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Fig. 10 High-throughput hearing gene screening in F0 knockouts using Yo-Pro-1 and acoustic evoked behavior response (AEBR) assay. (A) Representative fluorescent images of Yo-Pro-1 uptake for each F0 with an enlarged image of a neuromast placed in the bottom right corner. The images are shown in lateral view with the anterior to the left. A homozygous cdh23 knockout (cdh23-/-) was used as a positive control for the hearing loss phenotype. For example, neuromast morphology can be observed under bright-field, but no Yo-Pro-1-positive (Yo-Pro-1+) cells are present. Images for other hearing genes were presented in Supplementary Fig. S22 and Supplementary Fig. S23. (B) Quantification of the average number of Yo-Pro-1+ cells per neuromast (n = 4 neuromasts) of each F0 knockout (n = 5 larvae). Since the average number of Yo-Pro-1 + cells per neuromast was around 10 cells for uninjected controls across multiple experiments, we combined all numbers (n = 45 larvae). Error bars indicate the mean ± SD. Statistical significance was compared to the uninjected control group and calculated by Brown–Forsthye and Welch's ANOVA with Dunnett's T3 multiple comparisons test: not significant (ns) P ≥ 0.05, *P < 0.05, ***P < 0.001, and ****P < 0.0001. (C) A stable homozygous cdh23 knockout (cdh23-/-) was used as a positive control for the hearing loss phenotype. The AEBR assay results for other hearing genes were presented in Supplementary Fig. S24 and Supplementary Fig. S25. The percentage of responses to 12 sound stimuli for AEBR was calculated for each larva using AEBR. Statistical significance was determined using two-tailed unpaired Student's t test with Welch's correction: not significant (ns) P ≥ 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared to uninjected controls from the same batch.