Cloning Of Zebrafish Ca15b And Nanog-like CDNA And Its Expression In Primordial Germ Cells

Primordial germ cells （PGCs） are the progenitor cells of the gametes. During the early embryo development, PGCs migrate into the genital ridge through the accurate pathways, and then differentiate into female or male gametes. Migration and proliferation are the two essential events in PGCs development, which are the ideal model for the research on the mechanism of cell migration in vivo. In this study, using zebrafish as model, the expression patterns of CA15b and nanog-like were detected in the gonads and early embryo development. The results strongly indicate that these two genes are involved in the PGCs proliferention and development.Carbonic Anhydrase （CA） is the metalloznzyme, which catalyzes the reversible reaction of carbon dioxide and water to bicarbonate and a proton. The main function of CA is to regulat the acid-base in organisms. CAs are classified in five （α,β, γ,δ and ξ） genetically unrelated subfamilies, and the a-CA is the mainly class in the vertebrates. Sixteen members of the a-CA gene family have been identified in mammals. CAs are also existed in fish. In addition to their sequences data, little is known of their expression patterns and functions. In the present study, zebrafish CA15b sequence was identifed from the NCBI database （Accession No. NM₂13182）. The1716bp full-length cDNA of CA15b was obtained by3’and5’-RACE. The CA15b genomic sequence is located on zebrafish chromosome12, containing nine exons and eight introns. The zebrafish CA15b encodes a305amino acid residue protein, and contains a conserved alpha_CA_ⅣⅩⅤ_like domain. Multiple alignments showed that the zebrafish CA XVb protein shares52%,53%,36%,28%and28%identity with zebrafish CA XVa, zebrafish CA XVc, Xenopus CA XV, rat CA XV and mouse CA XV, respectively. RT-PCR analysis showed its expression in the ovaries, heart, brain and muscle, but not in the testis or liver. The results of in situ hybridization and immunohistochemistry analysis demonstrated that CA15b mRNA is transcribed in the ooplasm of stage Ⅰ to Ⅱ oocytes and is localized in the cortex of stage Ⅲ oocytes, but is not expressed in stage Ⅳ oocytes; the CA XVb protein is localized in the plasma membrane of oocytes. Based on whole-mount in situ hybridization, CA15b mRNA was found to be distributed in every blastomere of embryos from the1-cell stage to the blastula stage. Interestingly, strong signals of the transcripts were seen along the cleavage furrows of the2-and8-cell stage embryos, which then condensed into four clusters of cells in the blastula stage. During70%epiboly stages, the CA15b mRNA was existed in some cells which were moving towards the dorsal side of the embryos. In90%epiboly stages, the CA15b mRNA was located on either side of the midline, and was accumulated in the caudal portion of the24hours post-fertilization embryos. The expression patterns of CA15b mRNA are similar to those of vasa. Thus, we hypothesised that CA15b is critical in the development of PGCs and oocytes in zebrafish.NANOG is a transcriptional factor, which plays important roles to maintain the pluripotency in embryonic stem cells. In this study,1890bp full-length cDNA of zebrafish nanog-like was obtained by RACE analysis. By performing BLAST in NCBI zebrafish genomic sequence database, nanog-like was found on zebrafish chromosome24, containing four exons and three introns. The zebrafish nanog-like encodes a384amino acid residue protein, and contains a homeodomain. Multiple alignments showed that the zebrafish NANOG-like protein shares38%,51%,37%,49%and48%identity with the NANOG of medaka, chicken, rat, mouse and human, respectively. When restricted to the homeodomain, the identity rose to81%,63%,55%,52%and53%, compared to the species listed above. RT-PCR analysis showed that the nanog-like mRNA is expressed in ovaries, testis, liver and heart, but not in brain or muscle. The results of in situ hybridization, immunohistochemistry and fluorescence immunohistochemistry analysis demonstrated that nanog-like mRNA and protein are expressed in the stage Ⅰ and Ⅱ oocytes, but the expression level of the nanog-like mRNA is lower in the stage Ⅲ and Ⅳ oocytes. And the NANOG-like protein is located at the cortex in the stage Ⅲ and Ⅳ oocytes. The nanog-like mRNA and protein are also expressed in the spermatogonium. During the early embryo development, nanog-like mRNA is existed in every blastomere of embryos from the4-cell stage to the early gastrula stage, espeacially in the PGCs of the late blastula stage embryos. The double fluorescence immunohistochemistry analysis demonstrated that NANOG-like protein is also expressed in the PGCs of the late blastula stage embryos. A zebrafish NANOG-like protein binding DNA sequence was identified by protein-DNA binding experiment. We suggested that the nanog-like may involved in the development of germ cells and the proliferation of PGCs.