Role of Translational Regulators Nanos and Dazl in Preserving Totipotency

描述（由申请人提供）：在后生动物的早期发育中，从体细胞命运中排除生殖细胞是确保物种延续的必要过程。原始生殖细胞（PGCs）至少执行四种必需的活动，以保护它们免受体细胞分化并启动它们自己独特的基因表达程序：1)激活隔离的母种系mrna；2)母体体细胞信息的抑制；3)短暂的全基因组转录抑制，以确保胚胎其他部分的合子转录启动时，体细胞程序不会被激活；4)母体体细胞mrna降解后pgc特异性基因的转录激活。所有这些活动都发生在没有转录的情况下，因此必须主要在翻译水平上进行调节。该项目的目标是确定这个复杂程序中的关键参与者，揭示其活动的机械细节，并为保护和指定种系的网络构建一个可行的工作模型。rna结合蛋白Nanos和Dazl是多种物种（包括青蛙和人类）生殖系中的翻译调节因子。两者在PGCs中都通过一种未知的机制被翻译激活。Nanos和Dazl都与另一种（序列特异性）rna结合蛋白Pumilio （Pum）相互作用来调节翻译，但作用相反：Nanos抑制目标rna的翻译，而Dazl促进其翻译。我们在非洲爪蟾的研究表明，缺乏Nanos的PGCs过早启动Pol II转录，不适当地表达体细胞基因，并且无法存活。缺乏dazl的爪蟾PGCs不能迁移到原始性腺，并从种系中丢失。接下来的关键问题是，是什么激活了Nanos和dazl，以及它们的目标mrna的身份。初步研究支持rna结合蛋白Dead-end作为Nanos翻译激活剂的新作用。我们的工作模型是，生殖系的保存是由Dnd启动的，Dnd激活Nanos和可能包括dazl在内的其他生殖系mrna的翻译。然后，Nanos抑制体细胞命运所必需的母体mrna的翻译，而Dazl促进激活pgc特异性性状的rna的翻译。该项目的目的是：1)定义导致纳米和其他潜在RNA靶标的翻译激活的Dnd/RNA相互作用；相关性的最终测试将是在一个确定的体外系统中重建Nanos翻译，其中包含任何所需的蛋白质伴侣和Dnd；2)鉴定需要Nanos/Pum抑制的母体rna，以防止体细胞rna的异常表达；使用一种新的检测PGCs翻译调控的方法，我们将验证Nanos/Pum/RNA在体内的相互作用；3)鉴定PGC鉴定需要Dazl激活的母体rna；与Dazl共免疫沉淀的候选mrna将通过评估其在缺失Dazl的胚胎中恢复PGC特征的能力进行体内验证。有了这些信息，我们期望能够构建一个相对详细的工作模型，用于在发育的早期阶段调节种系命运。

DESCRIPTION (provided by applicant): The exclusion of germ cells from somatic cell fates in early development is an essential process in metazoans that ensures continuation of the species. Primordial germ cells (PGCs) execute at least four activities that are required to both protect them from somatic differentiation and to initiate their own unique gene expression programs: 1) activation of sequestered maternal germline mRNAs; 2) repression of maternal somatic messages; 3) transient genome-wide suppression of transcription to ensure that somatic programs are not activated when zygotic transcription initiates in the rest of the embryo; and 4) transcriptional activation of PGC-specific genes after degradation of maternal somatic mRNAs. All of these activities occur in the absence of transcription and therefore must be regulated primarily at the level of translation. The goal of the proposed project is to define key players in this intricate program, to uncover mechanistic details of their activities, and to construct a viable working model for the network that protects and specifies the germline. The RNA-binding proteins Nanos and Dazl are translational regulators in the germlines of diverse species, including frogs and humans. Both are translationally activated in PGCs by an unknown mechanism. Nanos and Dazl both interact with another (sequence-specific) RNA-binding protein, Pumilio (Pum), to regulate translation, but with opposite effects: Nanos represses translation of target RNAs, while Dazl promotes it. Our work in Xenopus has shown that PGCs lacking Nanos prematurely initiate Pol II transcription, inappropriately express somatic genes, and do not survive. Xenopus PGCs deficient in dazl fail to migrate to the primordial gonads and are lost from the germline. Key questions then are what activates Nanos and dazl, and the identities of their target mRNAs. Preliminary studies support a new role for the RNA-binding protein, Dead-end, as a translational activator of Nanos. Our working model is that preservation of the germline is initiated by Dnd, which activates the translation of Nanos and probably other germline mRNAs including dazl. Nanos then represses translation of maternal mRNAs essential for somatic fates, while Dazl promotes translation of RNAs that activate PGC-specific traits. The aims of this project are: 1) to define the Dnd/RNA interactions that result in translational activation of Nanos and other potential RNA targets; the definitive test for relevance will be to reconstitute Nanos translation in a defined in vitro system with any required protein partners and Dnd; 2) to identify the maternal RNAs whose repression by Nanos/Pum is required to prevent aberrant expression of somatic RNAs; using a novel assay for translational regulation in PGCs, we will authenticate Nanos/Pum/RNA interactions in vivo; 3) to identify the maternal RNAs whose activation by Dazl is required for PGC identity; candidate mRNAs that co-immunoprecipitate with Dazl will be validated in vivo by assessing their abilities to restore PGC traits in Dazl-depleted embryos. With this information, we expect to be able to construct a relatively detailed working model for the regulation of germ-line fate in the earliest stages of development.