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Mechanoreceptive hair cells are extremely sensitive to aminoglycoside antibiotics, including neomycin. Hair cell survival was assessed in larval wild-type zebrafish lateral line neuromasts 4 h after initial exposure to a range of neomycin concentrations for 1 h. Each of the lateral line neuromasts was scored in live fish for the presence or absence of hair cells using the fluorescent vital dye DASPEI to selectively label hair cells. All neuromasts were devoid of DASPEI-labeled hair cells 4 h after 500 lM neomycin exposure. Vital DASPEI staining was proportional to the number of hair cells per neuromast identified in fixed larvae using immunocytochemistry for acetylated tubulin and phalloidin labeling. The time course of hair cell regeneration in the lateral line neuromasts was also analyzed following neomycin-induced damage. Regenerated hair cells were first observed using live DASPEI staining 12 and 24 h following neomycin treatment. The potential role of proliferation in regenerating hair cells was analyzed. A 1 h pulse-fix protocol using bromodeoxyuridine (BrdU) incorporation was used to identify S-phase cells in neuromasts. BrdU incorporation in neomycin-damaged neuromasts did not differ from control neuromasts 4 h after drug exposure but was dramatically upregulated after 12 h. The proliferative cells identified during a 1 h period at 12 h after neomycin treatment were able to give rise to new hair cells by 24-48 h after drug treatment. The results presented here provide a standardized preparation for studying and identifying genes that influence vertebrate hair cell death, survival, and regeneration following ototoxic insults.

Environmental signals are important in the development of neural crest, during which process multipotent progenitor must choose from several fates. However, the nature of these environmental signals is unknown. A previous fate map of zebrafish cranial neural crest showed that lineage-restricted clones of pigment cells arise from medial cells near the neural keel, and that clones of neurons arise from lateral cells farther from the neural keel. Wnt-1 and Wnt-3a are candidate genes for influencing neural crest fate, as they are expressed next to medial, but not lateral, crest cells. Here we determine the role of Wnt signals in modulating the fate of neural crest by injecting messenger RNAs into single, premigratory neural crest cells of zebrafish. Lineage analysis of injected cells shows that activation of Wnt signalling by injection of mRNA encoding cytoplasmic beta-catenin promotes pigment-cell formation at the expense of neurons and glia. Conversely, inhibition of the Wnt pathway, by injection of mRNAs encoding either a truncated form of the transcription factor Tcf-3 or a dominant-negative Wnt, promotes neuronal fates at the expense of pigment cells. We conclude that endogenous Wnt signalling normally promotes pigment-cell formation by medial crest cells and thereby contributes to the diversity of neural crest cell fates.

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