The deletion of dhx38 caused inner ear development defects: (A) The different morphological specificities of the inner ears of different genotypes at distinct developmental stages. Scale bar: 40 µm. The black arrow indicates the otic vesicle, the white arrow indicates the otolith, and the red arrow indicates semicircular canal protrusion. (B) Statistical analysis of the otic area and otolith area in the embryos of different genotypes at 42 hpf, 45 hpf, 48 hpf, and 56 hpf. Individual embryos were picked casually from particular types for statistical analysis. The otic and otolith areas were measured using SPOT Advanced software (version 4.6) in the focal plane exhibiting the maximal area [16]. n, the number of checked embryos. n = 40. (C) The percentages of embryos that had semicircular canal protrusions in the sibling, the dhx38^+/− and dhx38^−/− groups at 48 hpf and 56 hpf. The data are presented as the mean ± SD. ns, p > 0.05; *, p < 0.05; ***, p < 0.001.

The expressions of some specification-related inner ear markers differed significantly between the sibling and dhx38^−/− mutant embryos: (A) The expressions of two otic matrix protein markers were normal at 36 hpf in the sibling and dhx38^−/− mutant embryos. Scale bar: 100 µm. (B) Two otic matrix protein markers displayed slight declines at 48 hpf and 56 hpf. Scale bar: 40 µm. (C) The in situ staining of markers for semicircular canal sensory cristae (bmp4), and saccular and utricular maculae marker (cahz), respectively. Scale bar: 40 µm. The epithelial pillar of the semicircular canal (ncs-1a), semicircular canal projections (dacha), and cilia (foxj1b). Scale bar: 100 µm. The red arrows indicate signal spots.

Apoptosis was increased in the inner ear epithelium cells of dhx38^−/− zebrafish: (A) TUNEL staining in the inner ears of dhx38 mutants at 36 hpf and 42 hpf. The white arrows indicate the signal spots.The n = 10 for each panel. Scale bar: 50 µm. (B) Quantitative analysis of apoptotic cells of the inner ear (the white, dotted, oval area) region between sibling and dhx38^−/− mutants. (C) The expression level of p53 pathway genes in sibling and dhx38^−/− mutants at 36 hpf and 42 hpf using qPCR. (D) TUNEL staining indicated that cell apoptosis was inhibited in the dhx38 zebrafish mutant embryos by inhibiting p53. The white arrows indicate the signal spots. Scale bar: 50 µm. (E) Quantitative analysis of TUNEL-positive cells in the inner ear (the white, dotted, oval area) region of siblings, dhx38^−/− homozygous embryos, and embryos with p53 inhibitor. (F) The inner ear morphology of different genotypes at 45 hpf and 48 hpf. (G) The statistical analysis of the otic lumen area at 45 hpf and 48 hpf. (H) The statistical proportion of protrusions at 45 hpf and 48 hpf. n = 30. The data are the mean ± SD. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Analysis of the accumulation of DNA damage in inner ears of dhx38^−/− mutants: (A) Whole-mount immunofluorescence analysis using the anti-γH2AX antibody in inner ear regions of siblings and dhx38^−/− at 36 hpf and 42 hpf. The white arrows indicate the signal spots. n = 10 for each panel. Scale bar: 50 µm. (B) Quantitative analysis of the γH2AX-positive cells exhibited in the inner ear (the white, dotted, oval area) region in (A). The data are presented as the mean ± SD. ***, p < 0.001.

Dhx38 regulates the alternative splicing of some genes involved in DNA damage repair: (A) Increased rate of transcripts with IR or ES among DNA repair in inner ears of dhx38 mutant embryos, as detected by PCR at 36 hpf. (B) The alternative splicing of these genes at 42 hpf. (C) The statistical data are presented as the mean ± SD of the PSI values at 36 hpf and 42 hpf from three biological replicates. PSI, percent splicing in. (D) The mRNA expression levels of some genes in inner ears of sibling and dhx38^−/− mutant embryos with DNA repair, as detected using RT-PCR. The data are presented as the mean ± SD. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

The abnormal splicing of genes involved inner ear development at 42 hpf: (A) The occurrence of an abnormal splicing event with IR and/or ES among genes associated with the development of the inner ear. (B) The statistical data of the PSI values at 42 hpf from three biological replicates. PSI, percent splicing in. (C) The downregulation of some genes with aberrant splicing in dhx38 mutants was detected using qRT-PCR at 42 hpf. The data are presented as the mean ± SD. ns, p > 0.05; *, p < 0.05; **, p < 0.01; ***, p < 0.001.