必不可少的；然而，关于在胚胎发育过程中指导其发育的分子机制，人们知之甚少。这项提案的长期目标是利用秀丽隐杆线虫遗传模型系统来描述这些机制。线虫特别适合于这项研究，因为在这种线虫中，可以连续跟踪生殖系从卵到成虫的发育。这一建议集中在基本生殖系因子PIE-1及其调节的两个进化保守的机制上。第一种机制涉及到胚胎生殖细胞中mRNA转录的整体抑制。我们以前的研究表明，PIE-1通过阻断早期生殖细胞的mRNA转录来保护生殖系免受体细胞转录因子的影响。我们将问以下问题：1)PIE-1是如何定位于生殖细胞谱系的？2)PIE-1中的哪些结构域抑制转录？3)转录抑制对生殖细胞命运有什么影响？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以及确定与这些结构域起作用的因素来解决。最近的技术进步使这些实验成为可能，这些技术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有的P颗粒相关的母体RNA的调节。我们已经鉴定出一个这样的RNA，nos-2，并表明在原始生殖细胞中NOS-2蛋白的表达依赖于PIE-1。NOS-2与果蝇Nanos相关，与另一种Nanos同源物NOS-1对于原始生殖细胞的发育是必不可少的。我们将确定1)NOS-2表达需要PIE-1的哪些方面的定位和结构，以及2)NOS-1和NOS-2控制生殖细胞命运的哪些方面。我们期望这些研究为基本的发育过程提供洞察力，包括蛋白质和mRNAs的不对称分离、转录抑制和生殖细胞命运的控制。线虫和脊椎动物种系之间的许多保守特征表明，在这个简单的模型系统中收集的原理将适用于其他动物，包括人类。