To elucidate the apparent patterning deficits, we next analyzed signaling molecules that are required for early patterning. Nodal, a member of the Tgfβ family of secreted molecules is required for the formation and maintenance of the primitive streak and induction of the AVE [25-27]. Nodal is normally expressed throughout the epiblast and overlying visceral endoderm at early post implantation stages [28], but later becomes restricted to the posterior of the embryo to the site of primitive streak with asymmetrical visceral endoderm expression marking the left-right axis. By E7.5, Nodal expression is restricted to the node. Nodal expression was assessed in Hdhex4/5/Hdhex4/5 embryos heterozygous for the Ndl lac Z allele [28,29]. Notably, heterozygous loss of nodal does not alter the Hdhex4/5/Hdhex4/5 phenotype, as determined by morphology of Hdhex4/5/Hdhex4/5:Ndllacz/Ndl+ embryos compared with Hdhex4/5/Hdhex4/5 embryos (data not shown). In contrast to wild-type embryos, which exhibit tight restriction of Nodal.LacZ expression to the node, Hdhex4/5/Hdhex4/5:Ndllacz/Ndl+ embryos express Nodal.LacZ throughout the endoderm overlying the epiblast, with higher levels in the posterior in an asymmetric pattern (Fig. 3A–D). The lack of tight restriction of nodal signal is consistent with a failure to form an organized node structure.