To investigate the morphology of Atrxnull embryos prior to death, embryos from the above crosses were initially dissected in their deciduas at 7.5 dpc, and paraffin sections were stained with haematoxylin (Figure 5A) or with an anti-ATRX antibody (Figure 5B–5E). Immunohistochemical staining revealed that Atrx was widely expressed in wild-type 7.5 dpc embryos (Figure 5B). Expression was highest in the embryonic region (Figure 5C) and the chorion (Figure 5D). Detectable but lower levels of expression were observed in the ectoplacental cone (Figure 5D) and surrounding decidual tissue. We also observed very high levels of Atrx expression in trophoblast giant cells (TGCs) surrounding the Reichert's membrane (Figure 5E). Within the large nuclei of these TGCs, the typical nuclear association of Atrx with blocks of pericentromeric heterochromatin [15] was clearly observable. Only background staining was seen in the corresponding Atrxnull embryonic tissues (Figure 5B–5D), while expression in the surrounding decidual tissue (of maternal origin) was normal and served as an antibody staining control (unpublished data). Morphologically, 7.5 dpc Atrxnull embryos were dramatically reduced in size and appeared developmentally retarded relative to stage-matched wild-type embryos (Figure 5A and 5B). However, despite their reduced size, the general morphology and organisation of embryonic structures in Atrxnull conceptuses appeared grossly normal. The amnion and chorion were clearly present and the amniotic, exocoelomic, and ectoplacental cavities were distinguishable, as were all three embryonic germ layers (Figure 5A–5C). At 8.5 dpc, embryos were dissected free of deciduas, and observed in whole mount. Individual conceptuses were genotyped by PCR using DNA isolated from yolk sac as described in Protocol S1. Consistent with observations at 7.5 dpc, the general morphology of the embryo proper of Atrxnull conceptuses also appeared grossly normal at 8.5 dpc. The head fold had clearly formed, and expression of the early mesoderm marker brachyury (T) [16] was detected in the primitive streak and emerging notochord by whole-mount in situ hybridisation (WMISH) (Figure 5F), indicating that Atrxnull embryos had gastrulated.