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.