Fig. 4

Fig. 4

Clarin 2 is required for the inner ear function in zebrafish. a RT-qPCR of clrn2 mRNA expression from 1 to 120 hpf of WT embryos/larvae. clarin 2 mRNA expression can be detected starting from 18 hpf and then increased throughout development. Data shown are mean ± SD and compared to 18 hpf. b RT-qPCR of clrn2 mRNA expression in different adult tissues. Data shown are mean ± SD and compared to muscle. c–d Whole-mount in situ hybridization (WISH) using antisense clrn2 probe reveals the inner ear expression of clrn2 mRNA (relative dark purple color, black arrowhead) at 3 (c) and 5 (d) dpf embryos. Sense clrn2 probe was used as negative control and relative light purple color is considered as background. clrn2 mRNA was consistently expressed in hair cells within inner ear macula (c–d) with lined and arrayed structure. A known hair cell marker pvalb9 was used as an indicator for hair cells in the inner ear of 3 dpf embryos (c). Cryosection was performed after clrn2 WISH at 5 dpf to confirm the small patch of signal on the macula is from hair cells rather than supporting cells (d, black arrow lower panel). Scale bar = 100 µm, except lower panel in D (20 µm). e RT-qPCR of clrn2 mRNA expression level was decreased 70% in clrn2 crispants compared to uninjected larvae, indicating clrn2 was successfully knocked out (p = 2.06E-06). Data shown are mean ± SD. ***p < 0.001, two-tailed unpaired Student’s t test. f Acoustically evoked behavioral responses (AEBR) in clrn2 wild type and crispants reveal significant reduction of sound induced responses. g Phalloidin staining on clrn2 crispants show that the hair cells in the inner ear anterior and posterior maculae display splayed, thin and split structures (purple arrowheads). A, anterior to the left. D, dorsal to the top. V, ventral to the top. Scale bar = 10 µm