392 that otic placode induction occurs normally in the absence of Wnt8 expression (Phillips et al. 2004). In that study, morpholino knockdown of the Wnt8 gene and misexpression of the Wnt antagonist dickkopf 1 failed to block otic induction. Phillips and colleagues proposed that Wnt serves to regulate the temporal expression of Fgf3 and Fgf8 in the hindbrain, hence playing an indirect role in otic induction (Phillips et al. 2004). Furthermore, Wnt signalling was proposed to affect the preplacodal state in chicks (Bailey and Streit 2006), and in mice, it was suggested to be required after Fgf-dependent induction to maintain otic fate (Ohyama et al. 2006). Here, we address the question whether Wnt signalling is involved in otic induction in medaka fish. We demonstrate that ectopic activation of this pathway affects otic development at the time of induction by juxtaposing regions normally separated in the embryo. This is in good agreement with hindbrain tissue as a source for otic-inducing signals. However our experiments suggest that signals in addition to Fgf3 and Fgf8 induce the formation of ectopic ear structures in medaka. Materials and methods Fish strain and transgenic lines Embryos of the medaka Cab inbred strain were used for all experiments. Stages were determined according to Iwamatsu (2004). All studies on heat-inducible Wnt1-transgenic embryos were carried out using three independent lines described previously (Bajoghli et al. 2007). Embryo injections Medaka embryos were microinjected into single blasto- meres at the one- to two-cell stage. mRNA of mouse Wnt1 was injected at 200 ng/μl. For transient experiments, a heat- inducible Wnt1 construct (Bajoghli et al. 2007) was injected at 10 ng/μl together with I-SceI meganuclease. Isolation of medaka EphA4 and Hoxb1a probes The primers used for the RT-PCR were as follows: for EphA4, 5- GAAAAGAACATCCCCATTCG-3 and 5-GAGTTGCG TTCCTCATATCCT-3; for Hoxb1a, 5-ATGGAAAACATG AACTCCTTTG-3 and 5-GTGCGGGACCGTTAGGTA-3. The DNA fragments were cloned into the pGEM-Teasy vector (Promega). Whole-mount in situ hybridisation Whole-mount in situ hybridisation was performed as described previously using DIG- or FITC-labelled probes (Aghaallaei et al. 2005). Dev Genes Evol (2009) 219:391–398 Results To elucidate the function of Wnt signalling during medaka otic induction, we decided to use gain-of-function analysis. Wnt1, Wnt8 and Wnt3a proteins define a functional class of Wnt ligands (Torres et al. 1996), which preferentially stimulate the canonical Wnt pathway (reviewed by Logan and Nusse 2004). We selected Wnt1 for our misexpression experiments. Injection of Wnt1 mRNA into medaka embryos resulted in a posteriorised phenotype characterised by severe truncations of anterior structures (Fig. 1b). Similar phenotypes have been observed upon ectopic activation of canonical Wnt signalling in various vertebrate species (Kelly et al. 1995; Stachel et al. 1993; van de Water et al. 2001; Yamaguchi and Shinagawa 1989). Since Wnt signalling has multiple functions during early embryonic development depending on the time window (Liu et al. 2005), we switched to induced expression using a heat-shock-inducible system (Bajoghli et al. 2004). Activa- tion of the injected heat-shock Wnt1 construct at early gastrulation resulted in the same posteriorisation phenotype seen for mRNA. However, 18% of the embryos (13 of 70) showed enlarged otic vesicles (data not shown), and in addition, 7% of the embryos (five of 70) exhibited small ectopic otic vesicles located anteriorly to the endogenous otic vesicle (Fig 1c, arrow). When we repeated these experiments with an expression construct for the zebrafish Wnt8 gene, we also observed ectopic otic vesicles in 20% of the embryos (11 of 56; data not shown). These experiments are consistent with data for injection of a Wnt8 expression construct in zebrafish (Phillips et al. 2004). In order to overcome the mosaic nature of the injection technique, we repeated the experiments with a heat-shock Wnt1 transgenic medaka line (Bajoghli et al. 2007). In agreement with transient misex- pression, uniform activation of Wnt1 in the whole embryo at early gastrulation led to posteriorisation. However, instead of the isolated ectopic vesicles observed before, one large vesicle appeared in the Wnt1-transgenic embryos, covering the complete anterior region starting from the position of the endogenous otic vesicles (Fig 1d). At later stages, the vesicle forms inner ear structures and contains multiple otoliths (Supplementary Fig. S1). We confirmed the identity of this ectopic otic structure by marker gene expression. The expression patterns of Starmaker (Fig. 1f), Eya1 and Six1 identified the structures as otic vesicles (Supplementary Fig. S1). Due its similarity with cyclopic eyes, we named this phenotype “cyclopic ear”. In order to learn more about the effect of timing, we performed a time-course experiment by systematically varying the time of heat-shock activation in Wnt1 trans- genic embryos from 30% epiboly to the three-somite stage (Fig. 2a). The experiments indicate a strict time dependence on the extent of the anterior truncations. Early activation of Wnt signalling leads to a severe truncation of the body axis