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Fig. 2. Wnt-dependent dorsal axis formation controls late, but not early, FoxI1e expression. Embryos injected with 40 ng β-Cat MO had reduced levels of direct targets Siamois and Xnr3 at stage 10 (A), and embryos injected with 50 pg β-Cat mRNA had increased levels (B). In control explants, the level of FoxI1e was higher in dorsal halves than ventral halves (C). The total level and distribution of FoxI1e was unchanged by β-Cat MO or mRNA (D–F). (G) Levels of FoxI1e mRNA at stages 9.5, 10, and 14 (compared to ODC mRNA levels at each stage) in embryos injected with either 100 pg BMP4 mRNA (upper panels), or 10, 40, or 160 pg of noggin mRNA (lower panels). Neither BMP4 overexpression, nor inhibition using Noggin, consistently affected the level of FoxI1e expression at the late blastula stage (stage 9.5). By the early gastrula stage (stage 10), BMP4 overexpression increased FoxI1e expression, but Noggin still had little effect. Expression of FoxI1e in the early neurula (stage 14) was increased by BMP4 overexpression and completely ablated by Noggin. This indicates that the early expression of FoxI1e is BMP-independent, but that the restriction of FoxI1e to the epidermis at neurulation is BMP-dependent. (H) Overall level of FoxI1e expression at stage 14 is unaffected by β-Cat MO. Embryos injected with 40 ng β-Cat MO at the 2-cell stage were injected with β-Gal at the 32-cell stage in the A1 blastomere. Red-gal staining, and FoxI1e in situ hybridization were carried out at stage 14. The anterior (A), posterior (P), dorsal (D), and ventral (V) regions of the embryo are marked in the control embryo, with the derivatives of the dorsal animal blastomere (A1), marked by the yellow arrowhead in the neural plate. Embryos lacking β-Cat (right panel) lack axes altogether. The red-gal positive cells (arrowed) mark the derivatives of the A1 blastomere. FoxI1e expression in β-Cat-depleted embryos persists in that clone of cells, indicating the β-Cat dependence of restriction of FoxI1e from the prospective CNS. The reddish cast toward the posterior end of the uninjected embryo is residual maternal pigment, not affected by bleaching. Scale bars represent 200 μm. |
