Fig. 6. Nodal mutants prematurely downregulate several determinants of pluripotency during implantation. (A) Whereas expression of Fgf4 and Sox2 is still unaffected, Oct4, Nanog, Foxd3 and Cripto are already downregulated or silenced in Nodal mutants between E5.0 and 5.5. (B) By contrast, molecular markers of the ExE, such as Pace4 and Bmp4, are not affected at this stage. (C) Epiblast explants of post-DVE embryos (E5.75) stripped of ExE and VE fail to maintain expression of Oct4/Pou5f1, unless they are cultured in the presence of recombinant Nodal. Administration of SB-431542 blocked the effect of exogenous Nodal protein, confirming that it is specific, even though in this experiment we used unpurified protein produced in human 293T cells (Beck et al., 2002). (D) Earlier explants isolated from DVE- and pre-DVE-stage embryos without VE did not survived well under the conditions examined (not shown). However, in the presence of VE, they maintained expression of Oct4, Nodal, as well as Furin (top row). By contrast, Oct4 and Nodal were downregulated in explants cultured with inhibitors of endogenous Nodal (SB) or Furin (CMK) activities. Furin remained expressed in the VE, suggesting that these treatments are not toxic.