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The recent availability in culture of embryo-derived pluripotential cells which exhibit both a normal karyotype and a high differentiative ability has encouraged us to assess the potential of these cells to form functional germ cells following their incorporation into chimaeric mice. We report here the results of blastocyst injection studies using three independently isolated XY embryo-derived cell lines (EK. CP1 , EK. CC1 .1 and EKCC1 .2) which produce a very high proportion (greater than 50%) of live-born animals that are overtly chimaeric. Seven chimaeric male mice, derived from these three lines, have, so far, proved to be functional germ-line chimaeras.

The earliest cell fate decision in the mammalian embryo separates the extra-embryonic trophoblast lineage, which forms the fetal portion of the placenta, from the embryonic cell lineages. The body plan of the embryo proper is established only later at gastrulation, when the pluripotent epiblast gives rise to the germ layers ectoderm, mesoderm and endoderm. Here we show that the T-box gene Eomesodermin performs essential functions in both trophoblast development and gastrulation. Mouse embryos lacking Eomesodermin arrest at the blastocyst stage. Mutant trophoectoderm does not differentiate into trophoblast, indicating that Eomesodermin may be required for the development of trophoblast stem cells. In the embryo proper, Eomesodermin is essential for mesoderm formation. Although the specification of the anterior-posterior axis and the initial response to mesoderm-inducing signals is intact in mutant epiblasts, the prospective mesodermal cells are not recruited into the primitive streak. Our results indicate that Eomesodermin defines a conserved molecular pathway controlling the morphogenetic movements of germ layer formation and has acquired a new function in mammals in the differentiation of trophoblast.

Abnormalities precipitated by a targeted truncation in the murine gene Brca2 define its involvement in DNA repair. In culture, cells harboring truncated Brca2 exhibit a proliferative impediment that worsens with successive passages. Arrest in the G1 and G2/M phases is accompanied by elevated p53 and p21 expression. Increased sensitivity to genotoxic agents, particularly ultraviolet light and methylmethanesulfonate, shows that Brca2 function is essential for the ability to survive DNA damage. But checkpoint activation and apoptotic mechanisms are largely unaffected, thereby implicating Brca2 in repair. This is substantiated by the spontaneous accumulation of chromosomal abnormalities, including breaks and aberrant chromatid exchanges. These findings define a function of Brca2 in DNA repair, whose loss precipitates replicative failure, mutagen sensitivity, and genetic instability reminiscent of Bloom syndrome and Fanconi anemia.

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Embryonic stem cells isolated directly from mouse embryos can be cultured for long periods in vitro and subsequently repopulate the germ line in chimaeric mice. During the culture period these embryonic cells are accessible for experimental genetic manipulation. Here we report the use of retroviral vectors to introduce exogenous DNA sequences into a stem-cell line and show that these modified cells contribute extensively to the somatic and germ-cell lineages in chimaeric mice. Compared with current methods for manipulation of the mouse genome, this approach has the advantage that powerful somatic-cell genetic techniques can be used to modify and to select cells with germ-line potential, allowing the derivation of transgenic strains with pre-determined genetic changes. We have by this means inserted many proviral vector sequences that provide new chromosomal molecular markers for linkage studies in the mouse and that also may cause insertional mutations.

The c-mos proto-oncogene encodes a 37-39K cytoplasmic serine/threonine kinase implicated in the meiotic maturation events during murine spermatogenesis and oogenesis. In Xenopus, ectopic expression of pp39mos can promote both the meiotic maturation of oocytes and also arrest the cleavage of blastomeres. To elucidate the role of pp39mos we have generated homozygous mutant mice by gene targeting in embryonic stem cells. These mice are viable and mutant males are fertile, demonstrating that pp39mos is not essential for spermatogenesis. In contrast, mutant females, have a reduced fertility because of the failure of mature eggs to arrest during meiosis. c-mos-/- oocytes undergo germinal vesicle breakdown and extrusion of both polar bodies followed in some cases by progression into cleavage. Mutant females also develop ovarian cysts. These results demonstrate that a major role for pp39mos is to prevent the spontaneous parthenogenetic activation of unfertilized eggs.

The differentiation ir vitro of clonal pluripotent teratocarcinoma cells is reported. The first stage of this process is the formation of simple embryoid bodies which are identical to those found in animals bearing intraperitoneal teratocarcinomas. They consist of an inner core of embryonal earcinoma cells surrounded by a layer of endodermal cells which produce Reichert's membrane. The endodermal cells become apparent shortly after the embryonal carcinoma cells have formed aggregates which are loosely attached to the substratum. One clonal teratocarcinoma line was found to produce complex cystic embryoid bodies in vitro. Following formation of the endodermal cells, extensive differentiation to a wide variety of cell types occurs. There are similarities between the process of embryoid body formation and the early events of differentiation of the mouse embryo.Mouse teratocarcinomas are a useful alternative to embryos for the study of mammalian cell determination (the process by which multipotential cells become committed to a particular developmental pathway), as well as for the study of subsequent terminal differentiation. The stem cells of these tumors, known as embryonal carcinoma cells, are pluripotent: like the cells of the early embryo, they can differentiate to form derivatives of all three primary germ layers. Kleinsmith and Pierce (1) first demonstrated this by showing that a single embryonal carcinoma cell injected intraperitoneally could give rise to a teratocarcinoma containing a wide variety of differentiated tissues. Embryonal carcinoma cells also have ultrastructural (2, 3), biochemical (4, 5), and antigenic (6) properties in common with early embryos. Unlike the cells of the early embryo, however, embryonal carcinoma cells are relatively easy to obtain in large numbers and to culture in vitro.Several clonal lines of embryonal carcinoma cells have been isolated in vitro (7)(8)(9)(10)(11). When these cells are injected into mice they give rise to teratocarcinomas containing a wide variety of both mature and immature tissues. Since subclones of these lines also give rise to teratocarcinomas on reinjection, it is evident that pluripotent cells can be maintained in vitro.We describe here the differentiation of clonal teratocarcinoma cells under defined conditions in vitro. The importance of differentiation of pluripotent cells in vitro is the opportunity it affords to study cell determination under defined conditions. We have, therefore, focussed our attention on the earliest stages of differentiation in vitro. The results described below indicate that it is an orderly process which mirrors the development of the early embryo. MATERIALS AND METHODSCeU Culture&. The pluripotent SIKR teratocarcinoma line from which subclones were isolated has previously been de-1441 scribed (9,12