Jennifer S. Stone

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

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. Atoh1‐positive nuclei emerged in the auditory epithelium by 15 hr post‐ototoxin administration, before overt hair cell damage and supporting cell re‐entry into the cell cycle. Subsequently, Atoh1 labeling was seen in 15% of dividing supporting cells. During cell division, Atoh1 was distributed symmetrically to daughter cells, but Atoh1 levels were dramatically regulated shortly thereafter. After cellular differentiation, Atoh1 labeling was confined to hair cells regenerated through either mitosis or direct transdifferentiation. However, Atoh1 expression in dividing progenitors did not necessarily predict hair cell fate specification in daughter cells. Finally, predominant modes of hair cell regeneration varied significantly across the radial axis of the auditory epithelium, with mitosis most frequent neurally and direct transdifferentiation most frequent abneurally. These observations suggest a role for Atoh1 in re‐specifying supporting cells and in biasing postmitotic cells toward the hair cell fate during hair cell regeneration in the mature chicken ear. Developmental Dynamics 236:156–170, 2007.

Inhibition Of Notch Activity Promotes Nonmitotic Regeneration of Hair Cells in the Adult Mouse Utricles

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) 3 d after neomycin treatment. By 18 d after 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.

RECENT INSIGHTS INTO REGENERATION OF AUDITORY AND VESTIBULAR HAIR CELLS

Advances in hair cell regeneration are progressing at a rapid rate. This review will highlight and critique recent attempts to understand some of the cellular and molecular mechanisms underlying hair cell regeneration in non-mammalian vertebrates and efforts to induce regeneration in the mammalian inner ear sensory epithelium.

Feathers and fins: Non-mammalian models for hair cell regeneration

Death of mechanosensory cells in the inner ear results in two profound disabilities: hearing loss and balance disorders. Although mammals lack the capacity to regenerate hair cells, recent studies in mice and other rodents have offered valuable insight into strategies for stimulating hair cell regeneration in mammals. Investigations of model organisms that retain the ability to form new hair cells after embryogenesis, such as fish and birds, are equally important and have provided clues as to the cellular and molecular mechanisms that may block hair cell regeneration in mammals. Here, we summarize studies on hair cell regeneration in the chicken and the zebrafish, discuss specific advantages of each model, and propose future directions for the use of non-mammalian models in understanding hair cell regeneration.

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

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 signaling (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, signaling 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.

Temporal, Spatial, and Morphologic Features of Hair Cell Regeneration in the Avian Basilar Papilla

Hair cell–selective antibodies were used in combination with the nucleotide bromodeoxyuridine (BrdU) to examine the temporal, spatial, and morphologic progression of auditory hair cell regeneration in chicks after a single gentamicin injection. New hair cells are first identifiable with an antibody to class III beta (β) tubulin (TuJ1) by 14 hours after BrdU incorporation, but progenitor cells in S phase and M phase are TuJ1‐negative. TuJ1 labeling reveals that new hair cells are first detected at 3 days after gentamicin, in the base, and the emergence and maturation of regenerating hair cells spreads apically over time. Differentiation of regenerating hair cells consists of a progressive series of morphologic changes. During early differentiation (14 hours to 1 day after BrdU), regenerating hair cells are round or fusiform and remain near the lumen, where they are generated. During intermediate differentiation (2–4 days after BrdU), regenerating hair cells resemble support cells; their somata are elongated, their nuclei are in the support cell layer, and they appear to contact both the lumenal surface and the basal lamina. The 275‐kDa hair cell antigen is first expressed in regenerating hair cells during this period. During late differentiation (7 days after BrdU and later), TuJ1‐positive cells acquire the globose shape of mature hair cells. Labeling with antibodies to hair cell antigen, calmodulin, and ribosomal RNA confirms this morphologic progression. Examination of sister cells born at 3 days post‐gentamicin reveals that there is equal likelihood that they will assume the hair cell or support cell fate (i.e., both asymmetric and symmetric differentiation occur). J. Comp. Neurol. 417:1–16, 2000. ©2000 Wiley‐Liss, Inc.

