Regeneration in avian hair cell epithelia: identification of intracellular signals required for S-phase entry.

Balance epithelia in birds closely resemble their mammalian counterparts, but their cells turnover rapidly and they quickly regenerate hair cells, leading to functional recovery from damage that would be permanent for a mammal. We isolated and cultured sheets of the chicken's utricular epithelium in bromo-deoxyuridine and specific inhibitors of different intracellular signalling pathways to identify signals that influence turnover and regeneration. Synthesis (S-phase) entry was effectively blocked by inhibition of PI3-K, TOR or MAPK, and significantly decreased by inhibitors of PKC. Comparisons indicate that activated PI3-K and TOR are required for S-phase entry in both avian and mammalian balance epithelia, but activation of the MAPK pathway appears to have a more significant role in avian utricles than in mammals. The dissimilarities in the requirements for these signalling pathways do not appear sufficient to explain the marked difference in regenerative capacity between the ears of birds and mammals.

Brief treatments with forskolin enhance s-phase entry in balance epithelia from the ears of rats.

In the ears of mammals, hair cell loss results in permanent hearing and balance deficits, whereas in fish, amphibians, and birds, the production of replacement hair cells can restore those modalities. In avian ears, continuous exposures to forskolin trigger cell proliferation and the regeneration of hair cells, so we investigated the effect of forskolin on sensory epithelia cultured from the ears of mammals. Continuous 72 hr exposures to forskolin failed to induce proliferation in neonatal rat utricles, but brief (

Intracellular signals that control cell proliferation in mammalian balance epithelia: key roles for phosphatidylinositol-3 kinase, mammalian target of rapamycin, and S6 kinases in preference to calcium, protein kinase C, and mitogen-activated protein kinase.

In fish, amphibians, and birds, the loss of hair cells can evoke S-phase entry in supporting cells and the production of new cells that differentiate as replacement hair cells and supporting cells. Recent investigations have shown that supporting cells from mammalian vestibular epithelia will proliferate in limited numbers after hair cells have been killed. Exogenous growth factors such as glial growth factor 2 enhance this proliferation most potently when tested on vestibular epithelia from neonates. In this study, the intracellular signaling pathways that underlie the S-phase entry were surveyed by culturing epithelia in the presence of pharmacological inhibitors and activators. The results demonstrate that phosphatidylinositol 3-kinase is a key element in the signaling cascades that lead to the proliferation of cells in mammalian balance epithelia in vitro. Protein kinase C, mammalian target of rapamycin, mitogen-activated protein kinase, and calcium were also identified as elements in the signaling pathways that trigger supporting cell proliferation.

Solitary hair cells are distributed throughout the extramacular epithelium in the bullfrog's saccule.

The frog inner ear contains eight sensory organs that provide sensitivities to auditory, vestibular, and ground-borne vibrational stimuli. The saccule in bullfrogs is responsible for detecting ground- and airborne vibrations and is used for studies of hair cell physiology, development, and regeneration. Based on hair bundle morphology, a number of hair cell types have been defined in this organ. Using immunocytochemistry, vital labeling, and electron microscopy, we have characterized a new hair cell type in the bullfrog saccule. A monoclonal antibody that is specific to hair cells revealed that a population of solitary hair cells exists outside the sensory macula in what was previously thought to be nonsensory epithelium. We call these extramacular hair cells. There are 80-100 extramacular hair cells in both tadpole and adult saccules, which extend up to 1 mm from the edge of the sensory macula. The extramacular hair cells have spherical cell bodies and small apical surfaces. Even in adults, the hair bundles of the extramacular cells appear immature, with a long kinocilium (6-9 microm) and short stereocilia (0.5-2 microm). At least 90% of extramacular hair cells are likely to be innervated as demonstrated by labeling of nerve fibers with an antineurofilament antibody. The extramacular hair cells may differentiate in regionsjust beyond the edge of the macula at an early stage in development and then be pushed out via the interstitial growth of the epithelium that surrounds the macula. It is also possible that they may be produced from cell divisions in the extramacular epithelium that has not been considered capable of giving rise to hair cells.

