We developed a transgenic mouse to permit conditional and selective ablation of hair cells in the adult mouse utricle by inserting the human diphtheria toxin receptor (DTR) gene into the Pou4f3 gene, which encodes a hair cell-specific transcription factor. In adult wild-type mice, administration of diphtheria toxin (DT) caused no significant hair cell loss. In adult Pou4f3 +/DTR mice, DT treatment reduced hair cell numbers to 6% of normal by 14 days post-DT. Remaining hair cells were located primarily in the lateral extrastriola. Over time, hair cell numbers increased in these regions, reaching 17% of untreated Pou4f3 +/DTR mice by 60 days post-DT. Replacement hair cells were morphologically distinct, with multiple cytoplasmic processes, and displayed evidence for active mechanotransduction channels and synapses characteristic of type II hair cells. Three lines of evidence suggest replacement hair cells were derived via direct (nonmitotic) transdifferentiation of supporting cells: new hair cells did not incorporate BrdU, supporting cells upregulated the pro-hair cell gene Atoh1, and supporting cell numbers decreased over time. This study introduces a new method for efficient conditional hair cell ablation in adult mouse utricles and demonstrates that hair cells are spontaneously regenerated in vivo in regions where there may be ongoing hair cell turnover.

The capacity of adult mammals to regenerate sensory hair cells is not well defined. To explore early steps in this process, we examined reactivation of a transiently expressed developmental gene, Atoh1, in adult mouse utricles after neomycin-induced hair cell death in culture. Using an adenoviral reporter for Atoh1 enhancer, we found that Atoh1 transcription is activated in some hair cell progenitors (supporting cells) three days after neomycin treatment. By 18 days post-neomycin, the number of cells with Atoh1 transcriptional activity increased significantly, but few cells acquired hair cell features (i.e., accumulated ATOH1 or myosin VIIa protein or developed stereocilia). Treatment with DAPT, an inhibitor of γ-secretase, reduced notch pathway activity, enhanced Atoh1 transcriptional activity, and dramatically increased the number of Atoh1-expressing cells with hair cell features, but only in the striolar/juxtastriolar region. Similar effects were seen with TAPI-1, an inhibitor of another enzyme required for notch activity (TACE). Division of supporting cells was rare in any control or DAPT-treated utricles. This study shows that mature mammals have a natural capacity to initiate vestibular hair cell regeneration and suggests that regional notch activity is a significant inhibitor of direct transdifferentiation of supporting cells into hair cells following damage.

Regeneration of sensory hair cells in the mature avian inner ear was first described just over 20 years ago. Since then, it has been shown that many other non-mammalian species either continually produce new hair cells or regenerate them in response to trauma. However, mammals exhibit limited hair cell regeneration, particularly in the auditory epithelium. In birds and other non-mammals, regenerated hair cells arise from adjacent non-sensory (supporting) cells. Hair cell regeneration was initially described as a proliferative response whereby supporting cells re-enter the mitotic cycle, forming daughter cells that differentiate into either hair cells or supporting cells and thereby restore cytoarchitecture and function in the sensory epithelium. However, further analyses of the avian auditory epithelium (and amphibian vestibular epithelium) revealed a second regenerative mechanism, direct transdifferentiation, during which supporting cells change their gene expression and convert into hair cells without dividing. In the chicken auditory epithelium, these two distinct mechanisms show unique spatial and temporal patterns, suggesting they are differentially regulated. Current efforts are aimed at identifying signals that maintain supporting cells in a quiescent state or direct them to undergo direct transdifferentiation or cell division. Here, we review current knowledge about supporting cell properties and discuss candidate signaling molecules for regulating supporting cell behavior, in quiescence and after damage. While significant advances have been made in understanding regeneration in nonmammals over the last 20 years, we have yet to determine why the mammalian auditory epithelium lacks the ability to regenerate hair cells spontaneously and whether it is even capable of significant regeneration under additional circumstances. The continued study of mechanisms controlling regeneration in the avian auditory epithelium may lead to strategies for inducing significant and functional regeneration in mammals.

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.

Birds respond to hair cell loss by stimulating cell division in the otherwise mitotically quiescent sensory epithelium and by generating new hair cells. We examined cell proliferation during hair cell regeneration in chick basilar papilla by using 5-bromo-2'-deoxyuridine (BrdU). Chicks were noise exposed for 4 or 24 hours and injected with BrdU, and cochleae were immunohistochemically labeled to detect BrdU. Immunoreactivity after short-term postinjection survival identified when cells entered S phase. For both 4 and 24 hour exposures, cells in S phase were first detected in the sensory epithelium after an injection at 18 hours after the onset of exposure and were also present after injections at 24, 30, 36, 42, 48, 72, 96, 120, and 144 hours. The most cells in S (or G2) phase were detected at 42 and 72 hours for 24 hour exposures and at 48 hours for 4 hour exposures. Chicks that survived for long periods after injection had BrdU-labeled hair cells, indicating that precursor cells that divided in the presence of BrdU generated new hair cells. Moreover, labeled hair cells and supporting cells were grouped into discrete clusters, suggesting that cells within each cluster are clonally related. Support for this hypothesis was provided by experiments showing that the number of labeled cells increased when chicks survived for longer periods after a single BrdU injection. These findings suggest that progenitors within the sensory epithelium may undergo several rounds of division to generate the appropriate number of new hair cells and supporting cells.