GENETIC ANALYSIS OF FETAL GERM CELL DEVELOPMENT

在哺乳动物胚胎中，原始生殖细胞(PGC)首先被分配到胚外组织中，然后在迁移到胎儿性腺的同时增殖。在性腺定植后不久，生殖细胞停止分裂，并经历性别特异性分化。携带关键生长因子或受体突变的小鼠表现出PGC缺陷，证明了躯体因素对PGC发育的必要性。使用支持PGC发育的多个方面的细胞培养实验，我们发现生殖细胞的增殖也可以被两个相关的配体，TGFβ和激活素所抑制。这一建议的目的是验证这样的假设，即这些信号通路在体内控制着PGCs的增殖，从而有助于确定每个胚胎的最终生殖细胞数量。我们的第一个具体目标是确定这些信号通路是否单独调节PGC的增殖。我们将测量体内和培养中缺乏激活素或转化生长因子β受体的胚胎的生殖细胞增殖。这两个受体系统共享一个共同的信号转导途径，这表明缺乏一个受体系统的胚胎中的生殖细胞缺陷可能会被另一个系统补偿。因此，我们的第二个特定目标是测量缺乏这两个受体系统的胚胎中PGC的增殖，以充分揭示潜在的缺陷。或者，这些信号通路可能不是PGC积累的正常调节所必需的，而只是用来抑制异位位置的高度多能性和迁移细胞类型的增殖。作为对这一假设的检验，在我们的第三个特定目标中，我们建议使用体内靶向表达TGFbeta来确定该配体是否可以过早地抑制PGC的增殖。先前的研究表明，PGCs在体外向TGFbeta的来源迁移，这表明这些信号通路是引导PGCs进入胎儿性腺的神秘过程的一部分。我们的第四个具体目标是利用细胞自主生殖细胞标记来确定缺乏这些受体系统的PGCs是否在异位位置聚集。

In mammalian embryos, primordial germ cells (PGCs) are first allocated into extraembryonic tissues and subsequently proliferate while migrating to the fetal gonad. Shortly after colonizing the gonad, germ cells cease dividing and undergo sex specific differentiation. Mice harboring mutations in key growth factors or receptors exhibit PGC defects, demonstrating the necessity of somatic factors for PGC development. Using a cell culture assay which supports several aspects of PGC development, we found that germ cell proliferation can also be inhibited by two related ligands, TGFbeta and activin. The goal of this proposal is to test the hypothesis that these signaling pathways control the proliferation of PGCs in vivo, and thereby contribute to determining the final number of germ cells per embryo. Our first specific aim is to determine whether these signaling pathways individually regulate PGC proliferation. We will measure germ cell proliferation both in vivo and in culture from embryos lacking either activin or TGFbeta receptors. These two receptor systems share a common signal transduction pathway, suggesting that germ cell defects in embryos lacking one receptor system may be functionally compensated by the other system. Therefore, our second specific aim to measure PGC proliferation in embryos lacking both receptors systems in order to fully expose potential defects. Alternatively, these signaling pathways may not be essential for the normal regulation of PGC accumulation, but serve only to inhibit the proliferation of a highly multipotent and migratory cell type in ectopic locations. As a test of this hypothesis, in our third specific aim we propose to use targeted expression of TGFbeta in vivo to determine whether this ligand can prematurely inhibit PGC proliferation. Previous work has shown that PGCs migrate toward a source of TGFbeta in vitro, suggesting that these signaling pathways are part of the mysterious process that guide PGCs to the fetal gonad. Our fourth specific aim is to take advantage of a cell autonomous germ cell marker to determine whether PGCs lacking these receptor systems accumulate in ectopic locations.