Identification of genes involved E-cadherin-dependent germ cell specification

生殖细胞系是由小鼠胚胎原肠胚形成时的多能外胚层细胞分化而来。原始生殖细胞（Primordial germ cells，PGCs）的前体细胞在胚胎后端的胚外中胚层中形成一簇，我们以前报道过前体细胞之间通过细胞粘附分子E-cadherin的相互作用对PGCs的最终特化是必不可少的。在这项研究中，我们试图确定所需的基因的PGC的规范，这应该是激活的信号通路参与E-钙粘蛋白。对于该实验，我们使用mil-1/GFP转基因小鼠的胚胎，其中绿色荧光蛋白（GFP）在PGC前体以及新生PGC中特异性表达。我们首先从PGC前体和新生PGC制备cDNA，这些前体和新生PGC分离自用或不用E-钙粘蛋白阻断抗体培养的早期原肠胚片段。我们通过使用cDNA进行差异杂交，获得了在新生PGCs中可能上调的候选基因。然而，我们没有发现明显的信号，这些基因的表达在PGCs的整体安装原位杂交。因此，我们直接从胚胎中获得PGC前体和新生PGC，并进行差异杂交，成功地鉴定了两个显示PGC显著表达的候选基因。目前，我们试图通过产生基因敲除小鼠来研究它们在PGC特异性上的可能功能。

Germ cell lineage is specified from pluripotential epiblast cells at the time of gastrulation in mouse embryos. The precursors of primordial germ cells (PGCs) form a cluster in the extraembryonic mesoderm at the posterior end of embryos and we previously reported that interaction among the precursor cells via a cell adhesion molecule, E-cadherin was essential for the final specification of PGCs. In this research project, we attempted to identify genes needed for specification of PGCs, which should be activated by a signaling pathway involved in E-cadherin. For this experiment, we used embryos of mil-1/GFP transgenic mice in which green fluorescent protein (GFP) was specifically expressed in the PGC precursors as well as nascent PGCs. We first prepared cDNA from PGC precursors and nascent PGC isolated from fragments of early gastrulating embryos cultured with or without a blocking antibody for E-cadherin. We performed differential hybridization by using the cDNAs and obtained candidate genes potentially upregulated in the nascent PGCs. However, we could not found significant signals of the expression of those genes in PGCs by whole mount in situ hybridization. Therefore, we then directly obtained PGC precursors and nascent PGCs form the embryos without culture and performed differential hybridization and successfully identified two candidate genes showing significant expression of PGCs. We currently attempt to examine their possible functions on PGC specification by generating knock-out mice.