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FIG. 3. SpAN ventralizes dorsal mesoderm during gastrulation. Dorsal marginal zones (DMZs) were isolated from both control and span
mRNA-injected (1.6 ng/embryo) early gastrulae, incubated until control embryos reached the indicated stages, and analyzed for the
expression of both dorsal- and ventral-specific genes by RNase protection (A). Levels of ornithine decarboxylase (ODC) mRNA were
determined as a loading control. The dorsal-specific genes goosecoid (gsc) and xnot are initially induced in span mRNA-injected DMZs, but
expression is subsequently down-regulated. In contrast, expression of the ventral-specific genes xhox3 and xwnt8 is increased in span
mRNA-injected embryos as gastrulation progresses. Gastrulae were also analyzed for xnot (B, C) and xwnt8 (D, E) expression by whole
mount in situ hybridization. In control stage 12 embryos, xnot was clearly localized to the dorsal blastopore lip (B) but was absent or very
diffuse in span mRNA-injected embryos (C), consistent with the loss of dorsal structures. In contrast, xwnt8 was expressed in ventral and
lateral mesoderm, but not dorsal mesoderm, of control stage 12 embryos (D), but expression expanded into the dorsal mesoderm of span
mRNA-injected embryos (E), demonstrating that ventralization of the dorsal marginal zone has occurred by this stage. |
