Regulation of Germ Cell Fate During Embryogenesis

描述（由申请人提供）：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。这项建议的长期目标是使用遗传模型系统秀丽隐杆线虫来定义这些机制。在这种透明的蠕虫中，生殖细胞在发育的前2小时内由不对称分裂的前体（生殖细胞卵裂球）产生。这个建议集中在三个进化上保守的机制至关重要的生殖系的建立。 第一种机制涉及母体蛋白质向新生生殖系的不对称分配。在上一个资助期，我们发现CCCH指蛋白（推定的RNA结合蛋白）是一种新型E3泛素连接酶在体细胞卵裂球中降解的靶向蛋白，该酶可识别CCCH指。CCCH蛋白在每次不对称分裂前在生殖系卵裂球中呈不对称分布，我们推测这种不对称性也是由局部蛋白质降解引起的。我们将使用转基因研究和活胚胎中的延时分析相结合来验证这一假设。第二种机制涉及CCCH指蛋白PIE-1对生殖系卵裂球中mRNA转录的全面抑制。我们以前的工作表明，PIE-1抑制磷酸化RNA聚合酶II的羧基末端结构域的激酶。我们将通过测定PIE-1突变体和PIE-1相互作用蛋白的体内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对原始生殖细胞发育至关重要，我们的初步研究表明CCCH蛋白通过控制RNA稳定性和翻译状态来调节nanos的表达。我们将使用体内结构/功能研究来验证这一假设，以确定将CCCH蛋白连接到纳米3 'UTR（3'非翻译区）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深入了解，包括蛋白质和RNA的不对称分配、转录抑制、翻译调节和生殖细胞命运的控制。如我们以前的研究所示，C.线虫和脊椎动物的生殖细胞使得在这个简单模型中收集的原理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.