Fig. 4. Overexpression of ß-catenin shows involvement in late blastula Wnt signalling. GFP, ß-catenin and marker gene expression in gastrulation stage Xenopus embryos. Using a double construct (hsp::ß-catenin::hsp::GFP) to generate transgenic embryos, both ß-catenin and GFP can be overexpressed to high levels (see Materials and Methods). This increase in levels can be seen in the western blot carried out with stage 10 embryos (A). GFP positive embryos (1-4) also have high levels of exogenous ß-catenin protein. In non-transgenic embryos (5-8) only low levels of GFP and exogenous ß-catenin protein is detectable, which is probably due to cytoplasmic expression from unincorporated plasmid DNA. (B-K) Detection of ubiquitous GFP expression under UV light (G) therefore allows transgenic embryos overexpressing ß-catenin (G-K) to be selected from control embryos (B-F). (G) GFP expressing embryo under UV-light (compare with non-GFP-expressing embryo in B). (H) Morphological phenotype of an embryo overexpressing ß-catenin (compare with control embryo in C and to Xwnt-8 overexpressing embryo in Fig. 7G). The embryos in D-F show normal molecular marker expression after whole-mount RNA in situ hybridisation is carried out at stage 11 and embryos I-K show molecular marker expression in ß-catenin overexpressing embryos. In all embryos dorsal is at the top. Expression of Xnot, a dorsal molecular marker (D) is repressed in ß-catenin overexpressing embryos (I), whereas the ventral and lateral mesodermal markers XmyoD and Xpo, are ectopically expressed in the dorsal midline of these ß-catenin overexpressing embryos (compare J and K with E and F). (L-Q) As a control, transgenic embryos using a single construct (hsp::GFP) to overexpress only GFP (O,Q) develop a normal morphology (P), as compared to non-transgenic siblings (L-N).
