Trophectoderm (TE), the first differentiated cell lineage of mammalian embryogenesis, forms the placenta, a structure unique to mammalian development. The differentiation of TE is a hallmark event in early mammalian development, but molecular mechanisms underlying this first differentiation event remain obscure. Embryonic stem (ES) cells can be induced to differentiate into the TE lineage by forced repression of the POU-family transcription factor, Oct3/4. We show here that this event can be mimicked by overexpression of Caudal-related homeobox 2 (Cdx2), which is sufficient to generate proper trophoblast stem (TS) cells. Cdx2 is dispensable for trophectoderm differentiation induced by Oct3/4 repression but essential for TS cell self-renewal. In preimplantation embryos, Cdx2 is initially coexpressed with Oct3/4 and they form a complex for the reciprocal repression of their target genes in ES cells. This suggests that reciprocal inhibition between lineage-specific transcription factors might be involved in the first differentiation event of mammalian development.

The pluripotency of embryonic stem (ES) cells is thought to be maintained by a few key transcription factors, including Oct3/4 and Sox2. The function of Oct3/4 in ES cells has been extensively characterized, but that of Sox2 has yet to be determined. Sox2 can act synergistically with Oct3/4 in vitro to activate Oct-Sox enhancers, which regulate the expression of pluripotent stem cell-specific genes, including Nanog, Oct3/4 and Sox2 itself. These findings suggest that Sox2 is required by ES cells for its Oct-Sox enhancer activity. Using inducible Sox2-null mouse ES cells, we show that Sox2 is dispensable for the activation of these Oct-Sox enhancers. In contrast, we demonstrate that Sox2 is necessary for regulating multiple transcription factors that affect Oct3/4 expression and that the forced expression of Oct3/4 rescues the pluripotency of Sox2-null ES cells. These results indicate that the essential function of Sox2 is to stabilize ES cells in a pluripotent state by maintaining the requisite level of Oct3/4 expression.

Knock-in embryonic stem (ES) cells, in which GFP or lacZ was expressed from the endogenous mouse vasa homolog (Mvh), which is specifically expressed in differentiating germ cells, were used to visualize germ cell production during in vitro differentiation. The appearance of MVH-positive germ cells depended on embryoid body formation and was greatly enhanced by the inductive effects of bone morphogenic protein 4-producing cells. The ES-derived MVH-positive cells could participate in spermatogenesis when transplanted into reconstituted testicular tubules, demonstrating that ES cells can produce functional germ cells in vitro. In vitro germ cell differentiation provides a paradigm for studying the molecular basis of germ line establishment, as well as for developing new approaches to reproductive engineering. G erm-line cells are responsible for transmitting genetic information and for reproducing totipotency from generation to generation. Pluripotent stem cell lines, embryonic stem (ES) cells, and embryonic germ cells are established from cells of the germ cell lineage. Therefore, germ cell specification must be linked to the maintenance of pluripotency, as well as to cell fate commitment leading to gametogenesis.Unlike many animal species, in which the germ line is predetermined by maternal factors, germ cell specification in mammals takes place at the onset of gastrulation, after implantation of the embryo. In the mouse, primordial germ cells (PGCs) are first distinguished at the base of the allantois in gastrulating embryos at embryonic day (E) 7.25 (1). Lineage studies of epiblast cells show that mouse PGCs are specified by inductive interactions at the onset of gastrulation (2, 3). Genetic analyses using targeted mutations have revealed that bone morphogenic protein (BMP) 4 and -8b, soluble growth factors belonging to the transforming growth factor ␤ superfamily that are produced by extraembryonic ectoderm close to the boundary with the proximal epiblast, are required for the generation of PGCs from epiblast cells (4,5). Moreover, primary cultures of epiblast fragments from embryos at E5.5-E6.0 generate migrating PGCs when they are cocultured with extraembryonic ectoderm (6), and culturing of whole epiblasts from E6.0 embryos on feeder cells expressing both BMP4 and BMP8b gives rise to PGCs (7). These results reveal that BMPs derived from the extraembryonic ectoderm play crucial roles in PGC determination in the proximal epiblast.Despite such developments, it is not yet known how the founder population of PGCs is segregated from other pluripotent epiblast cells that form somatic cells. To approach this question, we examined the production of germ cells by an established pluripotent ES cell line. ES cells can form all cell lineages when introduced into host blastocysts and give rise to various somatic cell lineages in culture. However, it is not known whether they can generate the germ cell lineage in culture in the absence of the morphogenetic events associated with gastrulation. It has been difficult to addr...

+ cells differentiated into cells of the somatic lineage more efficiently than non-fractionated ES cells in vitro and showed poor ability to contribute to chimera formation. These results confirmed that undifferentiated ES cell culture contains subpopulations corresponding to ICM, epiblast and PrE.KEY WORDS: ES cell, Reversibility, Subpopulation, Rex1 (Zfp42), Oct3/4 (Pou5f1), Mouse Development 135, 909-918 (2008)

To demonstrate the cellular and subcellular localization of mouse vasa homologue protein during germ cell development, specific antibody was raised against the full-length MVH protein. The immunohistochemical analyses demonstrated that MVH protein was exclusively expressed in primordial germ cells just after their colonization of embryonic gonads and in germ cells undergoing gametogenic processes until the post-meiotic stage in both males and females. The co-culture of EG cells with gonadal somatic cells indicated inductive MVH expression caused by an intercellular interaction with gonadal somatic cells. In adult testis, MVH protein was localized in the cytoplasm of spermatogenic cells, including chromatoid bodies in spermatids, known to be a perinuclear nuage structure which includes polar granules that contain VASA protein in Drosophila.