Analysis of small hair bundles in the utricles of mature guinea pigs.

HYPOTHESIS: This study aimed to determine whether hair cells with immature hair bundles exist in the normal utricular maculae of mature guinea pigs. BACKGROUND: A low rate of hair cell "turnover" occurs in the vestibular organs of normal adult birds. Small immature-appearing hair cells have been identified in the utricles of juvenile guinea pigs, but their existence in the vestibular system of mature mammals has not been confirmed. METHODS: Nine utricles from 14- to 16-week-old guinea pigs were processed for scanning electron microscopy. A systematic search for small hair bundles was made. These bundles were classified as either "newborn-like" or "intermediate" depending on specific characteristics of the apical cell surface, the stereocilia, and the kinocilium. RESULTS: A mean of 7 newborn-like and 41 intermediate hair bundles per utricle were identified. The average hair cell count for the guinea pig utricular macula was 7,200; thus, these small hair bundles comprise 0.7% of the total. CONCLUSIONS: The small hair bundles are interpreted as developing vestibular hair cells, produced to replace hair cells lost to normal processes. This is thought to represent a biologic phenomenon that is in some ways similar, but in other ways distinct, from hair cell regeneration after trauma.

Cell death, cell proliferation, and estimates of hair cell life spans in the vestibular organs of chicks.

We have examined the level of on-going cell death in the chick vestibular epithelia using the TUNEL method and compared this to the rate of on-going cell proliferation. Utricles contained 22.6 +/- 6.8 TUNEL-labeled cells (mean +/- s.e.m.) while saccules contained 15.1 +/- 4.0, with approximately 90% being labeled hair cells. In separate experiments, chicks were given a single injection of BrdU and killed 2 h later. Utricles contained 116.9 +/- 6.5 BrdU-labeled cells (mean +/- s.e.m.) and saccules contained 41.0 +/- 2.2. After 24 h in culture, utricles treated with 1 mM neomycin contained 115.5 +/- 38.9 TUNEL-labeled cells, an increase of 270% over controls. After 48 h, neomycin-treated saccules contained 40.9 +/- 7.8, an increase of 152% over controls. The majority of labeled cells were in the hair cell layer. Thus, neomycin exposure results in an apoptotic death of hair cells. The in vivo data measured here were used to estimate that the average life span of utricular hair cells in young chickens is approximately 20 days, in sharp contrast to the life spans assumed for hair cells in humans.

Regenerative proliferation in organ cultures of the avian cochlea: identification of the initial progenitors and determination of the latency of the proliferative response.

Sensory hair cells in the cochleae of birds are regenerated after the death of preexisting hair cells caused by acoustic over-stimulation or administration of ototoxic drugs. Regeneration involves renewed proliferation of cells in an epithelium that is otherwise mitotically quiescent. To determine the identity of the first cells that proliferate in response to the death of hair cells and to measure the latency of this proliferative response, we have studied hair-cell regeneration in organ culture. Cochleae from hatchling chicks were placed in culture, and hair cells were killed individually by a laser microbeam. The culture medium was then replaced with a medium that contained a labeled DNA precursor. The treated cochleae were incubated in the labeling media for different time periods before being fixed and processed for the visualization of proliferating cells. The first cells to initiate DNA replication in response to the death of hair cells were supporting cells within the cochlear sensory epithelium. All of the labeled supporting cells were located within 200 microns of the hair-cell lesions. These cells first entered S-phase approximately 16 hr after the death of hair cells. The results indicate that supporting cells are the precursors of regenerated hair cells and suggest that regenerative proliferation of supporting cells is triggered by signals that act locally within the damaged epithelium.

An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles.

Sensory epithelia from normal rat utricles and those cultured with and without neomycin treatment were assayed for the presence of growth factor receptor mRNAs by RT-PCR (reverse transcriptase-polymerase chain reaction). Both undamaged and damaged utricles showed mRNA for Insulin receptor, IGF-I receptor, FGF receptor 1, EGF receptor, and PDGF alpha receptor. Neomycin-damaged sensory epithelia showed less PDGF alpha receptor mRNA than undamaged epithelia, suggesting that this message by expressed at higher copy levels in hair cells than in supporting cells. Consistent with that hypothesis, immunohistochemistry revealed much stronger PDGF alpha receptor staining in the hair cells than in the supporting cells. Preliminary evidence suggests that IGF-I receptor message also may be lowered in neomycin-damaged epithelia.

