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Figure 4. Inhibition of SHH signaling with cyclopamine resulted in developmental defects in the midbrain of zebrafish embryos at 24 hpf. (A–D) Zebrafish embryos at 24 hpf which were treated 0.2% and 0.6% EtOH from 4 hpf. 0.6% EtOH-exposed embryos showed similar phenotypes of midbrain and MHB in comparison to those of 0.2% EtOH-exposed embryos. (E and F) Cyclopamine-treated embryos (20 μM) showed reduction in width of the midbrain and dorsally shrunken midbrain in comparison to those of EtOH controls. (G and H) 60 μM cyclopamine-treated embryos exhibited severe defects in the midbrain. (I–L) Spatiotemporal expression patterns of otx2b in the midbrain of cyclopamine-treated embryos at 24 hpf. CCP, cyclopamine. (A–L) Scale bars: 50 μm.