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Fig. 6. Entry of Smad2-Smad4 complexes into the nucleus does not occur until the
midblastula transition, irrespective of the stage of treatment with
Activin. (A) Animal pole cells derived from embryos injected with
RNA encoding VNm9-Smad4 and VC155-Smad2 and ECFP lineage marker were treated
with Activin at early blastula stage 7 (left-hand column) or late blastula
stage 9 (right-hand column), and were cultured for the indicated times. Note
that nuclear translocation of Smad complexes in cells treated with Activin at
stage 7 occurs only at 75 minutes after treatment, corresponding to stage 8.5
(c), but that the delay in cells treated with Activin at stage 9 is less than
10 minutes (f). (d,h) Control cells that were not treated with Activin.
(B) Gene activation in response to Activin also only occurs after stage
9. Animal pole regions were dissected from Xenopus embryos at stage
7, 8 or 9 and cultured in the presence or absence of Activin for either 30
minutes or until control embryos reached stage 9 before being assayed for
expression of chordin and goosecoid. Following treatment at stage 7, gene
activation does not occur within 30 minutes of culture, but is clearly
detectable by stage 9. The same is true of animal caps treated at stage 8, but
treatment of animal pole regions at stage 9 results in gene activation within
30 minutes. (C) Lack of nuclear BiFC in embryos at stage 7 is not a
consequence of the slow maturation of BiFC constructs or delayed complex
formation. Compared with uninjected cells (a), weak cytoplasmic fluorescence
is clearly visible in cells expressing VC155-Smad2 and VNm9-Smad4 as early as
stage 7 (b). (D) Phosphorylation of endogenous Smad2 occurs within 30
minutes of treatment with Activin even at stage 7. The western blot was
performed with protein extracts of animal pole regions at the indicated
stages. (E) VC155-Smad2 is expressed and can be phosphorylated in
response to Activin as early as stage 7. Positions of VC155-Smad2 and of
endogenous Smad2 are indicated. Protein extracts are derived from animal pole
regions excised from embryos previously injected with RNA encoding VC155-Smad2
and VNm9-Smad4.
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