FIGURE 2

Effect of Loxhd1b knockdown on decreasing the number of hair cells in the otic vesicle and posterior lateral line of zebrafish. (A) The targeting site of Loxhd1b splice blocking morpholino and the PCR primer design for validating the knockdown analysis. The wild type mature transcripts indicate the natural splicing product of Loxhd1b mRNA. The splicing MO mature transcripts indicate the abnormal splicing product of Loxhd1b mRNA with Exon 2 deletion caused by morpholino injection. (B) Effectiveness of Loxhd1b knockdown confirmed by PCR. (C) The schematic for three clusters of cristae hair cells in the otic vesicle. ACHC, anterior cristae hair cells; LCHC, lateral cristae hair cells; PCHC, posterior cristae hair cells. (D) Fluorescence microscopic imaging analysis of Loxhd1b knockdown line at 3 dpf. Arrowheads indicate hair cell clusters. Scale bar = 500 μm. (E) Confocal imaging analysis of cristae hair cells in the otic vesicle of control and Loxhd1b deficiency zebrafish at 72 and 96 hpf. Scale bar = 50 μm. (F) WISH experiments of the eya1 gene and the imaging analysis of control, claudin h morphants, and rescued zebrafish at 96 hpf in bright field. Scale bar = 500 μm. (G) Statistical analysis of zebrafish lateral line neuromast at 72 hpf. (H) Statistics of zebrafish lateral line neuromasts at 96 hpf. (I) Statistical analysis of the number of different cristae hair cells in the inner ear of control and Loxhd1b-MO in 72 hpf. (J) Statistical analysis of the number of different cristae hair cells in the inner ear of control and Loxhd1b-MO in 96 hpf. (K) C-startle response in Loxhd1b morphants zebrafish larvae was significantly lower than that in control zebrafish. **P < 0.01, ***P < 0.001, and ****P < 0.0001.