Elizabeth C. Oesterle

Sox2 and Jagged1 Expression in Normal and Drug-Damaged Adult Mouse Inner Ear

Inner ear hair cells detect environmental signals associated with hearing, balance, and body orientation. In humans and other mammals, significant hair cell loss leads to irreversible hearing and balance deficits, whereas hair cell loss in nonmammalian vertebrates is repaired by the spontaneous generation of replacement hair cells. Research in mammalian hair cell regeneration is hampered by the lack of in vivo damage models for the adult mouse inner ear and the paucity of cell-type-specific markers for non-sensory cells within the sensory receptor epithelia. The present study delineates a protocol to drug damage the adult mouse auditory epithelium (organ of Corti) in situ and uses this protocol to investigate Sox2 and Jagged1 expression in damaged inner ear sensory epithelia. In other tissues, the transcription factor Sox2 and a ligand member of the Notch signaling pathway, Jagged1, are involved in regenerative processes. Both are involved in early inner ear development and are expressed in developing support cells, but little is known about their expressions in the adult. We describe a nonsurgical technique for inducing hair cell damage in adult mouse organ of Corti by a single high-dose injection of the aminoglycoside kanamycin followed by a single injection of the loop diuretic furosemide. This drug combination causes the rapid death of outer hair cells throughout the cochlea. Using immunocytochemical techniques, Sox2 is shown to be expressed specifically in support cells in normal adult mouse inner ear and is not affected by drug damage. Sox2 is absent from auditory hair cells, but is expressed in a subset of vestibular hair cells. Double-labeling experiments with Sox2 and calbindin suggest Sox2-positive hair cells are Type II. Jagged1 is also expressed in support cells in the adult ear and is not affected by drug damage. Sox2 and Jagged1 may be involved in the maintenance of support cells in adult mouse inner ear.

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.

Transforming growth factor α with insulin stimulates cell proliferation in vivo in adult rat vestibular sensory epithelium

Hair cells, the sensory receptors of the mammalian inner ear, have long been thought to be produced only during embryogenesis, and postnatal hair cell loss is considered to be irreversible and is associated with permanent hearing and balance deficits. Little is known about the factors that regulate hair cell genesis and differentiation. The mitogenic effects of insulin and transforming growth factor α (TGFα) were assayed in vivo in normal and drug‐damaged rat inner ear. Tritiated thymidine and autoradiographic techniques were used to identify cells synthesizing DNA. Simultaneous infusion of TGFα and insulin directly into the inner ear of adult rats stimulated DNA synthesis in the vestibular sensory receptor epithelium. New supporting cells and putative new hair cells were produced. Infusion of insulin alone or TGFα alone failed to stimulate significant DNA synthesis. These results suggest that exogenous growth factors may have utility for therapeutic treatment of hearing and balance disorders in vivo. J. Comp. Neurol. 399:413–423, 1998.

Induction of Cell Proliferation in Avian Inner Ear Sensory Epithelia by Insulin-Like Growth Factor-I and Insulin

Postembryonic production of inner‐ear hair cells occurs both normally and after insult in lower vertebrates and avians. To determine how this proliferation is controlled, several growth factors were tested for effects on progenitor‐cell division in cultured avian vestibular sensory epithelium. Mitogenic effects of bombesin, epidermal growth factor, insulin‐like growth factor‐I (IGF‐I), insulin, and transforming growth factor‐α were assayed in organotypic cultures of utricles from the mature, undamaged (normal) chicken inner ear. Tritiated thymidine and autoradiographic techniques and 5‐bromo‐2′‐deoxyuridine (BrdU) immunocytochemistry were used to identify cells synthesizing DNA. IGF‐I stimulated DNA synthesis in the vestibular sensory receptor epithelium in a dose‐dependent manner. DNA synthesis was also stimulated by insulin. These results suggest that stimulation of the IGF‐I receptors by IGF‐I or insulin binding stimulates cell proliferation in the mature avian vestibular sensory epithelium. J. Comp. Neurol. 380:262–274, 1997.

Macrophage and microglia‐like cells in the avian inner ear

Recent studies suggest that macrophages may influence early stages of the process of hair cell regeneration in lateral line neuromasts; numbers of macrophages were observed to increase prior to increases in hair cell progenitor proliferation, and macrophages have the potential to secrete mitogenic growth factors. We examined whether increases in the number of leukocytes present in the in vivo avian inner ear precede the proliferation of hair cell precursors following aminoglycoside insult. Bromodeoxyuridine (BrdU) immunohistochemistry was used to identify proliferating cells in chicken auditory and vestibular sensory receptor epithelia. LT40, an antibody to the avian homologue of common leukocyte antigen CD45, was used to label leukocytes within the receptor epithelia. Macrophages and, surprisingly, microglia‐like cells are present in normal auditory and vestibular sensory epithelia. After hair cell loss caused by treatment with aminoglycosides, numbers of macrophage and microglia‐like cells increase in the sensory epithelium. The increase in macrophage and microglia‐like cell numbers precedes a significant increase in sensory epithelial cell proliferation. The results suggest that macrophage and microglia‐like cells may play a role in releasing early signals for cell cycle progression in damaged inner ear sensory epithelium. J. Comp. Neurol. 398:241–256, 1998.

