FIGURE 5.

Concerted deployment of IWP and IWR compounds distinguishes β-catenin-dependent and -independent responses in vivo. A, identification of an IWP compound with in vivo activity in zebrafish. IWP12 inhibits the expression of an EGFP fluorescent protein reporter driven by synthetic TCF-binding elements in a transgenic line (Tg(7xTCF-Xla.Siam:GFP)ia4]. A ∼10-fold excess of IWP12 is equivalent in activity to IWR1 compound. Fluorescence intensity was quantified (below) in an area that covers most of the posterior region (box). Data are mean ± S.E. from three animals. B, IWR and IWP compounds inhibit Wnt signaling in zebrafish primary embryonic fibroblasts. Embryonic fibroblasts isolated from 6 hpf Tg(7xTCF-Xla.Siam:GFP)ia4 embryos were cultured in the presence or absence of indicated compound. GFP expression was visualized 20 h later. C, IWP compounds inhibit tailfin regeneration, a Wnt-dependent process. Tailfins of zebrafish larvae at 3 days post fertilization were resected and the larvae subsequently reared in medium containing DMSO, IWR1 (10 μm), or IWP12 (50 μm) for an additional 4 days. D, IWP12 inhibits embryonic convergent extension by targeting β-catenin-independent Wnt signaling. Zebrafish embryos were treated with GSK3β inhibitor (a Wnt/β-catenin pathway activator), IWP12 compound, or both starting 4 hpf followed by whole mount in situ analysis at 24 hpf with probes and the respective developmental structures they label indicated: hgg1 (ctsl1b) [prechordal plate (pcp)], ntl [prospective notochord (n) and germ ring blastopore margin], and dlx3b [anterior edge of the neural plate (np)]. Changes in the distance between the neural plates and prechordal plates, as well as the notochord were quantified based upon the severity of the phenotype as represented. Number of animals examined under each condition is indicated above each plot. E, inactivation of GSK3β rescues Wnt/β-catenin pathway activity in animals treated with IWP12. F, engrailed expression in the midbrain-hindbrain boundary (MHB) is suppressed by chemical inhibition of Porcn. Zebrafish embryos (4 hpf) treated with IWP12 for 20 h were subjected to in situ analysis with a probe for eng1a. Number of animals examined in each condition is indicated within each plot.