Characterization of the carbonic anhydrases 15b expressed in PGCs during early zebrafish development

Abstract

The main function of carbonic anhydrases is to regulate acid-base balance. In the present study, the zebrafish CA15b sequence was identified from the National Center for Biotechnology Information database (accession No. NM_213182). The 1716 base pair full-length cDNA of CA15b was obtained by 3' and 5'-rapid amplification of cDNA ends analysis. It was expressed (reverse transcription polymerase chain reaction and Western blot analysis) in the ovary, heart, brain, and muscle, but not in testis or liver. Based on in situ hybridization, CA15b mRNA was transcribed in the ooplasm of stage I to stage II oocytes, in the cortex of stage III oocytes, and along the periphery of stage IV oocytes. Furthermore, this protein was localized (immunohistochemistry) in the plasma membrane of oocytes. Based on whole-mount in situ hybridization, CA15b mRNA was present in every blastomere of embryos from one-cell to blastula stages. Strong signals of the transcripts were present along cleavage furrows of two- and eight-cell stage embryos, which subsequently condensed into four clusters of cells during the blastula stage. During subsequent stages, the four groups of CA15b-expressing cells appeared to move toward the dorsal side of the embryos, clustered into two groups on either side of the midline, and remained visible as they migrated toward the region of the gonad in embryos at 24 hours postfertilization. Expression patterns of CA15b were similar to those of vasa, a marker of primordial germ cells. Thus, we hypothesized that CA15b might be necessary for development of primordial germ cells and female germ cells in zebrafish.