Hair Cell Staining, Counting, and Regeneration

Hair cell staining and counting were as described.^{63 (link)} For analysis of hair cell development, WT or mutant embryos at 5 dpf were placed in a cell strainer (BD Falcon, San Jose, CA, USA) and stained with 2 μmol/l YoPro-1 (Life Science) for 5–15 min and then lateral line neuromast hair cells were counted using fluorescent imaging (inverted Zeiss Axiophot, ×10 magnification, Bethesda, MD, USA). For hair cell sensitivity analysis, WT or mutant embryos at 5 dpf were treated with the ototoxic drugs copper sulfate (Sigma) at 10 μmol/l for 30 min or neomycin (Sigma) at 200 μmol/l for 30 min, and then were immediately stained with YoPro-1 and used for hair cell counting. For hair cell regeneration analysis, WT or mutant embryos at 5 dpf were treated with the ototoxic drugs copper sulfate at 10 μmol/l for 2 h or neomycin at 200 μmol/l for 1 h, allowed to recover for 2 days and then regenerated hair cells were counted at posterior lateral line positions of P1, P2, P4 and P5.^{64 (link)} For each of the above experiments, ~10 embryos were used for the analysis and repeated three times. Hair cells from four neuromasts in each embryo were counted. The average number of hair cells and the s.e.m. were presented in the graphs.