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The homeodomain transcription factor Nanog plays crucial roles in the embryonic/gonadal development and maintenance of embryonic stem cells (ESCs), interacting with transcription factors such as Oct4 and Sox2 in mammals. Nevertheless, knowledge of its exact pathways to molecular mechanism remains weak in teleosts, and unexploited in Pearlscale angel sh Centropyge vrolikii. The role of Nanog in the gonadal development and sex reversal of C. vrolikii is worth focusing. In this study, we isolated and characterized a Nanog homolog in C. vrolikii named as Cv-Nanog to understand the expression pattern of the gonadal development. The full-length cDNA sequence of Cv-Nanog was 2,136 bp in length and encodes a homeodomain protein of 436 amino acid residues. The analysis of phylogeny, the gene structure and western blot provided the evidence that Cv-Nanog was homologous to Nanog gene of mammalian. The protein sequence comparison demonstrated that Cv-Nanog shared the high similarity with orthologs of other vertebrates in the conserved homeodomain. The analysis of quantitative real-time PCR (qRT-PCR) and western blot showed that Cv-Nanog was maternally expressed detectable merely in gonads, and the expression was signi cantly high in ovaries than in testis. In situ hybridization revealed that the transcripts were located in the cytoplasm and membrane of the oocytes in ovaries, and in the testis, Cv-Nanog mRNA expression was weakly expressed in sertoli cells, but highly expressed in germ cells. After overexpression of Cv-Nanog, the expression levels of pluripotent factors Sox2 and Oct4 increased signi cantly with 21.5-fold and 12.2-fold, respectively. At the same time, the TGF-beta signaling pathway was activated, and the gonadal cells' growth was promoted, and the expression of ovary-bias genes Cyp19a and Foxl2 was up-regulated, the expression of testis-bias genes Sox9 and Dmrt1 was down-regulated, and nally the ovarian development was promoted. These results implied that Nanog gene may play a crucial role in the process of gonadal development and sexual reversion in C. vrolikii. This study is helpful to further understand the molecular regulatory mechanism of Nanog, also provides important clues for the future studies in gonadal development. HighlightsThe Cv-Nanog was signi cantly expressed in the gonads.The expression level of Cv-Nanog in the ovaries was signi cantly higher than that in the testis.The functional domain of Cv-Nanog was highly conserved among teleosts.In situ hybridization revealed that the transcript was located in the germ cells in the gonads.The TGF-beta signaling pathway was activated, and the gonadal cells' growth was promoted after overexpression Cv-Nanog in the gonads.

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