The phylogenetic and expression analysis of zebrafish claudin h. (A) The alignment of claudin h amino acid sequences from different species, and the identical aa residues among all the aligned sequences are labeled with color. (B) Phylogenetic analysis of claudin h. Neighbor-joining tree was produced with the Mega 5.0 software and the red star marked the zebrafish. (C,D) At 72 hpf, the in situ hybridization signal of claudin h is localized in the otic vesicle and neuromast. The red dotted line marked the boundary of the otic vesicle and the red arrow head marked the neuromast in the head and posterior lateral line. (C′,D′) The magnified figure of the positive signals in otic vesicle and neuromast line.

Loss function of claudin h caused the defects of otic vesicle and otoliths. (A,D) Imaging analysis of otic vesicle and otoliths in control and claudin h knocking down groups at 72 and 96 hpf. The yellow dotted line marked the boundary of the otic vesicle. Scale bar = 50 μm. (B,C) The statistical analysis of otic vesicle area in the control and claudin h morphants at 72 and 96 hpf. (E) Quantification of zebrafish embryos with abnormal otolith (defects in both number and shape of otoliths: claudin h morphants lost the utricle otolith or had a unnormal saccular otolith). Each bar represents the mean ± SE. Values with **** above the bars are significantly different (P < 0.0001).

Loss function of the claudin h caused the vestibular dysfunction and hearing defects. (A) The schematic diagram shows the rotatory trajectory of the larva during VOR. (B) The heads and eyes of control and claudin h morphants acquired during VOR test at extreme tilting positions. (C,D) The vestibular function of zebrafish larvae at 5 dpf is evaluated by vestibular head tilt response measurement (right and left eyes, respectively). (E) The schematic diagram shows the startle response testing equipment. (F) The swimming trajectory of the control and claudin h morphants. (G,H) Swimming distance and peak velocity of zebrafish larvae at 5 dpf that reflected the auditory function of zebrafish larvae by examining the startle response. Values with *, ***, and ****above the bars are significantly different (P < 0.05, P < 0.001, and P < 0.0001, respectively).

Claudin h deficiency suppressed cristae hair cells development. (A) The schematic for three different cristae hair cells in the otic vesicle. ACHC, anterior cristae hair cells; LCHC, lateral cristae hair cells; PCHC, posterior cristae hair cells. (B) Confocal imaging analysis of cristae hair cells in the otic vesicle of control and claudin h deficiency zebrafish at 72 and 96 hpf. The red dotted circle line marked the three different cristae hair cell clusters and magnified lateral cristae hair cell clusters (yellow arrow head) was shown in right and the yellow dotted square line marked the cilia of cristae hair cells. (C,D) The statistical analysis of the numbers of different cristae hair cells in the control and claudin h morphants at 72 and 96 hpf. (E,F) The statistical analysis of the cilia lengths of different cristae hair cells in the control and claudin h morphants at 72 and 96 hpf. Values with *, ***, and ****above the bars are significantly different (P < 0.05, P < 0.001, and P < 0.0001, respectively).

Claudin h knockdown decreased hair cell in the posterior lateral line of zebrafish. (A) The imaging analysis of control and claudin h morphants at 72 and 96 hpf in bright field and fluorescent field. Scale bar = 500 μm. (B,C) Quantification of the number of hair cell clusters in the posterior lateral line of control and claudin h morphants at 72 and 96 hpf. (D) The schematic for different hair cell clusters in the posterior lateral line. Scale bar = 10 μm. (E) Confocal imaging analysis of L1 hair cell clusters in the posterior lateral line of control and claudin h deficiency zebrafish at 72 and 96 hpf. (F,G) Quantification of the number of hair cells per L1 neuromast in the control and claudin h morphants at 72 and 96 hpf. Values with **, ***, and ****above the bars are significantly different (P < 0.01, P < 0.001, and P < 0.0001, respectively).

Overexpression of claudin h could rescue the development defects of hair cells in claudin morphants. (A) Imaging analysis of otic vesicle and cristae hair cells in control, claudin h morphants, and rescue group at 96 hpf. The yellow dotted line marked the boundary of the otic vesicle. Scale bar = 50 mm. (B,C) The statistical analysis of otic vesicle area in different groups at 96 hpf. (C,D) The statistical analysis of the number of different cristae hair cells and the cilia lengths in different groups at 96 hpf. (E) WISH results of the eya1 gene and the imaging analysis of control, claudin h morphants and rescue zebrafish at 96 hpf in bright field and fluorescent field. Scale bar = 500 μm. (F) Quantification of the number of hair cell clusters in the posterior lateral line of different groups at 96 hpf. (G) Confocal imaging analysis of L1 hair cell clusters in the posterior lateral line of different groups at 96 hpf. (H) Quantification of the number of hair cells per L1 neuromast in the control, claudin h morphants, and rescue zebrafish at 96 hpf. Values with *, **, ***, and ****above the bars are significantly different (P < 0.05, P < 0.01, P < 0.001, and P < 0.0001, respectively). (I) At 72 hpf, the in situ hybridization signal of claudin h in the control zebrafish, claudin h morphants, and rescue zebrafish.

Claudin h deficiency caused the hair cells apoptosis. (A) DAPI and cleaved caspase-3 staining for the L1 hair cell clusters in the posterior lateral line of the control zebrafish and claudin h morphants. Scale bar = 10 μm. (B) Quantification of zebrafish embryos with the hair cell apoptosis in the control and claudin h morphants. Values with **** above the bars are significantly different (P < 0.0001).

Knocking out of claudin h caused the defects of otic vesicle and impaired the development of cristae hair cells and neuromast hair cells. (A) Imaging analysis of otic vesicle and cristae hair cells in control and claudin h mutants at 96 hpf. The yellow dotted line marked the boundary of the otic vesicle. (B) The statistical analysis of otic vesicle area in control and claudin h mutants at 96 hpf. (C,D) The statistical analysis of the number of different cristae hair cells and the cilia lengths in control and claudin h mutants at 96 hpf. (E) The imaging analysis of control and claudin h mutants at 96 hpf in bright field and fluorescent field. (F) Confocal imaging analysis of L1 hair cell clusters in the posterior lateral line of control and claudin h mutants at 96 hpf. (G,H) Quantification of the number of hair cell clusters and the number of hair cells per L1 neuromast in the posterior lateral line of control and claudin h mutants at 96 hpf. Values with *, ***, and ****above the bars are significantly different (P < 0.05, P < 0.001, and P < 0.0001, respectively).