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

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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.