Notch regulation of progenitor cell behavior in quiescent and regenerating auditory epithelium of mature birds

Abstract

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.

Gene-expression analysis of hair cell regeneration in the zebrafish lateral line

“…It is possible that active Notch or Fgf signaling induces cdk inhibitors, causing cells to exit the cell cycle or become quiescent (66). However, manipulation of Notch signaling does not affect the cell cycle in chick or murine inner ears (13,30), suggesting that signaling pathways other than Wnt/β-catenin and Notch are involved in triggering or inhibiting proliferation.…”

“…It is well established that the inhibition of Notch signaling causes an increase in the number of hair cells in uninjured and injured chicken, guinea pig, and mouse utricles and cochleae and in injured zebrafish ears and lateral line neuromasts (52,53,(55)(56)(57)88). The presence of Notch is detrimental to hair cell differentiation, because it blocks the proneural gene atoh1a and thereby maintains support cells (9,13,65,89,90). The ability of support cells to transdifferentiate into hair cells after down-regulation of Notch signaling was exploited recently to restore a modest amount of hair cells in the injured adult mouse cochlea that led to some promising functional recovery of hearing (30).…”

“…Work in birds has shed light on the mechanisms regulating sensory hair cell regeneration in vertebrates (2,4,(11)(12)(13)(14)(15)(16)(17)(18). For instance, during sensory hair cell regeneration in the chick, the majority of hair cells arise by proliferation of neighboring support cells (19).…”

Hair Cell Generation by Notch Inhibition in the Adult Mammalian Cristae

“…Pearson's correlation where *** denotes p≤0.001. chick basilar papilla (Ma et al 2008;Daudet et al 2009). Due to the damage in our adult cultures, we cannot preclude the possibility that damage is necessary for DAPT-induced hair cell generation.…”

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.…”