Dev Genes Evol (2009) 219:391–398 395 Fig. 3 Time-lapse analysis of the cyclopic ear phenotype. The otic vesicle morphology was analysed in Wnt1-transgenic embryos from stage 20 to stage 30. Heat shock was performed at 30% epiboly for 2 h two genes are expressed in the more anteriorly positioned midbrain–hindbrain boundary (mhb). The fate of the region anterior to r3 in group B embryos therefore remains unclear. A combination of local competence factors together with signals from a distance is thought to induce otic structures in vertebrates (Riley and Phillips 2003; Whitfield et al. 2002). Fgf ligands are thought to mediate the signals originating from r4 (Maves et al. 2002; Phillips et al. 2001), and Dlx3b and Foxi1 are the best candidates for the competence factors (Esterberg and Fritz 2009; Lee et al. 2003; Solomon et al. 2004). Dlx3b is a preplacodal marker gene (Liu et al. 2003) initially expressed in a horseshoe- shape surrounding the entire neural plate (Supplementary Fig. S3; Hochmann et al. 2007; Kaji and Artinger 2004). Due to the anterior truncations, the neural plate was shortened in the Wnt1 embryos, but Dlx3b expression in the adjacent ectoderm remained in both groups (Fig. 4k, l and Supplementary Fig. S3). In group A embryos, the expression was upregulated in the anterior region (Fig. 4k), anticipating the development of the ectopic otic structures. In group B, strong Dlx3b expression was restricted to regions flanking r4, coinciding with the normal position of otic structures (Fig. 4l). The forkhead-domain containing transcription factor Foxi1 is expressed in two domains positioned laterally to the neural plate (Hochmann et al. 2007), which we also found in group B embryos (Fig. 4o). at 39°C. Ectopic otic vesicles are indicated by black arrows. The fusion of vesicles is indicated by yellow arrows Interestingly we detected strong Foxi1 expression in group A embryos at the position where the ectopic otic structures develop (Fig. 4n), contrary to group B embryos, which do not show any Foxi1 expression in this region (Fig. 4o). Sox3 is a marker for both the epibranchial and the otic preplacodal region, whereas Pax2 marks otic tissue specifi- cally (Nikaido et al. 2007). In order to see whether the ectopic placodes of Wnt1 expressing embryos are restricted to otic development, we analysed group A embryos for expression of the two marker genes (Supplementary Fig. S4). We found both genes activated in overlapping anterior regions, indicating a clear bias towards ear development. This is in agreement with data from chick and mouse experiments, where Wnt signalling was found to partition the progenitor region, positively into inner ear and negatively into epibranchial placodes (Freter et al. 2008; Ohyama et al. 2006). Group B embryos did not show expression of either marker gene in the anterior region (data not shown). Discussion Ectopic expression of Wnt1 during early development of vertebrates is known to result in anterior truncations (Christian and Moon 1993). The heat-shock transgenic line allowed us to perform highly specific time-course experi- ments for ectopic Wnt1 activity in medaka fish. By