Fig. 1

Phenotypes of zebrafish cyb5d1 mutants. (A and B) Whole-mount in situ hybridization results showing cyb5d1 expression in 14 somite (14 s) and 24 hpf embryos. OV, otic vesicle; KV, Kupffer’s vesicle; FL, floor plate; PD, pronephric duct; OP, olfactory placode. (C) Diagram showing the protein and genomic structures of zebrafish Cyb5d1. The corresponding nucleic acid sequences of wild type (wt) and mutant alleles are shown at the bottom. The target sequence of the sgRNA is underlined, and the PAM sequence is indicated in red. Arrow points to the truncation site of Cyb5d1 in the mutant. (D) Phenotypes of otoliths in the OV of wt and cyb5d1 mutants at different stages as indicated. Arrowheads indicate otoliths in the OV of 24 hpf embryos. (Scale bar, 100 μm.) (E) Bar graphs showing the percentage of embryos with two (normal; N) or three otoliths (three separate, 3S; three with two fused together, 3F) in different groups of embryos as indicated. Examples of embryos with 3S or 3F otoliths are shown in SI Appendix, Fig. S1. n = 447 for cyb5d1 mutants and n = 449 for wild type larvae. (F) Kymographs showing the beating pattern of multiple cilia bundles in the olfactory pit of wild type (cMO) and cyb5d1 morphants (cyb5d1 MO) in 72 hpf. (G) Beat frequency of otic vesicle motile cilia in wild type and cybd51 mutant embryos at the 23 somite stage. (H) Beat frequency of olfactory cilia in wild type (cMO) or cyb5d1 morphants (cyb5d1 MO) at 72 hpf. n.s., not significant; ***P < 0.001. Error bars are SDs for the means.