ErbB Expression: The Mouse Inner Ear and Maturation of the Mitogenic Response to Heregulin

In humans, hair cell loss often leads to hearing and balance impairments. Hair cell replacement is vigorous and spontaneous in avians and nonmammalian vertebrates. In mammals, in contrast, it occurs at a very low rate, or not at all, presumably because of a very low level of supporting cell proliferation following injury. Heregulin (HRG), a member of the epidermal growth factor (EGF) family of growth factors, is reported to be a potent mitogen for neonatal rat vestibular sensory epithelium, but its effects in adults are unknown. We report here that HRG-α stimulates cell proliferation in organotypic cultures of neonatal, but not adult, mouse utricular sensory epithelia. Our findings support the idea that the proliferative capabilities of the adult mammalian vestibular sensory epithelia differ significantly from that seen in neonatal animals. Immunohistochemistry reveals that HRG-binding receptors (erbBs 2–4) and erbB1 are widely expressed in vestibular and auditory sensory epithelia in neonatal and adult mouse inner ear. The distribution of erbBs in the neonatal and adult mouse ear is consistent with the EGF receptor/ligand family regulating diverse cellular processes in the inner ear, including cell proliferation and differentiation.

Postnatal production of supporting cells in the chick cochlea

The auditory receptor organ in birds, the basilar papilla, is mitotically active after acoustic overstimulation or pharmacological insult and is capable of self-repair. The damaged epithelium is repopulated with new hair cells and supporting cells. The cell production that underlies this regenerative self-repair is believed to be a response evoked by damage in populations of cells that normally become mitotically quiescent even before hatching. In contrast, regeneration in the vertebrate nervous system is often correlated with continued or recent neurogenesis in the tissue concerned. The hypothesis that there may be ongoing postnatal production of cells in the normal avian basilar papilla was investigated. Autoradiographic analysis of tritiated-thymidine-injected animals was used to look for the existence of newly formed cells in the basilar papilla of normal posthatch chickens. Several types of supporting cells, namely, organ supporting cells, border cells and hyaline cells, are produced postnatally in the normal chicken. Typically, they are added interstitially to the apical (distal) half of the basilar papilla.

Hair-cell regeneration in organ cultures of the postnatal chicken inner ear

The sensory epithelium of the avian inner ear retains into adulthood progenitor cells for inner-ear hair cells and other cell types in the epithelium. Hair cells are produced normally on an ongoing basis in the vestibular sensory epithelium, and hair-cell production is increased after insult in both auditory and vestibular sensory epithelia. The details of postnatal hair-cell production are not understood. In particular, molecular factors involved in the initiation and regulation of hair-cell genesis and differentiation are not known. Studies of this phenomena have been hampered by the lack of cell culture models. An organ culture system was developed which encourages generation and differentiation of hair cells in mature inner-ear sensory epithelia. Continuous labeling with tritiated thymidine showed genesis of both supporting cells and hair cells in normal vestibular epithelia grown in culture, and an increase in hair-cell and supporting-cell proliferation in damaged sensory epithelia grown in culture as compared to undamaged controls. This demonstrates, in vitro, both the division and differentiation of hair-cell progenitor cells in normal vestibular epithelia, and the maintenance of the hair-cell regeneration process in damaged inner-ear epithelia. This culture system should be useful for studies of hair-cell genesis and differentiation as well as studies of hair-cell and supporting-cell functioning in general.

p27Kip1 is required to maintain proliferative quiescence in the adult cochlea and pituitary

Cell cycle inhibitors, such as the cyclin-dependent kinase (Cdk) inhibitor proteins and retinoblastoma (Rb) family members, control exit from the cell cycle during the development of a variety of terminally differentiated tissues. It is unclear whether sustained expression of these proteins is required to prevent cell cycle re-entry in quiescent and terminally differentiated cells. The organ of Corti (cochlear sensory epithelium) and pars intermedia (intermediate lobe of the pituitary) are two tissues that share the characteristic of ongoing cell division in mice lacking either the p27Kip1 Cdk inhibitor, Ink4 proteins, or Rb. Here, we use tamoxifen-inducible mouse models to delete p27Kip1 in postnatal animals and show this is sufficient to induce proliferation in both the organ of Corti and pars intermedia. Thus, these tissues remain sensitive to the presence of p27Kip1 even after their developmental exit from the cell cycle. The neonatal cochlea displayed heightened sensitivity to changes in p27Kip1 expression, with a proliferative response higher than that of constitutive null mice. In adults, the proliferative response was reduced but was accompanied by increased cell survival. In contrast, re-establishment of normal p27Kip1 expression in animals with established pituitary tumors, in an inducible “knock-on” model, led to cessation of pituitary tumor growth, indicating the cells had maintained their susceptibility to p27-mediated growth suppression. Although restoration of p27Kip1 did not induce apoptosis, it did lead to resolution of pathological features and normalization of gene expression. Our data underscore the importance of p27Kip1 expression in the maintenance of cellular quiescence and terminal differentiation.