Nanog suppresses the expression of vasa by directly regulating nlk1 in the early zebrafish embryo.

Nanog is a homeodomain transcription factor that is essential for maintenance of pluripotency and self-renewal of embryonic stem cells (ESCs). In the present study, we demonstrate that zebrafish Nanog (zNanog) directly binds to the promoter region of zebrafish nlk1 (znlk1) by ChIP-Seq analysis and that it up-regulates the expression of znlk1 in fibroblast-like embryonic cells of Danio rerio (ZEM-2S cells) and in zebrafish embryos at 30% epiboly both at the mRNA and protein levels. In addition, compared with control (MO-C) embryos at 30% epiboly, the mRNA and protein expression of vasa and the numbers of vasa-positive cells were increased in embryos injected with zNanog morpholino (MO-zNanog). Further, injection of znlk1 mRNA into zNanog-depleted embryos restored the expression of vasa and the number of vasa-positive cells. These data indicated that zNanog up-regulates the expression of znlk1 through directly binding to the znlk1 promoter, thereby suppressing the expression of vasa. Vasa is a marker gene for PGCs. Our results suggest that zNanog plays a role in restraint of PGC cell number through regulating the expression of znlk1 in the early embryonic development. The current results provide fundamental information to support further investigation regarding the regulatory mechanism of zNanog during the development of PGCs.