Dev Genes Evol (2009) 219:391–398 R Fig. 4 Analysis of hindbrain and otic marker genes in the Wnt1- transgenic lines. Dorsal views of embryos at two somites (a–r). Anterior is to the left. The positions of rhombomeres (r) and the mid- hindbrain boundary (mhb) are indicated. The heat shock of Wnt1- transgenic embryos was performed at 30% epiboly in group A and 50% epiboly in group B, respectively. The asterisks indicate anterior expression of Fgf3 and Fgf8, respectively, in group B embryos, despite the fact that they lack ectopic ear structures. The arrow indicates weak En2 expression. mhb. mid-hindbrain boundary systematically varying the time of activation, we observed a strict dependence of timing on the extent of the anterior truncations (Fig. 2a). These phenotypes can be interpreted by a gradient of Wnt signalling being responsible for patterning the anteroposterior axis (Kiecker and Niehrs 2001). The critical influence of timing in our experiments is in good agreement with lithium exposure experiments (Yamaguchi and Shinagawa 1989). Induction of canonical Wnt signalling in the transgenic embryos at 30% epiboly resulted in a novel phenotype. Extensive otic tissue developed and finally covered the complete anterior region of the embryos. Interestingly, these effects on otic induction seemed to be uncoupled from the axis truncations. Injection of both Wnt1 mRNA and DNA resulted in truncations, but only the uniform misexpression at 30% epiboly in the transgenic line produced the cyclopic ear phenotype. In addition, the number of ectopic otic vesicles was dose dependent, whereas the axis truncations appeared indistinguishable from low to high Wnt1 doses (Fig. 2b). In the zebrafish model system, activation of canonical Wnt signalling at the midblastula transition did not produce ectopic otic vesicles. This experiment was used as an argument against a role of canonical Wnt signalling in otic induction (Phillips et al. 2004). In support of these data, we also failed to induce ectopic otic tissue with constitutive active DNA constructs in medaka. However, in contrast to the latter study, we demonstrate a strict time dependence for Wnt signalling in the otic induction process, namely at 30% epiboly. Fgf signalling plays a major role in otic induction (reviewed in Riley and Phillips 2003; Whitfield et al. 2002), and we recently could demonstrate an inducing effect of Fgf8 misexpression during early gastrulation in medaka. In these experiments, the formation of ectopic otic vesicles was further boosted by coexpression of the competence factors Dlx3b and Foxi1 (Aghaallaei et al. 2007). Here we compared groups of Wnt1-transgenic embryos induced at 30% epiboly (group A), with those induced at 50% epiboly (group B). In both groups of Wnt1- transgenic embryos, we found similar expression of Fgf8 (Fig. 4e, f); however, only group A embryos developed ectopic otic structures. Fgf3 expression was stronger in the anterior region of group A embryos (Fig. 4h, k, n) and weaker and more variable in group B (Fig. 4i, l, o). In 397 addition, we observed Dlx3b activity in both groups (Fig. 4k, l and Supplementary Fig. S3). Therefore Fgf signalling activity and Dlx3b expression, representing two prerequisites for otic induction, were present in the anterior regions of both groups. However, in group B embryos, no Foxi1 expression was detected in this region (Fig. 4o), contrary to group A (Fig. 4n). The differential Foxi1 expression in the two groups of embryos could be critical for the induction of ectopic otic development (Fig. 4n, o). Indeed we previously found a central role for this competence factor in the preplacodal gene regulatory network in medaka (Aghaallaei et al. 2007). Loss-of- function experiments in zebrafish also confirm a critical role of Foxi1 (Lee et al. 2003; Solomon et al. 2003). The main result of pulsed Wnt activation is the truncation of anterior structures. In order to study the role of canonical Wnt signalling on inner ear formation, it would be ideal to uncouple the effects on the otic induction process from those on axis formation. However, this is not possible with our experimental approach. Due to the axis truncations, an expanded r4 region is positioned next to the anterior expression domains of Dlx3b and Foxi1 in group A embryos. The close contact between this rhombomere and ectoderm positive for the competence factors is a hallmark of normal otic development. The ectopic activation of Foxi1 in group A embryos could be mediated by signals of this r4 region. In agreement with this, we previously described the activation of Foxi1 by Fgf8 in medaka embryos (Aghaallaei et al. 2007). However, we found equal expression of Fgf8 in both group A and group B embryos (Fig. 4e, f). Anterior Fgf3 expression was variable in group B (Fig. 4i, l, o), but since none of these embryos developed ectopic ear structures, Fgf3 expression is unlikely to be responsible for the differences. Therefore signals in addition to these Fgf ligands seem to be essential for the ectopic otic development in group A embryos. Alternatively, signals could be secreted from the hindbrain structures in group B embryos, which block the formation of otic tissue, but instead promote the midbrain–hindbrain boundary gene Engrailed 2. Interestingly, recent experiments with retinoic acid (RA) signalling in zebrafish revealed widespread ectopic otic induction upon excess activation of this pathway (Hans and Westerfield 2007). Foxi1 was ectopically activated in these embryos, whereas other marker genes for otic induction were not altered significantly. It is therefore possible that a genetic network of different signalling pathways (Fgf/RA/ Wnt) controls otic induction and in particular Foxi1. Further experiments will be necessary to clarify the role of Wnt signalling in this process. Acknowledgements We thank Jochen Wittbrodt and Mirana Ramialison for the medaka Starmaker probe, which was kindly provided before publishing. The work was supported by the Austrian Science Fund (FWF, grant 19571-B11).