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The extra-embryonic endoderm lineage plays a major role in the nutritive support of the embryo and is required for several inductive events, such as anterior patterning and blood island formation. Blastocyst-derived embryonic stem (ES) and trophoblast stem (TS) cell lines provide good models with which to study the development of the epiblast and trophoblast lineages,respectively. We describe the derivation and characterization of cell lines that are representative of the third lineage of the blastocyst –extra-embryonic endoderm. Extra-embryonic endoderm (XEN) cell lines can be reproducibly derived from mouse blastocysts and passaged without any evidence of senescence. XEN cells express markers typical of extra-embryonic endoderm derivatives, but not those of the epiblast or trophoblast. Chimeras generated by injection of XEN cells into blastocysts showed exclusive contribution to extra-embryonic endoderm cell types. We used female XEN cells to investigate the mechanism of X chromosome inactivation in this lineage. We observed paternally imprinted X-inactivation, consistent with observations in vivo. Based on gene expression analysis, chimera studies and imprinted X-inactivation, XEN cell lines are representative of extra-embryonic endoderm and provide a new cell culture model of an early mammalian lineage.

At the blastocyst stage of pre-implantation mouse development, close contact of polar trophectoderm with the inner cell mass (ICM) promotes proliferation of undifferentiated diploid trophoblast. However, ICM/polar trophectoderm intimacy is not maintained during post-implantation development, raising the question of how growth of undifferentiated trophoblast is controlled during this time. The search for the cellular basis of trophoblast proliferation in post-implantation development was addressed with an in vitro spatial and temporal analysis of fibroblast growth factor 4-dependent trophoblast stem cell potential. Two post-implantation derivatives of the polar trophectoderm – early-streak extra-embryonic ectoderm and late-streak chorionic ectoderm – were microdissected into fractions along their proximodistal axis and thoroughly dissociated for trophoblast stem cell culture. Results indicated that cells with trophoblast stem cell potential were distributed throughout the extra-embryonic/chorionic ectoderm, an observation that is probably attributable to non-coherent growth patterns exhibited by single extra-embryonic ectoderm cells at the onset of gastrulation. Furthermore, the frequency of cells with trophoblast stem cell potential increased steadily in extra-embryonic/chorionic ectoderm until the first somite pairs formed, decreasing thereafter in a manner independent of proximity to the allantois. Coincident with occlusion of the ectoplacental cavity via union between chorionic ectoderm and the ectoplacental cone, a decline in the frequency of mitotic chorionic ectoderm cells in vivo, and of trophoblast stem cell potential in vitro, was observed. These findings suggest that the ectoplacental cavity may participate in maintaining proliferation throughout the developing chorionic ectoderm and, thus, in supporting its stem cell potential. Together with previous observations, we discuss the possibility that fluid-filled cavities may play a general role in the development of tissues that border them.

At least one polar body, almost invariably the second, persists intact to the early blastocyst stage in nearly two-thirds of mouse conceptuses of the PO strain. The distribution in early blastocysts of these surviving polar bodies was highly non-random. Most not only lay in the midregion of the embryonic-abembryonic axis but, on discovering that early blastocysts are bilaterally rather than radially symmetrical about this axis, were found to align with the bilateral axis. Cell marking experiments failed to detect movement of polar bodies relative to the surface of the conceptus during either cleavage or blastulation. That the distribution of degenerating polar bodies and their presumed debris was similar to intact ones also argued against their motility, as did the finding that at all stages second polar bodies were attached to conceptuses by a thin, extensible, weakly elastic ‘tether’. Although the transfer of small fluorochromes between them was rarely observed beyond second cleavage, the second polar body and conceptus could remain coupled ionically up to the blastocyst stage. It is concluded that the second polar body normally remains attached to the conceptus through persistence of the intercellular bridge formed during its abstriction, and therefore provides an enduring marker of the animal pole of the zygote. Hence, according to the distribution of polar bodies, the axis of bilateral symmetry of the early blastocysts is normally aligned with the animalvegetal axis of the zygote and its embryonic-abembryonic axis is orthogonal to it. Such relationships suggest that, at least in undisturbed development, specification of the axes of the blastocyst depends on spatial patterning of the zygote.

Culture of postimplantation conceptuses was used in conjunction with microsurgery to investigate the timing, the mechanism and the developmental regulation of chorioallantoic fusion in the mouse. The timing of fusion was determined in both freshly recovered conceptuses and in those that had been cultured from as early as the mid-streak stage. Attachment of the allantois to the chorion was found to have occurred in most conceptuses by the 6-somite stage, irrespective of whether they had been cultured. In investigating the mechanism of fusion, we wished to determine whether it depended on directed growth of the allantoic bud or on its differential adhesion to the chorion. Microsurgery was used to transplant allantoic tissue into the exocoelomic cavity of conceptuses from which the resident allantois had been removed. In synchronous grafting experiments, transplanted allantoises typically attached to the chorion despite loss of their connection with the hindgut region of the fetus. Hence selective attachment of the allantois to the chorion clearly cannot depend simply on its directed growth. While the transplanted allantoic tissue attached to the chorion selectively, it did not attach to it precociously, despite being favourably positioned to do so. These findings argue that the initial attachment of the allantois to the chorion depends on a selective adhesive mechanism that is developmentally regulated. Further grafting experiments in which donor conceptuses were either more or less advanced than hosts revealed that attachment of the allantois to the chorion depends primarily on the stage of the allantois rather than on the stage of the chorion. Collectively, these findings support the hypothesis that the initial stage of chorioallantoic fusion depends on selective adhesion between regionally differentiated mesodermal surfaces which is governed principally by the stage of development of the allantois.