In situ hybridization
Developmentally staged zebrafish (13, 18 and 24 hpf) embryos were fixed with 4% paraformaldehyde (Sigma) during an over-night incubation at 4℃. Whole-mount in situ hybridization was performed using the conventional alkaline-phosphatase-based single-color method (Kudoh et al., 2001). We used a gene paired box 2a (pax2a ) (Sanek et al., 2009; Lee et al., 2008) and paired box 6a (pax6a ) (Sanek et al., 2009; Lee et al., 2008) at 18 and 24 hpf, neuronal differentiation 4 (neurod4 ; previously known as zath3 ) (Miyake et al., 2012) at 24 hpf.