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Fig. 1. Hex anteriorises the phenotypes induced by ß-catenin and
regulates its downstream targets. (A) Axis duplication phenotypes
induced by Hex and ß-catenin. Embryos were injected with 500 pg Hex,
ß-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 and
ß-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 ß-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-
VP2 were used. Ventral injections are indicated with `V' where 500
pg Hex and 500 pg ß-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 in
ß-catenin and ß-catenin co-injection with Hex show Xnr3 staining in
the animal hemisphere. Arrowheads indicate the site of injection. (F)
Expression of ß-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 ß-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 ß-catenin.
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