Atoh1 expression defines activated progenitors and differentiating hair cells during avian hair cell regeneration

Abstract

In the avian inner ear, nonsensory supporting cells give rise to new sensory hair cells through two distinct processes: mitosis and direct transdifferentiation. Regulation of supporting cell behavior and cell fate specification during avian hair cell regeneration is poorly characterized. Expression of Atoh1, a proneural transcription factor necessary and sufficient for developmental hair cell specification, was examined using immunofluorescence in quiescent and regenerating hair cell epithelia of mature chickens. In untreated birds, Atoh1 protein was not detected in the auditory epithelium, which is quiescent. In contrast, numerous Atoh1-positive nuclei were seen in the utricular macula, which undergoes continual hair cell turnover.

Supporting Cell Division Is Not Required for Regeneration of Auditory Hair Cells After Ototoxic Injury In Vitro

“…If this were true, we should be able to detect cells in the process of conversion that possess features of both SCs and HCs, as has been demonstrated in the chicken BP after in vivo HC damage (Cafaro et al 2007). To test this hypothesis, we labeled BPs that were cultured with streptomycin for 3 days with antibodies to Sox2 or ß-tectorin precursor, both of which are selective markers for SCs (Goodyear et al 1996;Cafaro et al 2007;Daudet et al 2009), and antibodies to Atoh1, an early marker of differentiating HCs (Cafaro et al 2007). In these cultures, we indeed found examples of cells that labeled for Atoh1 and either Sox2 or ß-tectorin precursor, suggestive of cells transferring from an SC to an HC state ( Supplementary Fig.…”

“…These observations suggest many HCs were regenerated by direct transdifferentiation, during which an SC converts phenotypically into an HC without dividing. Further evidence for this phenomenon, which was proposed in the mid-1990s (Baird et al 1996;Adler and Raphael 1996;Roberson et al 1996), was recently provided by the observation that SCs upregulate the HC-specific protein Atoh1 shortly after HC damage is initiated in chickens in vivo (Cafaro et al 2007). We provide additional evidence for "transitional" cells here.…”

“…However, dividing SCs were focused in two stripes-along the neural and abneural edges-that did not reflect the widespread loss of HCs in a given region. A neural stripe of SC division was described for the chicken BP after in vivo gentamicin treatment (Cafaro et al 2007), although the significance of this pattern is not understood. It is unclear if the abneural stripe of SC division is a culture artifact or a normal phenomenon that is amplified by culture conditions.…”

“…In support of this, we found that BrdU-negative HCs appeared more highly differentiated (had developed stereociliary bundles) at 8 days than BrdU-positive HCs. In addition, during in vivo avian auditory HC regeneration, direct transdifferentiation precedes cell division by 24-48 h (Roberson et al 2004;Cafaro et al 2007). Alternative explanations for these findings are that differentiation of postmitotic cells into HCs is delayed in culture and/ or that incorporated BrdU specifically delays or inhibits HC differentiation.…”

“…In chickens, some regenerated auditory HCs are unlabeled for a proliferation marker ( 3 H-thymidine or bromodeoxyuridine (BrdU)) despite its continual perfusion into the inner ear after damage (Roberson et al 1996(Roberson et al , 2004. Cells with features of both HCs and SCs were seen in regenerating BPs, suggesting such cells were SCs converting into HCs (Adler et al 1997;Cafaro et al 2007). Also, attenuation of SC division did not prevent the reemergence of HCs in the BP (Adler and Raphael 1996).…”