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Fig. 4. FoxD3 function is required for axis formation. (A) The
sequence of FoxD3 flanking the initiator methionine with the sequence
of the morpholino antisense oligonucleotide (181-158) highlighted in yellow.
(B) Western analysis of animal explants prepared from embryos injected
with FoxD3 RNAs (2 ng) alone, or in combination with antisense
(FoxD3MO) or mismatch (misMO) morpholino oligonucleotides (50 ng). Translation
of a FoxD3 RNA containing the 5'UTR and the complete antisense
target sequence (FoxD3+utr) was inhibited by FoxD3MO, but not misMO.
Translation of FoxD3 lacking the 5'UTR (FoxD3-utr) was
unaffected by either oligonucleotide. Equal protein loading was confirmed by
blotting for the ubiquitous MAPK. (C) RT-PCR analysis of Muscle
Actin (M. Actin) induction in animal explants injected with
FoxD3 RNAs containing or lacking the 5'UTR (200 pg) and FoxD3MO
or misMO (50 ng). PCR controls are as described in
Fig. 2. (D) At the
four-cell stage each blastomere was injected in the marginal zone with FoxD3MO
or misMO (25 ng), and extracts prepared at the midgastrula stage (stage 11)
were analyzed by western blotting for the accumulation of endogenous FoxD3
protein. A single major band, migrating at the same position as overexpressed
Xenopus FoxD3, was detected in uninjected and misMO-injected samples,
and was reduced 
tenfold in FoxD3MO-injected samples. The exposure of the
western blot in panel D was approximately eight times longer than that shown
in panel B. (E-M) At the four-cell stage each blastomere was injected
in the marginal zone with 250 pg of VP16-FoxD3 RNA (H,I), 15 ng of FoxD3MO
(J,K) or 15 ng of misMO (L,M). At the tailbud stage (stage 30), embryos were
sectioned (transverse, dorsal up) to examine the formation of axial
structures, including notochord (nc), somitic muscle (sm) and neural tube (nt)
(G,I,K,M). (E) Quantification of the combined results of five independent
experiments.
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