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ETCHbox genes are fast-evolving homeobox genes present only in eutherian (placental) mammals which originated by duplication and divergence from a conserved homeobox gene, Cone-rod homeobox (CRX). While expression and function of CRX are restricted to the retina in eutherian mammals, ETCHbox gene expression is specific to preimplantation embryos. This dramatic difference could reflect the acquisition of new functions by duplicated genes or subfunctionalization of pleiotropic roles between CRX and ETCHbox genes. To resolve between these hypotheses, we compared expression, sequence and inferred function between CRX of metatherian (marsupial) mammals and ETCHbox genes of eutherians. We find the metatherian CRX homeobox gene is expressed in early embryos and in eyes, unlike eutherian CRX, and distinct amino acid substitutions were fixed in the metatherian and eutherian evolutionary lineages consistent with altered transcription factor specificity. We find that metatherian CRX is capable of regulating embryonically expressed genes in cultured cells in a comparable way to eutherian ETCHbox. The data are consistent with CRX having a dual role in eyes and embryos of metatherians, providing an early embryonic function comparable to that of eutherian ETCHbox genes; we propose that subfunctionalization of pleiotropic functions occurred after gene duplication along the placental lineage, followed by functional elaboration. [ABSTRACT FROM AUTHOR]

Generation of inside cells that develop into inner cell mass (ICM) and outside cells that develop into trophectoderm is central to the development of the early mouse embryo. Critical to this decision is the development of cell polarity and the associated asymmetric (differentiative) divisions of the 8-cell-stage blastomeres. The underlying molecular mechanisms for these events are not understood. As the Par3/aPKC complex has a role in establishing cellular polarity and division orientation in other systems, we explored its potential function in the developing mouse embryo. We show that both Par3 and aPKC adopt a polarized localization from the 8-cell stage onwards and that manipulating their function re-directs cell positioning and consequently influences cell fate. Injection of dsRNA against Pars or mRNA for a dominant negative form of aPKC into a random blastomere at the 4-cell stage directs progeny of the injected cell into the inside part of the embryo. This appears to result from both an increased frequency by which such cells undertake differentiative divisions and their decreased probability of retaining outside positions. Thus, the natural spatial allocation of blastomere progeny can be over-ridden by downregulation of Par3 or aPKC, leading to a deceased tendency for them to remain outside and so develop into trophectoderm. In addition, this experimental approach illustrates a powerful means of manipulating gene expression in a specific clonal population of cells in the preimplantation embryo. [ABSTRACT FROM AUTHOR]

Cell differentiation during pre-implantation mammalian development involves the formation of two extra-embryonic lineages: trophoblast and primitive endoderm (PrE). A subset of cells within the inner cell mass (ICM) of the blastocyst does not respond to differentiation signals and forms the pluripotent epiblast, which gives rise to all of the tissues in the adult body. How this group of cells is set aside remains unknown. Recent studies documented distinct sequential phases of marker expression during the segregation of epiblast and PrE within the ICM. However, the connection between marker expression and lineage commitment remains unclear. Using a fluorescent reporter for PrE, we investigated the plasticity of epiblast and PrE precursors. Our observations reveal that loss of plasticity does not coincide directly with lineage restriction of epiblast and PrE markers, but rather with exclusion of the pluripotency marker Oct4 from the PrE. We note that individual ICM cells can contribute to all three lineages of the blastocyst until peri-implantation. However, epiblast precursors exhibit less plasticity than precursors of PrE, probably owing to differences in responsiveness to extracellular signalling. We therefore propose that the early embryo environment restricts the fate choice of epiblast but not PrE precursors, thus ensuring the formation and preservation of the pluripotent foetal lineage. [ABSTRACT FROM AUTHOR]

The first two lineages to differentiate from a pluripotent cell population during mammalian development are the extraembryonic trophectoderm (TE) and the primitive endoderm (PrE). Whereas the mechanisms of TE specification have been extensively studied, segregation of PrE and the pluripotent epiblast (EPI) has received comparatively little attention. A current model of PrE specification suggests PrE precursors exhibit an apparently random distribution within the inner cell mass of the early blastocyst and then segregate to their final position lining the cavity by the late blastocyst. We have identified platelet-derived growth factor receptor alpha (Pdgfrα) as an early-expressed protein that is also a marker of the later PrE lineage. By combining live imaging of embryos expressing a histone H2B-GFP fusion protein reporter under the control of Pdgfra regulatory elements with the analysis of lineage-specific markers, we investigated the events leading to PrE and EPI lineage segregation in the mouse, and correlated our findings using an embryo staging system based on total cell number. Before blastocyst formation, lineage-specific factors are expressed in an overlapping manner. Subsequently, a gradual progression towards a mutually exclusive expression of PrE- and EPI- specific markers occurs. Finally, cell sorting is achieved by a variety of cell behaviours and by selective apoptosis. [ABSTRACT FROM AUTHOR]