Embryonic stem (ES) cells are important tools in the study of gene function and may also become important in cell therapy applications. Establishment of stable XX ES cell lines from mouse blastocysts is relatively problematic owing to frequent loss of one of the two X chromosomes. Here we show that DNA methylation is globally reduced in XX ES cell lines and that this is attributable to the presence of two active X chromosomes. Hypomethylation affects both repetitive and unique sequences, the latter including differentially methylated regions that regulate expression of parentally imprinted genes. Methylation of differentially methylated regions can be restored coincident with elimination of an X chromosome in early-passage parthenogenetic ES cells, suggesting that selection against loss of methylation may provide the basis for X-chromosome instability. Finally, we show that hypomethylation is associated with reduced levels of the de novo DNA methyltransferases Dnmt3a and Dnmt3b and that ectopic expression of these factors restores global methylation levels.

The specification of a subset of epiblast cells to acquire a neural fate constitutes the first step in the generation of the nervous system. Little is known about the signals required for neural induction in the mouse. We have analysed the role of BMP signalling in this process. We demonstrate that prior to gastrulation, Bmp2/4 signalling via Bmpr1a maintains epiblast pluripotency and prevents precocious neural differentiation of this tissue, at least in part by maintaining Nodal signalling. We find that during gastrulation, BMPs of the 60A subgroup cooperate with Bmp2/4 to maintain pluripotency. The inhibition of neural fate by BMPs is independent of FGF signalling, as inhibition of FGF signalling between 5.5 and 7.5 days post-coitum does not block neural differentiation in the mouse embryo. Together, our results demonstrate that inhibition of BMP signalling has a central role during neural induction in mammals and suggest that FGFs do not act as neural inducers in the post-implantation mouse embryo.

The anterior visceral endoderm (AVE), a signalling centre within the simple epithelium of the visceral endoderm (VE), is required for anterior-posterior axis specification in the mouse embryo. AVE cells migrate directionally within the VE, thereby properly positioning the future anterior of the embryo and orientating the primary body axis. AVE cells consistently come to an abrupt stop at the border between the anterior epiblast and extra-embryonic ectoderm, which represents an end-point to their proximal migration. Little is known about the underlying basis for this barrier and how surrounding cells in the VE respond to or influence AVE migration. We use high-resolution 3D reconstructions of protein localisation patterns and time-lapse microscopy to show that AVE cells move by exchanging neighbours within an intact epithelium. Cell movement and mixing is restricted to the VE overlying the epiblast, characterised by the enrichment of Dishevelled-2 (Dvl2) to the lateral plasma membrane, a hallmark of Planar Cell Polarity (PCP) signalling. AVE cells halt upon reaching the adjoining region of VE overlying the extra-embryonic ectoderm, which displays reduced neighbour exchange and in which Dvl2 is excluded specifically from the plasma membrane. Though a single continuous sheet, these two regions of VE show distinct patterns of F-actin localisation, in cortical rings and an apical shroud, respectively. We genetically perturb PCP signalling and show that this disrupts the localisation pattern of Dvl2 and F-actin and the normal migration of AVE cells. In Nodal null embryos, membrane localisation of Dvl2 is reduced, while in mutants for the Nodal inhibitor Lefty1, Dvl2 is ectopically membrane localised, establishing a role for Nodal in modulating PCP signalling. These results show that the limits of AVE migration are determined by regional differences in cell behaviour and protein localisation within an otherwise apparently uniform VE. In addition to coordinating global cell movements across epithelia (such as during convergence extension), PCP signalling in interplay with TGFβ signalling can demarcate regions of differing behaviour within epithelia, thereby modulating the movement of cells within them.