Figure 2.

DiAsp staining and blockade of RA activity with DEAB confirm that RAR activity is required for hair cell regeneration. A, Hair cell regeneration was evaluated by labeling hair cells with DiAsp, which labels mature hair cells. Schematic representation of experimental procedure: a 45 min 1 h heat-shock was performed 1 h after lateral crista laser ablation. Before laser ablation and imaging (48 hpa and 6 dpa), a DiAsp solution was injected into the inner ear lumen. B, Inner ear reconstructed view of the three cristae labeled with DiAsp. C, D, Hair cell regeneration in ablated crista is significantly impaired at 48 hpa in dnRAR-expressing larvae (D). In nonablated crista, overexpression of dnRAR does not affect the number of hair cells (C). E, F, RA synthesis was pharmacologically inhibited in 4.5-d-old tg(brn3c-mGFP) larvae using DEAB, an inhibitor of aldh1 activity. Brn3c GFP-positive hair cells were imaged and quantified at 2, 24, and 48 hpa and 6 dpa in DMSO- and DEAB-treated larvae. F, Blockade of RA synthesis significantly impairs HC regeneration at 48 hpa and 6 dpa. In nonablated crista, DEAB treatment does not affect the number of hair cells (E). *p < 0.05. **p < 0.01. ac, anterior crista; lc, lateral crista; pc, posterior crista. Scale bars: A, 50 μm; F, 10 μm.