A brief history of hair cell regeneration research and speculations on the future

Millions of people worldwide suffer from hearing and balance disorders caused by loss of the sensory hair cells that convert sound vibrations and head movements into electrical signals that are conveyed to the brain. In mammals, the great majority of hair cells are produced during embryogenesis. Hair cells that are lost after birth are virtually irreplaceable, leading to permanent disability. Other vertebrates, such as fish and amphibians, produce hair cells throughout life. However, hair cell replacement after damage to the mature inner ear was either not investigated or assumed to be impossible until studies in the late 1980s proved this to be false. Adult birds were shown to regenerate lost hair cells in the auditory sensory epithelium after noise- and ototoxic drug-induced damage. Since then, the field of hair cell regeneration has continued to investigate the capacity of the auditory and vestibular epithelia in vertebrates (fishes, birds, reptiles, and mammals) to regenerate hair cells and to recover function, the molecular mechanisms governing these regenerative capabilities, and the prospect of designing biologically-based treatments for hearing loss and balance disorders. Here, we review the major findings of the field during the past 25 years and speculate how future inner ear repair may one day be achieved.

Hair Cell Replacement in Adult Mouse Utricles after Targeted Ablation of Hair Cells with Diphtheria Toxin

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.

Progenitor cell cycling during hair cell regeneration in the vestibular and auditory epithelia of the chick.

We investigated nucleotide-labeling patterns during ongoing hair cell regeneration in the avian vestibular epithelium and during drug-induced regeneration in the avian auditory epithelium. For utricle experiments, post-hatch chicks received an injection of bromodeoxyuridine (BrdU) and were allowed to survive from 2 hours to 110 days after the injection. Utricles were fixed and immunoreacted to detect BrdU. The number of BrdU-labeled nuclei in the hair cell and support cell layers of the utricular sensory epithelium changes significantly between 2 hours and 110 days post-BrdU. At 2 hours, most labeled cells are isolated, while by 5–10 days, the majority of labeled cells are organized in pairs that are most frequently composed of a hair cell and a support cell. Pairs of labeled cells are seen as late as 110 days. Clusters of more than 3 labeled cells are uncommon at all time-points. The total number of labeled cells increases approximately 1.5-fold between 5 and 60 days post-BrdU. This increase is due primarily to a rise in the number of labeled support cells, and it is likely that it represents additional rounds of division by a subset of cells that were labeled at the time of the BrdU injection. There is a significant decrease in labeled nuclei in the hair cell layer between 60 and 110 days post-BrdU, suggesting that hair cells die during this period. To investigate support cell recycling in the drug-damaged auditory epithelium, we examined nucleotide double labeling after separate injections of BrdU and tritiated thymidine. A small number of support cells that incorporate BrdU administered at 3 days post-gentamicin treatment also label with tritiated thymidine administered between 17 and 38 hours later. We conclude that a small population of support cells recycles during regeneration in both the normal utricle and the drug-damaged basilar papilla.

Dynamic Studies of Ototoxicity in Mature Avian Auditory Epithelium

ABSTRACT: Hearing loss induced by ototoxicity is a worldwide problem despite the development of newer antibiotics and chemotherapy agents. The cellular mechanisms responsible for aminoglycoside‐induced hearing loss are still poorly understood. We have developed two different methods of studying the dynamic cellular and subcellular changes in the chick auditory sensory epithelium that occur during hair cell death. The first study was performed in mature chicks after a single, high dose injection of gentamicin, which results in the rapid loss of all hair cells in the basal third of the cochlea. Chicks were sacrificed at discrete time points after drug treatment, and transmission electron microscopy was performed to study the ultrastructural changes in basal hair cells during the course of cell death. We noted various changes in the cell morphology including accumulation of cytoplasmic inclusion bodies, dispersion of the cytoplasmic polyribosomes, mitochondrial swelling, and cellular extrusion by 24 h after injection. The next two studies were performed using tissue cultures from mature avian auditory sensory epithelium. Cultured cells were labeled using vital fluorescent markers, and levels of intracellular calcium and reactive oxygen species within hair cells were studied following aminoglycoside exposure. We identified a dose‐dependent increase in the levels of intracellular calcium, which was blocked by an inhibitor of voltage‐gated calcium channels. We also found that levels of reactive oxygen species in hair cells greatly increased after exposure to gentamicin, and this response was blocked by two different antioxidants. These studies serve to identify key cellular and molecular changes in hair cells in response to ototoxic drugs. Further study of these processes may lead to a better understanding of how ototoxicity is induced and to potential preventative interventions.