Regulation of Germ Cell Fate During Embryogenesis

描述(申请人提供)：生殖系对于物种的繁殖和永久存在是必不可少的；然而，人们对首先将生殖细胞与所有其他胚胎细胞(体细胞)区分开来的分子机制知之甚少。这项提案的长期目标是使用秀丽隐杆线虫遗传模型系统来定义这些机制。在这种透明的蠕虫中，生殖细胞是在发育的头2小时内不对称地分裂的前体(生殖系卵裂球)产生的。这项建议侧重于三种进化上保守的机制，这些机制对于建立生殖系至关重要。 第一种机制涉及母体蛋白质不对称地分配到新生的生殖系。在之前的资助期间，我们发现CCCH指蛋白(可能的RNA结合蛋白)是一种识别CCCH指的新型E3泛素连接酶在体细胞分裂球中降解的靶点。CCCH蛋白在每次不对称分裂之前在生殖系卵裂球中变得不对称分布，我们假设这种不对称也是局部蛋白质降解的结果。我们将结合转基因研究和活胚胎的延时分析来验证这一假设。第二种机制涉及CCCH Finger Protein PIE-1对胚系卵裂球中mRNA转录的全局抑制。我们以前的工作表明，PIE-1抑制了一种使RNA聚合酶II的羧基末端结构域磷酸化的激酶。我们将通过测定PIE-1突变体和PIE-1相互作用蛋白在体内的活性来检验这一假设。第三种机制涉及原始生殖细胞中Nanos RNA的翻译激活。Nanos是原始生殖细胞发育所必需的，我们的初步研究表明，CCCH蛋白通过控制RNA的稳定性和翻译状态来调节Nanos的表达。我们将通过体内结构/功能研究来验证这一假设，以确定CCCH蛋白与Nanos 3‘UTR(3’非翻译区)之间的功能和物理相互作用。 这些研究将提供对基本发育过程的见解，包括蛋白质和RNA的不对称分配、转录抑制、翻译调控和生殖细胞命运的控制。正如我们之前的研究表明的那样，线虫和脊椎动物生殖细胞之间的许多保守特征使得这个简单模型中收集的原理很可能适用于其他动物，包括人类。

DESCRIPTION (provided by applicant): The germ line is essential for reproduction and the perpetuation of species; yet little is known about the molecular mechanisms that first distinguish germ cells from all other embryonic cells (somatic cells). The long-term goal of this proposal is to define these mechanisms using the genetic model system Caenorhabditis elegans. In this transparent worm, the germ cells arise from asymmetrically dividing precursors (germline blastomeres) during the first 2 hours of development. This proposal focuses on three evolutionarily conserved mechanisms essential for the establishment of the germline. The first mechanism involves asymmetric partitioning of maternal proteins to the nascent germline. In the previous funding period, we showed that CCCH finger proteins (putative RNA binding proteins) are targeted for degradation in somatic blastomeres by a novel E3 ubiquitin ligase, which recognizes CCCH fingers. CCCH proteins become asymmetrically distributed in germ line blastomeres before each asymmetric division, and we hypothesize that this asymmetry also results from localized protein degradation. We will test this hypothesis using a combination of transgenic studies and time-lapse analyses in live embryos. The second mechanism involves global inhibition of mRNA transcription in germline blastomeres by the CCCH finger protein PIE-1. Our previous work suggests that PIE-1 inhibits a kinase that phosphorylates the carboxy-terminal domain of RNA polymerase II. We will test this hypothesis by determining the activity in vivo of PIE-1 mutants and PIE-1 interacting proteins. The third mechanism involves translational activation of nanos RNA in primordial germ cells. Nanos is essential for primordial germ cell development, and our initial studies indicate that CCCH proteins regulate nanos expression by controlling both RNA stability and translational status. We will test this hypothesis using structure/function studies in vivo to define the functional and physical interactions connecting the CCCH proteins to the nanos 3'UTR (3' untranslated region). These studies will provide insights into basic developmental processes, including asymmetric partitioning of proteins and RNAs, transcriptional repression, translational regulation, and the control of germ cell fate. As our previous studies indicate, the many conserved characteristics between C. elegans and vertebrate germ cells make likely that principles gathered in this simple model will be applicable to other animals, including humans.