Unlike mammals, birds regenerate auditory hair cells (HCs) after injury. During regeneration, mature non-sensory supporting cells (SCs) leave quiescence and convert into HCs, through non-mitotic or mitotic mechanisms. During embryogenesis, Notch ligands from nascent HCs exert lateral inhibition, restricting HC production. Here, we examined whether Notch signalling (1) is needed in mature birds to maintain the HC/SC pattern in the undamaged auditory epithelium or (2) governs SC behavior once HCs are injured. We show that Notch pathway genes are transcribed in the mature undamaged epithelium, and after HC injury, their transcription is upregulated in the region of highest mitotic activity. In vitro treatment with DAPT, an inhibitor of Notch activity, had no effect on SCs in the undamaged epithelium. Following HC damage, DAPT had no direct effect on SC division. However, after damage, DAPT caused excessive regeneration of HCs at the expense of SCs, through both mitotic and non-mitotic mechanisms. Conversely, overexpression of activated Notch in SCs after damage caused them to maintain their phenotype and inhibited HC regeneration. Therefore, signalling through Notch is not required for SC quiescence in the healthy epithelium or to initiate HC regeneration after damage. Rather, Notch prevents SCs from regenerating excessive HCs after damage.

In chickens, nonsensory supporting cells divide and regenerate auditory hair cells after injury. Anatomical evidence suggests that supporting cells can also transdifferentiate into hair cells without dividing. In this study, we characterized an organ culture model to study auditory hair cell regeneration, and we used these cultures to test if direct transdifferentiation alone can lead to significant hair cell regeneration. Control cultures (organs from posthatch chickens maintained without streptomycin) showed complete hair cell loss in the proximal (high-frequency) region by 5 days. In contrast, a 2-day treatment with streptomycin induced loss of hair cells from all regions by 3 days. Hair cell regeneration proceeded in culture, with the time course of supporting cell division and hair cell differentiation generally resembling in vivo patterns. The degree of supporting cell division depended upon the presence of streptomycin, the epithelial region, the type of culture media, and serum concentration. On average, 87% of the regenerated hair cells lacked the cell division marker BrdU despite its continuous presence, suggesting that most hair cells were regenerated via direct transdifferentiation. Addition of the DNA polymerase inhibitor aphidicolin to culture media prevented supporting cell division, but numerous hair cells were regenerated nonetheless. These hair cells showed signs of functional maturation, including stereociliary bundles and rapid uptake of FM1-43. These observations demonstrate that direct transdifferentiation is a significant mechanism of hair cell regeneration in the chicken auditory after streptomycin damage in vitro.