GENETIC ANALYSIS OF FETAL GERM CELL DEVELOPMENT

在哺乳动物胚胎中，原始生殖细胞（PGCs）首先被分配到胚胎外组织中，随后在迁移到胎儿性腺时增殖。在定植性腺后不久，生殖细胞停止分裂并进行性别特异性分化。携带关键生长因子或受体突变的小鼠表现出PGC缺陷，表明PGC发育的必要性。通过支持PGC发育的几个方面的细胞培养实验，我们发现生殖细胞增殖也可以被两个相关的配体tgf β和激活素抑制。本研究的目的是验证这些信号通路在体内控制生殖细胞增殖的假设，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增殖。我们将在体内和培养中测量缺乏激活素或tgf -受体的胚胎的生殖细胞增殖。这两种受体系统具有共同的信号转导途径，这表明胚胎中缺乏一种受体系统的生殖细胞缺陷可能由另一种受体系统在功能上补偿。因此，我们的第二个具体目标是在缺乏这两个受体系统的胚胎中测量PGC增殖，以充分暴露潜在的缺陷。或者，这些信号通路可能不是正常调节PGC积累所必需的，而只是抑制一种高度多能和迁移的细胞类型在异位位置的增殖。作为对这一假设的检验，在我们的第三个具体目标中，我们建议使用TGFbeta在体内的靶向表达来确定该配体是否可以过早地抑制PGC增殖。先前的研究表明，PGCs在体外向tgf - β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物来确定缺乏这些受体系统的PGCs是否在异位位置积聚。

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.