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Fig. 1. Hex anteriorises the phenotypes induced by β-catenin and regulates its downstream targets. (A) Axis duplication phenotypes induced by Hex and β-catenin. Embryos were injected with 500 pg Hex,β -catenin, or both RNAs into a single-ventral blastomere at the four-cell stage. In situ hybridisation was performed for the anterior neural marker BF1 at stage 35. The inset and arrowheads indicate a small ventral outgrowth produced by Hex injection. (B) Phenotypes induced by Hex andβ -catenin in ventral marginal zone explants. Embryos were injected with the indicated RNA into both blastomeres at the two-cell stage, cultured to gastrulation, VMZ explants dissected and cultured until staging control embryos reached stage 35. BF1 in situ hybridisation was performed to highlight the phenotypes obtained. Hex RNA was injected at 500 pg. (C,D) Real-time RT-PCR analysis of Siamois and Xnr3 expression in VMZ explants analysed at stage 10.5. Embryos were injected as in B with the indicated RNA. Values were normalised to the expression level of Odc and the relative change in gene expression for the genes analysed was calculated by dividing the values from injected samples by the values from the uninjected. Data are based on three independent experiments. (E) Cell automonous induction of β-catenin targets Siamois and Xnr3. RNA was injected into a single blastomere at the four-cell stage with the indicated RNA alongside nucGFP RNA. Dorsal injections are indicated with `D' where either 250 pg Hex or 100 pg Hex-λVP2 were used. Ventral injections are indicated with `V' where 500 pg Hex and 500 pg β-catenin were used. Embryos were processed by double in situ hybridisation and stained for both nucGFP to indicate the injected cells (light blue) and Xnr3 (dark blue). The schematic diagram in the lower right-hand corners of the lower panels indicates that the injection was carried out in both blastomeres at the two-cell stage. The insets inβ -catenin and β-catenin co-injection with Hex show Xnr3 staining in the animal hemisphere. Arrowheads indicate the site of injection. (F) Expression of β-catenin targets Siamois and Xnr3 in embryos depleted of endogenous Hex. Embryos were injected with a total of 40 ng Hex MO or control MO at the two-cell stage, either alone or in combination with 500 pg β-catenin. 500 pg mouse Hex (mHex) was used to rescue the phenotypes. Siamois (upper panel) and Xnr3 (lower panel) expression was analysed by in situ hybridisation at stage 10.5. Arrowheads indicate the ectopic expression induced by β-catenin. |
