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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.
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