Regulation of Germ Cell Fate During Embryogenesis

 描述（由申请人提供）：该项目的长期目标是表征指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）显示出两个保守的特征：它们具有生殖颗粒，生殖系特异性的RNA颗粒，并且它们表达RNA结合蛋白的Nanos家族成员。该提案的目标是了解生殖颗粒和Nanos如何共同作用以指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过早地激活了通常在卵母细胞和体细胞中表达的约1000个基因的表达，这种表型也见于缺乏Polycomb抑制复合物的突变体中。具体目标我将确定Nanos用于防止这种大规模基因错误调节的机制。我们已经确定了一个有希望的候选Nanos靶点：LIN-15 B，它可能是DRM转录因子复合物的一个成分，可以激活卵母细胞中的基因表达。具体目标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，MEG，而不是核心中的蛋白质，是PGC命运所必需的。我们将研究如何MEG，预测是本质上无序的，没有可识别的结构域，功能与纳米指定PGC。两项技术突破支持了这一应用。首先，我们已经制定了分离足够数量的PGC的方法，以通过RNAseq确定它们的转录组。我们将使用这种方法来定义Nanos和胚芽颗粒蛋白如何在全基因组水平上影响PGC转录组。其次，我们开发了一种高效的基因组编辑方法，使用CRISPR/Cas9，使我们能够在短短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基因和生物化学途径，这些基因和蛋白质是这种应用的重点。

 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.