Sebox overexpression inhibits mesodermal gene expression. A: RT-PCR analysis of gene expression in animal cap explants injected with 400 pg β-gal, Xenopus sebox, or zebrafish mezzo mRNA. Animal caps were dissected at stage 8.5 and cultured to the equivalent stage 11, followed by RT-PCR analysis. The marker odc served as a loading control. B: Western blot analysis showing that both the microinjected Xenopus sebox and zebrafish mezzo mRNAs were translated into proteins at comparable levels. β-actin served as a loading control. C: Representative embryos at the indicated stages are viewed from the dorsal side. A total of 5 ng RLDx alone or combined with 400 pg sebox mRNA was microinjected into the marginal zone of two blastomeres at the four-cell stage and recorded at stage 15 and stage 24. D: RT-PCR analysis of the expression of the indicated genes in the animal caps with the indicated treatments. The animal caps were isolated at stage 8.5 and harvested at stage 11. The marker odc served as a loading control. E: Activin-treated animal cap explants were recorded at the equivalent stage 16. Animal caps from wild-type (ctrl) or sebox (400 pg)-overexpressing embryos were isolated at st8.5 and cultured with or without Activin (5 ng/ml). Scale bar = 1 mm. F: WISH for wnt5a, wnt5b and wnt11b expression in stage 11 embryos injected with 400 pg sebox mRNA into the dorsal part at the four-cell stage. RLDx (5 ng per embryo) was co-injected as a lineage tracer. The correctly injected embryos were screened at st10–10.25 under a fluorescence dissecting microscope. All embryos were viewed from the vegetal pole with the dorsal side upward. G: qRT-PCR analysis of the expression of wnt5a, wnt5b, and wnt11b in control and sebox-injected embryos at stage 11. The relative expression levels of genes were normalized to the level of odc.