Fig. 7. TSG is required for efficient inhibition of BMP signaling by chordin. (A-H) The TSG-MO blocks secondary axis induction by chordin, but not by wnt8 or noggin. 0.6-6 pg of 5'-Chd mRNA, 0.5-1.5 pg of Xenopus wnt8 mRNA, or 5-10 pg of Xenopus noggin mRNA was microinjected into the ventral marginal zone of four-cell stage embryos, without or with 10 ng TSG-MO. While the TSG-MO had no effect on the secondary axis induction by wnt8 (D,E) or noggin (G,H), it inhibited secondary axis induction by chordin (B,C). Control embryos were uninjected embryos from each experiment. Coinjection with 10 ng control MO per embryo has no effect on secondary axis induction by any of these RNAs (data not shown). `2°' designates the induced secondary axis. All embryos are oriented with their 1° axes to the right. (I) The TSG-MO blocks chordin function in explanted animal caps. Increasing doses (lanes 6-8, and 9-11, are derived from embryos injected with 0.6, 2 and 6 pg, respectively) of 5'-Chd mRNA, with or without 20 ng TSG-MO, were injected into the animal poles of four-cell stage embryos. The ectodermal explants (animal caps) were removed at blastula stages and incubated until the sibling control embryos reached the midgastrula stages (stage 11). Total RNA was then extracted from the caps and RT-PCR was performed to assay for gene expression. While chordin induced the anterior neural marker otx2 and repressed the direct BMP target genes vent2 and msx1 in a dose-dependent fashion (lanes 6-8), chordin lost its activity in the presence of the TSG-MO (lanes 9-11). (J) The TSG-MO significantly shifts the response to ectopically expressed chordin in animal caps. Increasing doses of chordin RNA (2, 5, 10, 20, 50, 100, 500 and 1000 pg in lanes 2-9 and 11-18) were injected, without or with 20 ng TSG-MO, into animal poles of two-cell stage embryos. Animal caps were obtained and processed as described above and assayed for gene expression by radioactive RT-PCR. While 10 pg chordin was sufficient to induce otx2 and repress msx1 (lane 4), a 10- to 50-fold higher dose of chordin (lanes 16 and 17) was required to achieve comparable levels of induction/repression in the presence of TSG-MO. (K) The TSG-MO has a minor effect on the activity of noggin in gastrula animal caps. Increasing doses of noggin (0.2, 0.5, 1, 2, 5 and 10 pg in lanes 2-7 and 9-14) were injected, without or with 20 ng TSG-MO, into animal poles of two-cell stage embryos. Animal caps were obtained and processed as above and gene expression was assayed by RT-PCR. TSG-MO has a minor influence on repression of msx1 and vent2 and shifts the induction of otx2 by noggin slightly.

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