394 Dev Genes Evol (2009) 219:391–398 Fig. 2 Temporal and dosage effects of Wnt1 activation during early development. Dorsal views of Wnt1-transgenic embryos. a The heat shock was performed at different stages for 2 h at 39°C. b Wnt1- transgenic embryos were heat-shocked at early gastrulation for different durations. The Gfp activity 1 day after heat shock was used as a marker. Ectopic otic vesicles are indicated by arrows. n the number of examined embryos, ep. epiboly, ey eye, fb forebrain, md midbrain, hb hindbrain, ov otic vesicle difference was the number of ectopic otic vesicles, which increased upon longer heat treatment (Fig. 2b, arrows). Next, we performed a time-lapse analysis. The experi- ments revealed that the cyclopic ear phenotype is an “end- product” of the fusion of the endogenous otic vesicles with several small ectopic structures into a single otic vesicle (Fig. 3, yellow arrows). The fusion of the vesicles started immediately after their formation (1-day-old embryos) and ended when the embryos where 3 days old and further development was arrested. Our observations show that the fusion process is dynamic and does not occur in a reproducible manner. However, the number of ectopic otic vesicles at the beginning is Wnt1 dosage dependent. To understand the molecular events responsible for the cyclopic ear phenotype, we analysed the expression pattern of some hindbrain and otic marker genes (for schematic presentation, see supplementary Fig. 2S online). For this purpose, one group of Wnt1-transgenic embryos (group A) was induced at 30% epiboly. For comparison, another group of Wnt1-transgenic embryos (group B) was induced at 50% epiboly, which did not develop ectopic otic vesicles. The embryos were fixed at a preplacodal stage (two-somite stage). For analysis of the hindbrain structures, we used probes directed against EphA4 and Hoxb1a, expressed in r3/r5 and r4, respectively (see “Materials and methods”; Fig 4a). The expression patterns of the hindbrain marker genes confirmed the truncations of the anteroposterior axis in the Wnt1 transgenic embryos. In group B embryos, r3 and all structures posterior to this rhombomere appeared normal (Fig. 4c, f, i, r), whereas in group A embryos, the last distinguishable rhombomere was r5 (Fig. 4b, e, h). Interestingly, the expression patterns of the marker genes suggest that the remaining anterior structures of the embryos are of one uniform fate. In group A embryos, this region anterior to r5 expressed Hox1a, Fgf8 and Fgf3 (Fig. 4b, e, h), which would be consistent with an r4 fate. In group B embryos, the region anterior to r3 expressed Fgf8 (Fig. 4f), indicating an r2 fate. However, we also found expression of Fgf3 and Engrailed 2 (En2; Fig. 4i, r), normally not expressed in r2. In wild-type embryos, these