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

生殖细胞谱系是由小鼠胚胎原肠胚形成时的多能外胚层细胞确定的。原始生殖细胞（PGCs）的前体细胞在胚胎后端的胚外中胚层形成簇状，我们之前报道过前体细胞之间通过细胞粘附分子e -钙粘蛋白的相互作用对PGCs的最终形成至关重要。在本研究项目中，我们试图确定PGCs规范所需的基因，这些基因应该是由E-cadherin参与的信号通路激活的。在本实验中，我们使用了mil-1/GFP转基因小鼠胚胎，其中绿色荧光蛋白（GFP）在PGC前体和新生PGCs中特异性表达。我们首先从含有或不含E-cadherin阻断抗体的早期原肠胚胚片段中分离的PGC前体和新生PGC制备cDNA。我们利用这些cdna进行了差异杂交，获得了在新生PGCs中可能上调的候选基因。然而，通过原位杂交，我们没有发现这些基因在PGCs中表达的显著信号。因此，我们直接从未经培养的胚胎中获得PGC前体和新生PGCs，并进行差异杂交，成功鉴定出两个显著表达PGCs的候选基因。我们目前试图通过产生敲除小鼠来检查它们对PGC规范的可能功能。

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.