Retinoic acid signaling mediates hair cell regeneration by repressing p27kip and sox2 in supporting cells

During development, otic sensory progenitors give rise to hair cells and supporting cells. In mammalian adults, differentiated and quiescent sensory cells are unable to generate new hair cells when these are lost due to various insults, leading to irreversible hearing loss. Retinoic acid (RA) has strong regenerative capacity in several organs, but its role in hair cell regeneration is unknown. Here, we use genetic and pharmacological inhibition to show that the RA pathway is required for hair cell regeneration in zebrafish. When regeneration is induced by laser ablation in the inner ear or by neomycin treatment in the lateral line, we observe rapid activation of several components of the RA pathway, with dynamics that position RA signaling upstream of other signaling pathways. We demonstrate that blockade of the RA pathway impairs cell proliferation of supporting cells in the inner ear and lateral line. Moreover, in neuromast, RA pathway regulates the transcription of p27(kip) and sox2 in supporting cells but not fgf3. Finally, genetic cell-lineage tracing using Kaede photoconversion demonstrates that de novo hair cells derive from FGF-active supporting cells. Our findings reveal that RA has a pivotal role in zebrafish hair cell regeneration by inducing supporting cell proliferation, and shed light on the underlying transcriptional mechanisms involved. This signaling pathway might be a promising approach for hearing recovery. SIGNIFICANCE STATEMENT: Hair cells are the specialized mechanosensory cells of the inner ear that capture auditory and balance sensory input. Hair cells die after acoustic trauma, ototoxic drugs or aging diseases, leading to progressive hearing loss. Mammals, in contrast to zebrafish, lack the ability to regenerate hair cells. Here, we find that retinoic acid (RA) pathway is required for hair cell regeneration in vivo in the zebrafish inner ear and lateral line. RA pathway is activated very early upon hair cell loss, promotes cell proliferation of progenitor cells, and regulates two key genes, p27(kip) and sox2. Our results position RA as an essential signal for hair cell regeneration with relevance in future regenerative strategies in mammals.This work was supported by Fundación Areces XVI CN Grants BFU2011-27006 and BFU2014-55738 REDT to B.A., A.R.-M. was supported by a Juan de la Cierva fellowship. D.R. holds an FI Generalitat de Cataluña fellowship. We thank the Advanced Light Microscopy Unit and A. Mallabiabarrena for helping with the laser ablation experiments; K. Poss and H. Roehl for providing the tg (hsp70:zdnRAR) and tg (hsp70:fgf3) transgenic lines, respectively; C. Cañestro for sharing unpublished data; and members of the laboratory for helpful discussions and suggestions