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Fig. 6. Induction of Chordin by Smad3 is indirect and requires Smicl. (A)
RNA (500 pg) encoding Smad2 or Smad3 was injected into embryos of Xenopus
laevis at the one-cell stage and animal pole regions were dissected at
mid blastula stage 8 (before the mid blastula transition). The animal pole
regions were cultured for 3 hours in the presence or absence of cycloheximide
and expression of Chordin was assayed by quantitative RT-PCR. Smad3
is a more efficient inducer of Chordin than is Smad2, and the action
of both is indirect. In the same experiment, Xwnt8 and eFGF
proved to be induced to higher levels by Smad2 than by Smad3, confirming that
the two Smad family members have differential effects in the Xenopus
embryo. Additional experiments showed that eFGF and
Goosecoid are direct targets of Smad2 and Smad3, respectively (data
not shown). (B) Inhibition of Smicl function prevents induction of
Chordin by Smad3 and by Xnr1. Embryos of Xenopus laevis were
injected with morpholino oligonucleotides coMO or XlMO (80 ng) either alone or
in the presence of RNA encoding Smad3 (500 pg) or Xnr1 (100 pg). Animal caps
were dissected at mid blastula stage 8 and they were cultured to the
equivalent of stage 10.5 before being analysed for Chordin expression
by quantitative RT-PCR. Both Smad3 and Xnr1 induced expression of
Chordin, and this was inhibited by a morpholino oligonucleotide
directed against Smicl. (C) A model based on the data presented so far: the
induction of Chordin by Xnr1 and Smad3 is indirect and requires Smicl
and the synthesis of another factor X.
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