Identification of Pluripotency Genes in the Fish Medaka

Abstract

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.

Klf4 is required for germ-layer differentiation and body axis patterning duringXenopusembryogenesis

“…This might be because of the divergence of gene regulation between lower and high vertebrates during evolution. Zygotic transcription of Klf4 is detected in both lower vertebrate and mammalian early embryos, but the abundance of zygotic transcripts is lower than maternal ones, as revealed in Xenopus and medaka fish (Wang et al, 2011). Ubiquitous transcription of Klf4 in early cleavage, blastula and gastrula embryos fits well with its function in germ-layer formation described in the present study.…”

“…Xenopus Klf4 is maternally expressed. In mammals, there is little maternal transcript of Klf4 according to microarray data (Hamatani et al, 2008); however, significant maternal inheritance of Klf4 is observed in medaka and zebrafish (Wang et al, 2011;Li et al, 2011;Luo et al, 2011). This might be because of the divergence of gene regulation between lower and high vertebrates during evolution.…”

nanog 5′-upstream sequence, DNA methylation, and expression in gametes and early embryo reveal striking differences between teleosts and mammals

“…The protein sequence of this gene was shown to be poorly conserved among vertebrates with the exception of the homeodomain region. In contrast to mammals, the nanog transcript was found to be maternally inherited in medaka (Camp et al, 2009;Wang et al, 2011). In addition, the specific role of Nanog in cell lineage commitment was suggested to be different between medaka and mammals.…”

Characterization ofDanio rerio Nanogand Functional Comparison toXenopus Vents

“…It was postulated that Nanog is an amniote-specific gene and is absent from anamniotes and invertebrates [25]. However, Nanog homologs have recently been reported for the teleost fish (medaka) [27,28] and for the urodele amphibian axolotl [29]. It was suggested that the mechanisms governing pluripotency might be conserved from urodele amphibians to mammals [29].…”