Figure 5.

Activation of the Wnt/β-catenin pathway requires nuclear localization of Dsh. (a) Axis-inducing activity of Dsh constructs. One ventral vegetal blastomere of 8-cell embryos was injected with 1 ng Dsh-GFP, DsNLSm, or DsSNLS mRNA as indicated. Uninjected sibling embryos are also shown. (b) Activation of the Siamois reporter gene. The reporter -833pSia-Luc plasmid (20 pg) was coinjected with Dsh-GFP, DsNLSm or DsSNLS mRNA (0.5 ng each) into a single animal ventral blastomere of 8-cell embryos. Injected embryos were lysed at stage 10+ for luciferase activity determination. Results are shown in relative light units as the mean +/- standard deviation from triplicate samples. (c) Requirement for Dsh NLS for the stabilization of β-catenin. Flag-β-catenin mRNA (0.4 ng) was coinjected with Dsh, DsNLSm, DsSNLS or ΔRGS-Axin mRNA (2 ng each) into four animal blastomeres of 4–8-cell embryos. Levels of β-catenin and Dsh constructs were assessed in stage 10 embryo lysates with anti-Flag antibodies and anti-Xdsh antibodies; β-tubulin serves as a loading control. Dsh and DsSNLS, but not DsNLSm, are able to stabilize β-catenin. ΔRGS-Axin was used as a control for an activator of the Wnt pathway.