NANOG IN THE TWIN FISH MODELS MEDAKA AND ZEBRAFISH: FUNCTIONAL DIVERGENCE OR PLEIOTROPY OF VERTEBRATE PLURIPOTENCY GENE

Abstract

Pluripotency maintenance and embryo patterning are key events of early embryogenesis. Transcription factors Nanog, Oct4 and Sox2 constitute a core circuit for regulating pluripotency in mouse embryonic stem (ES) cells and early developing embryos. However, zebrafish Pou2, homolog of mammalian Oct4, is a maternal determinant for dorsoventral patterning. Nanog shows extensive sequence divergence, producing an as low as 26% identity between mammals and chicken. Whether nanog exists and plays a conserved role in pluripotency or patterning in lower vertebrates, in particular in fish, the ancient vertebrate lineage, has been unclear. This work was aimed at the identification, expression and function of nanog in the medaka and zebrafish as excellent models for analyzing pluripotency and patterning. The medaka and zebrafish nanog termed Ong and Zng respectively, encode proteins of 420 amino acids (aa, Ong) and 384 aa (Zng), which exhibit a best but 16-18% low sequence identity to tetrapod Nanog and lacks chromosomal synteny to tetrapod vertebrates. It has, however, the conserved 4-exon structure and a unique motif. The homology between fish and mouse nanog genes was established by the experiments where the mouse nanog can produce gain-of-function phenotype and rescue the loss-of-function phenotype in both fish species. In vivo, Ong is expressed throughout the pluripotency cycle, including the zygote and germline. In vitro, Ong RNA and protein are high in ES cells and down-regulated upon differentiation. Importantly, forced Ong expression supported ES cell proliferation under differentiation conditions. Upon zygotic RNA injection, Ong overexpression affected blastomeres proliferation, whereas Ong interference by dominant-negative mutants or morpholino-based knockdown compromised cell divisions and lineage commitment, leading to gastrula arrest as well as to the loss of yolk vein and tail defects. Strikingly, despite extensive sequence divergence and chromosome rearrangements, medaka nanog possesses the conserved role in pluripotency maintenance at early stages and previously unidentified roles in late stages. Zng exhibits a similar expression pattern to Ong with a salient difference: in contrast to restricted Ong expression in the central blastomeres, Zng distributes in all blastomeres. Zng knockdown led to strongly dorsalized embryos and other profound defects including eyeless phenotype, loss of yolk vein and tail defects. Zng knockdown led to severe reduction in the early zygotic expression of ventralizing genes (vox, ved and vent), dorsoventral patterning gene pou2 and neuroectodermal genes (pax2.1 and pax6.1) and to the expanded expression of the mesendodermal gene ntl (no tail). Therefore, similar to zebrafish Pou2, Zng becomes another determinant for dorsoventral patterning in early embryogenesis. In conclusion, nanog plays an essential role in pluripotency maintenance in medaka but in dorsoventral patternining in zebrafish. This striking finding provides direct evidence for functional conservation and divergence or pleiotropism of a patterning or pluripotency gene.