Dev Genes Evol (2009) 219:391–398 Fig. 1 Activation of the canonical Wnt pathway in medaka embryos. Dorsal views of embryos at 2dpf (a, c, d) and 5dpf (b). A non-transgenic sibling (a) is compared to embryos with transient injections of Wnt1 mRNA (b), a heat-inducible Wnt1 construct (c) and Wnt1-transgenic lines (d). Starmaker is strongly expressed in normal otic vesicles (e) and in cyclopic ear structures (f). An ectopic otic vesicle is indicated by an arrow. The heat-shock treatment was performed for 2 h at 39°C for c, d and f and 3 h at 30% for a. ey eye, ov otic vesicle, pf pectoral fin 393 at the level of the hindbrain, whereas posterior parts of the embryos are not affected. The extent of the axis truncations does not differ dramatically between the one cell stage (mRNA injection, see Fig 1b) and early gastrulation (Fig. 2a). However between early and late gastrulation, the truncations are gradually reduced, with midbrain structures appearing at heat shocks at 60% epiboly and forebrain structures at 80% epiboly. Ectopic Wnt activation at neurula stages only affects eye development, whereas embryos heat-shocked during somitogenesis appear normal. These phenotypes would correlate with the timing of neural plate induction by the migrating mesoderm, which might be blocked at different time points by the ectopic Wnt signals. The gradual loss of anterior structures can also be observed with hindbrain marker gene expression (see below). In the time-course experiment, the cyclopic ear pheno- type was only observed when Wnt signalling was induced at 30% epiboly. Slightly later activation of the pathway at 40% epiboly generates embryos with normal otic vesicle morphology. In previous work, we identified early gastru- lation as a critical phase for otic induction in medaka (Aghaallaei et al. 2007). The ectopic Wnt activity therefore affects otic development during the induction phase. To examine whether the effect of Wnt signalling is dosage dependent, we varied the time of heat treatment in embryos at early gastrulation from 5 min to 2 h. We used the Gfp signals originating from the bidirectional heat- shock promoter as a marker for the expression level (Fig. 2b). Interestingly, all embryos heat-treated for more than 10 min showed the same phenotype; the only