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Phenotype of the persephone mutant. (A) Hair cell protection in homozygous persephone mutants. 5 dpf zebrafish (progeny of a heterozygous incross) were treated with or without 200 μM neomycin for 30 min, and then rinsed. After 1 hr recovery in fresh embryo media, hair cells were labeled with the vital dye DASPEI. Left panels are differential interference contrast (DIC) images and right panels are corresponding fluorescent images. Top, wildtype sibling (+/+), mock-treated, Middle, wildtype sibling (+/+), treated with 200 μM neomycin, and Bottom, persephone homozygote treated with 200 μM neomycin. Red arrows indicate examples of neuromasts present in the untreated wildtype and treated persephone larvae. Mutant larvae show dramatic retention of hair cells relative to their siblings. (B) Parvalbumin antibody staining of hair cells in representative fish from an in-cross of persephone heterozygotes. Wildtype siblings show loss of hair cells when treated with 200 μM neomycin. Homozygous persephone mutants show a dramatic retention of parvalbumin-stained hair cells following neomycin treatment, consistent with DASPEI hair cell staining results. (C) Protection observed in persephone is not due to a delay in neomycin-induced hair cell death. persephone mutants (blue line) exposed to neomycin for 1 hr, rinsed and maintained for 6 or 24 hr in fresh EM, and then assessed for hair cell death, do not show significantly greater hair cell death than those assayed after 1 hr. Hair cell death is not delayed in persephone. Wildtype siblings treated in parallel (green line) are shown for comparison. (n = 10 fish, 10 neuromasts per fish; Error bars: S.D.; p value<0.001).
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