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. Author manuscript; available in PMC: 2008 Jun 24.
Published in final edited form as: Development. 2007 Oct 24;134(23):4209–4218. doi: 10.1242/dev.010645

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.

Fig. 6

(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 min after treatment, corresponding to stage 8.5 (panels 3), but that the delay in cells treated with Activin at stage 9 is less than 10 minutes (panel 6). Panels 4 and 8 represent control cells that have not been 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 min 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; weak cytoplasmic fluorescence is readily detectable in such cells. (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 equivalent of stage 10.5 (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.