Cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus).

The Deleted in Azoospermia (DAZ) family of RNA binding proteins consists of highly conserved genes boule, daz and daz-like (dazl) essential for germ cell development. boule is known for its unisexual meiotic expression in invertebrates and mammals, but meiotic-specific female expression plus meiosis-preferential male expression in trout, and meiosis-preferential bisexual expression in medaka. dazl shows highly conserved bisexual expression throughout gametogenesis in diverse species. Here we report the cloning and expression of boule and dazl in the Nile tilapia (Oreochromis niloticus), an important aquaculture fish. Molecular cloning and sequence analysis led to the identification of tilapia boule and dazl cDNAs. The predicted partial Boule contains a conserved RRM motif and Dazl has the C-terminal sequence. On a phylogenetic tree, tilapia Boule and Dazl are in separate clades of Boule and Dazl homologs from other species, indicating their divergence during early vertebrate evolution. By RT-PCR analysis, boule and dazl showed bisexual gonad-specific expression. By in situ hybridization analysis, both boule and dazl RNAs were restricted to female and male germ cells of adult gonads but absent in gonadal soma. In the ovary, boule and dazl RNAs were abundant in oocytes. In the testis, boule and dazl RNAs were prominent in meiotic spermatocytes but barely detectable in meiotic products. These data show that boule and dazl are expressed bisexually in germ cells and provide useful markers to study gametogenesis in the adult tilapia.

Medaka cleavage embryos are capable of generating ES-like cell cultures.

Mammalian embryos at the blastocyst stage have three major lineages, which in culture can give rise to embryonic stem (ES) cells from the inner cell mass or epiblast, trophoblast stem cells from the trophectoderm, and primitive endoderm stem cells. None of these stem cells is totipotent, because they show gene expression profiles characteristic of their sources and usually contribute only to the lineages of their origins in chimeric embryos. It is unknown whether embryos prior to the blastocyst stage can be cultivated towards totipotent stem cell cultures. Medaka is an excellent model for stem cell research. This laboratory fish has generated diploid and even haploid ES cells from the midblastula embryo with ~2000 cells. Here we report in medaka that dispersed cells from earlier embryos can survive, proliferate and attach in culture. We show that even 32-cells embryos can be dissociated into individual cells capable of producing continuously growing ES-like cultures. Our data point to the possibility to derive stable cell culture from cleavage embryos in this organism.

Identification of pluripotency genes in the fish medaka.

Stem cell cultures can be derived directly from early developing embryos and indirectly from differentiated cells by forced expression of pluripotency transcription factors. Pluripotency genes are routinely used to characterize mammalian stem cell cultures at the molecular level. However, such genes have remained unknown in lower vertebrates. In this regard, the laboratory fish medaka is uniquely suited because it has embryonic stem (ES) cells and genome sequence data. We identified seven medaka pluripotency genes by homology search and expression in vivo and in vitro. By RT-PCR analysis, the seven genes fall into three groups of expression pattern. Group I includes nanog and oct4 showing gonad-specific expression; Group II contains sall4 and zfp281 displaying gonad-preferential expression; Group III has klf4, ronin and tcf3 exhibiting expression also in several somatic tissues apart from the gonads. The transcripts of the seven genes are maternally supplied and persist at a high level during early embryogenesis. We made use of early embryos and adult gonads to examine expression in stem cells and differentiated derivatives by in situ hybridization. Strikingly, nanog and oct4 are highly expressed in pluripotent blastomeres of 16-cell embryos. In the adult testis, nanog expression was specific to spermatogonia, the germ stem cells, whereas tcf3 expression occurred in spermatogonia and differentiated cells. Most importantly, all the seven genes are pluripotency markers in vitro, because they have high expression in undifferentiated ES cells but dramatic down-regulation upon differentiation. Therefore, these genes have conserved their pluripotency-specific expression in vitro from mammals to lower vertebrates.