Fig. 6. N-XTcf-3 inhibits dorsalising and ventrolateral-promoting Wnt signalling when expressed in Xenopus embryos. (A) N-XTcf-3 construct used in this experiment. The morphology and molecular marker expression of uninjected control embryos are shown (B-F). N-XTcf-3 RNA was injected into the dorsal marginal zone (DMZ; G-K) or the ventral marginal zone (VMZ; L-P) at the two-cell stage and embryos were analysed by morphology at stage 31 (G and L) and molecular marker expression at stage 11 (H-K,M-P). Dorsally injected embryos have a ventralised phenotype with reduced dorsal mesodermal structures (G). Analysis of molecular markers show that there is a repression of the dorsal marker (chordin, H; compare with C) and a reduction of Xnot (I; compare with D). Ectopic expression of the ventral and lateral markers XmyoD (J) and Xpo (K) in the dorsal midline is also seen (compared with E,F). Ventrally injected embryos are affected by injection of N-XTcf-3 with normal dorsal mesodermal structures but reduced ventral structures. This can be seen both morphologically (L compared with B) and with molecular markers (M-P compared with C-F). The expression of the dorsal marker Chd is comparable with the control expression (M,C) and Xnot, a notochord specific dorsal marker shows a slight expansion (N) into the ventral cells, which express N-XTcf-3 (compare with normal expression pattern, D). Reduced ventral development is also shown by the reduction of ventrolateral gene expression of XmyoD and Xpo in the ventral cells which express N-XTcf-3 (O,P compared with E,F). (Q) Western blot detecting HA-tagged N-XTcf-3 protein at stages 6 and 10 of development after injection of the RNA at the four-cell stage into the DMZ or the VMZ. As a control, uninjected embryos show no N-XTcf-3 protein present, however, injected embryos show comparable levels of protein at stages 6 and 10 with both ventral and dorsal injection of N-XTcf-3 RNA.