Regulation of Germ Cell Fate During Embryogenesis

 描述(由申请人提供)：这个项目的长期目标是描述决定生殖系命运的机制，这是发育生物学中的一个基本问题。在所有动物中，生殖系的创始细胞(原始生殖细胞)表现出两个保守的特征：它们拥有生殖系特有的生殖粒和RNA颗粒，它们表达RNA结合蛋白Nanos家族的成员。这项提议的目标是了解生殖细胞颗粒和纳米细胞如何共同发挥作用，以确定原始生殖细胞的命运。在初步工作中，我们发现缺乏Nanos活性的PGCs过早激活了在卵母细胞和体细胞中正常表达的~1000个基因的表达，这一表型也见于缺乏Polycomb抑制复合体的突变体。具体目的我将确定Nanos用来防止这种大规模基因错误调控的机制。我们已经确定了一个有希望的候选Nanos靶点：LIN-15B，它是DRM转录因子复合体的一个可能成分，可以激活卵母细胞中的基因表达。特殊目的II将研究我们最近表征的一组新的胚芽颗粒蛋白的作用。MEG蛋白在每个胚芽颗粒的中心核心周围形成稳定的支架。我们的遗传分析表明，对PGC命运至关重要的是Megs，而不是核心中的蛋白质。我们将研究MEGs是如何与Nanos一起作用于特定的PGC的。支持这一应用的两项技术突破。首先，我们已经设计出方法来分离足够多的PGC，以便通过RNAseq来确定它们的转录组。我们将使用这种方法在全基因组水平上定义Nanos和Germ颗粒蛋白如何影响PGC转录组。第二，我们开发了一种高效的基因组编辑方法，使用CRISPR/Cas9，允许我们在短短4天内突变、标记、删除和替换任何感兴趣的基因。这种方法为我们提供了前所未有的遗传和生化途径来获取基因和蛋白质，这些基因和蛋白质是这一应用的重点。

 DESCRIPTION (provided by applicant): The long-term goal of this project is to characterize the mechanisms that specify the fate of the germline, a fundamental problem in developmental biology. In all animals, the founder cells of the germline (primordial germ cells) display two conserved characteristics: they possess germ granules, RNA granules specific to the germline, and they express members of the Nanos family of RNA-binding proteins. The goal of this proposal is to understand how germ granules and Nanos function together to specify the fate of primordial germ cells. In preliminary work, we have found that PGCs that lack Nanos activity prematurely activates the expression of ~1000 genes normally expressed in oocytes and somatic cells, a phenotype also seen in mutants that lack the Polycomb Repressive Complex. Specific Aim I will identify the mechanisms used by Nanos to prevent this massive gene mis-regulation. We have already identified one promising candidate Nanos target: LIN-15B, a likely component of the DRM transcription factor complex that activates gene expression in oocytes. Specific Aim II will examine the role of a new group of germ granule proteins we recently characterized. The MEG proteins form stabilizing scaffolds around the central cores of each germ granule. Our genetic analyses indicate that the MEGs, but not the proteins in the core, are essential for PGC fate. We will investigate how the MEGs, predicted to be intrinsically-disordered with no recognizable domain, function with Nanos to specify PGCs. Two technical breakthroughs support this application. First we have worked out methods to isolate PGCs in large enough numbers to determine their transcriptome by RNAseq. We will use this method to define how Nanos and germ granule proteins affect the PGC transcriptome at a genome-wide level. Second we have developed a highly efficient genome editing method, using CRISPR/Cas9, that allows us to mutate, tag, delete and replace any gene of interest in just 4 days. This method gives us unprecedented genetic and biochemical access to the genes and proteins that are the focus of this application.