The mouse Crxos gene encodes three structurally related homeoproteins, EGAM1, EGAM1N, and EGAM1C, as transcription and splicing variants. Recently, we identified the functions of EGAM1 and EGAM1N in the regulation of differentiation in mouse embryonic stem cells. However, the function of EGAM1C remains unknown. To explore the additional roles of these proteins, the ontogenic expression of the respective mRNAs in post implantation mouse embryos and extraembryonic tissues, particularly from embryonic day (E) 10.5 to E18.5, was analyzed. The expression of Egam1n mRNA was specifically detected in embryos throughout this period, whereas that of Egam1 was undetectable in any of the tissues examined. However, in the placenta, Egam1c mRNA and its encoded protein were detected after E16.5, and these expression levels increased by E18.5 immediately before partum. Quantitative RT-PCR and in situ hybridization analyses in placentae revealed that the spatial and temporal expression patterns of the Egam1c mRNA were related to some extent with those of Prl3a1 and Prl5a1 and partially overlapped that of Prl3b1, which are members of the placental prolactin (PRL) gene family. When EGAM1C was overexpressed moderately in mouse trophoblast stem cells as a model for undifferentiated and differentiating placental cell types, the expression levels of endogenous Prl3b1 and Prl5a1 were enhanced under both undifferentiated and differentiating culture conditions. These results indicated that EGAM1C may play a role in the expression of members of the placental PRL gene family, such as Prl3b1 and Prl5a1.
The homeoprotein EGAM1C was identified in preimplantation mouse embryos and embryonic stem (ES) cells. To explore the impact of EGAM1C on the hallmarks of mouse ES cells, MG1.19 cells stably expressing EGAM1C at levels similar to those in blastocysts were established using an episomal expression system. In the presence of leukemia inhibitory factor (+LIF), control transfectants with an empty vector formed flattened cell colonies, whileEgam1ctransfectants formed compacted colonies with increased E-CADHERIN expression. InEgam1ctransfectants, the cellular contents of POU5F1 (OCT4), SOX2, TBX3, and NANOG increased. Cell growth was accelerated in an undifferentiated state sustained by LIF and in the course of differentiation. During clonal proliferation, EGAM1C stabilized the undifferentiated state. In adherent culture conditions, EGAM1C partly inhibited the progression of differentiation at least within a 4-day culture period in the presence of retinoic acid by preventing the downregulation of LIF signaling with a robust increase in TBX3 expression. Conversely, EGAM1C enhanced the expression of lineage marker genesFgf5(epiblast),T(mesoderm),Gata6(primitive endoderm), andCdx2(trophectoderm) in −LIF conditions. In embryoid bodies expressing EGAM1C, the expression of marker genes for extraembryonic cell lineages, includingTpbpa(spongiotrophoblast) andPlat(parietal endoderm), increased. These results demonstrated that the ectopic expression of EGAM1C is capable of affecting the stabilization of an undifferentiated state and the progression of differentiation in MG1.19 ES cells, in addition to affecting cellular morphology and growth.
Fibroblast growth factor 4 (FGF4) is considered as a crucial gene for the proper development of bovine embryos. However, the complete nucleotide sequences of the structural genes encoding FGF4 in identified breeds are still unknown. In the present study, direct sequencing of PCR products derived from genomic DNA samples obtained from three Japanese Black, two Japanese Shorthorn and three Holstein cattle, revealed that the nucleotide sequences of the structural gene encoding FGF4 matched completely among these eight cattle. On the other hand, differences in the nucleotide sequences, leading to substitutions, insertions or deletions of amino acid residues were detected when compared with the already reported sequence from unidentified breeds. We cannot rule out a possibility that the structural gene elucidated in the present study is widely distributed in cattle. To the best of our knowledge, this is the first determination of the complete nucleotide sequence of the structural gene encoding bovine FGF4 in identified breeds.
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