nanog 5'-upstream sequence, DNA methylation, and expression in gametes and early embryo reveal striking differences between teleosts and mammals.

The nanog gene plays a major role in vertebrate development and was only recently discovered in teleosts. In order to gain new insight into its regulation in gametes and early embryo in teleost fish, the present study aimed at characterizing nanog upstream sequence features and DNA methylation, as well as early embryonic expression pattern in a Cyprinid fish, the goldfish. Using an in silico approach, we were able to demonstrate that despite the existence of conserved regulatory motifs in the promoter region of the nanog gene, specific features known to play a major role in the regulation of Nanog in mammals were missing in teleosts. The analysis of CpG sites in the upstream region of the nanog genes in goldfish revealed a significant DNA methylation state in oocytes while a hypomethylated state was observed in sperm. Using both quantitative PCR and whole mount in situ hybridization, we were able to clearly demonstrate the maternal inheritance of the nanog transcript in goldfish. Corresponding mRNA levels subsequently decreased during early gastrulation. Together, our results reveal striking differences in expression and DNA methylation patterns in gametes and during early development and in upstream region features between teleosts and mammals that are consistent with the hypothesis of a rapid evolution of the Nanog gene in vertebrates, at least in some lineages.