Regulation of Germ Cell Fate During Embryogenesis

描述（由申请人提供）：该项目的长期目标是描述生殖细胞和体细胞的区别机制，这是发育生物学中的一个基本问题。在秀丽隐杆线虫中，在第一次分裂之前，通过细胞质中蛋白质和蛋白质/RNA复合物的不对称分配，在合子中建立了体细胞-种系不对称。细胞质分配由保守极性调节因子（PAR蛋白）调控，该蛋白不对称地定位于受精卵皮层。这项提议的目标是揭示PAR在皮层的活动如何调节细胞质的极性。特异性目的1 （SA1）将重点关注rna结合蛋白MEX-5分离到前细胞质的机制。初步数据表明，MEX-5的不对称性取决于PAR-1的磷酸化，PAR-1是后皮层上的一种激酶，它增加了MEX-5在后细胞质中的局部扩散。SA2将集中于种系决定因子PIE-1，其分离到后部，与MEX-5相对，且梯度更陡。我们将研究PIE-1如何整合皮质和细胞质线索以形成不同的梯度。最后，SA3将聚焦于将P颗粒定位于后部的机制。P颗粒是进化上保守的rna -蛋白复合物，特异于种系。我们发现了一种新的P颗粒成分PPTR-1，用于有丝分裂过程中P颗粒的完整性；我们的初步研究结果表明，P颗粒的种系特异性取决于受调节的组装，而不需要细胞质分配。我们将使用生物化学，遗传和活体显微镜方法的组合来测试这些假设，并确定所涉及的基因和生物化学相互作用。与其他研究充分的胚胎极性模型不同，我们的系统不依赖于预定位的rna来产生不对称，从而为我们探索细胞质中直接分离蛋白质的机制提供了独特的机会。

DESCRIPTION (provided by applicant): The long-term goal of this project is to characterize the mechanisms that distinguish germ cells from somatic cells, a fundamental problem in developmental biology. In C. elegans, soma- germline asymmetries are established in the zygote before the first division, through the asymmetric partitioning of proteins and protein/RNA complexes in the cytoplasm. Cytoplasmic partitioning is regulated by conserved polarity regulators (PAR proteins), which localize asymmetrically in the zygote cortex. The goal of this proposal is to uncover how PAR activity at the cortex regulates polarity in the cytoplasm. Specific Aim I (SA1) will focus on the mechanisms that segregate the RNA-binding protein MEX-5 to the anterior cytoplasm. Preliminary data suggest that MEX-5 asymmetry depends on phosphorylation by PAR-1, a kinase on the posterior cortex, which increases MEX-5 diffusion locally in the posterior cytoplasm. SA2 will focus on the germline determinant PIE-1, which segregate to the posterior, opposite MEX-5 and in a steeper gradient. We will investigate how PIE-1 integrates both cortical and cytoplasmic cues to form a distinct gradient. Finally, SA3 will focus on the mechanisms that localize P granules to the posterior. P granules are evolutionarily conserved RNA-protein complexes specific to the germline. We have discovered a new P granule component PPTR-1 for P granule integrity during mitosis; our initial findings suggest that the germline specificity of P granules depends on regulated assembly and does not require cytoplasmic partitioning. We will use a combination of biochemical, genetic, and live microscopy approaches to test each of these hypotheses, and identify the genes and biochemical interactions involved. Unlike other well-studied embryonic polarity models, our system does not rely on pre-localized RNAs to generate asymmetry, and thus gives us the unique opportunity to explore the mechanisms that directly segregate proteins in the cytoplasm. PUBLIC HEALTH RELEVANCE: Cell polarity is essential to generate cell diversity during development, to prevent uncontrolled cell growth, and for the every-day functioning of many polarized cell types (epithelial cells, neurons, and lymphocytes). By taking advantage of a simple model system, our studies will illuminate the mechanisms that build and maintain the architecture of many cell types and prevent tumor formation.