Regeneration of sensory cells after laser ablation in the lateral line system: hair cell lineage and macrophage behavior revealed by time-lapse video microscopy.

The regeneration of sensory hair cells in lateral line neuromasts of axolotls was investigated via nearly continuous time-lapse microscopic observation after all preexisting hair cells were killed by a laser microbeam. The laser treatments left neuromasts with one resident cell type, which was supporting cells. Over the course of 1 week, replacement hair cells arose either directly via differentiation of cells present in the epithelium from the beginning of the time-lapse period or via the development of cells produced after one or two divisions of supporting cells. All of the cell divisions that produced hair cells were asymmetrical. During the first hour after the treatment, macrophages and smaller leukocytes were attracted to the laser-treated neuromasts. The smaller leukocytes returned to control levels 48-60 hr after the treatment, whereas macrophages remained active there throughout the period of hair cell replacement. Macrophage incidence peaked 36-48 hr after the laser treatment. Macrophages phagocytosed damaged hair cells and supporting cells, as well as new cells and preexisting cells without recognizable damage. The results provide direct evidence of hair cells arising as progeny produced from the divisions of supporting cells, evidence of hair cells and supporting cells arising from the same cell division, evidence relating to the timing of hair cell differentiation, and indirect evidence pertaining to proposals that hair cells sometimes arise via conversion of cells without an intervening division. The results also suggest that macrophages may influence early stages in the process of hair cell regeneration.

Growth factors as potential drugs for the sensory epithelia of the ear.

The highly ordered structures of the hearing and balance organs of vertebrate ears go through a coordinated sequence of cellular and morphogenetic events. It is to be expected that protein growth factors and other extracellular signals will regulate many events during embryonic development of the ear, including the induction of the ear, the specific induction of sensory epithelia, the proliferation of the cells that form the sensory epithelia, the differentiation of the sensory and supporting cells, and the attraction and maintenance of innervation. After embryonic development, growth factors will support cell survival and innervation of new sensory cells. In damaged sensory epithelia, supplementation of the normal growth factors in these tissues has the potential to influence cellular responses to trauma, to reduce cell death and to promote the replacement of dead cells through renewed proliferation and differentiation, so as to improve hearing and balance health via preventive and restorative treatments. Assessment of the influences of specific growth factors on the sensory epithelia of vertebrate ears is at an early stage: this paper provides a brief account of what we know from studies of normal and experimentally manipulated epithelia, discusses the current questions and suggests directions for future studies.

Replacement of hair cells after laser microbeam irradiation in cultured organs of corti from embryonic and neonatal mice.

This study examined the potential for hair cell regeneration in embryonic and neonatal mouse organs of Corti maintained in vitro. Small numbers of hair cells were killed by laser microbeam irradiation and the subsequent recovery processes were monitored by differential interference contrast (DIC) microscopy combined with continuous time-lapse video recordings. Replacement hair cells were observed to develop in lesion sites in embryonic cochleae and on rare occasions in neonatal cochleae. In embryonic cochleae, replacement hair cells did not arise through renewed proliferation, but instead developed from preexisting cells that changed from their normal developmental fates in response to the loss of adjacent hair cells. In cochleae established from neonates, lost hair cells usually were not replaced, but 11 apparently regenerated hair cells and a single hair cell labeled by 3H-thymidine were observed as rare responses to the creation of hair cell lesions in these organs. The results indicate that the organ of Corti can replace lost hair cells during embryonic and on rare occasions during early neonatal development. The ability of preexisting cells to change their developmental fates in response to hair cell death is consistent with the hypothesis that during embryonic development hair cells may inhibit neighboring cells from specializing as hair cells. In neonatal cultures, the rare occurrence of apparently regenerated hair cells indicates that some cells in the postembryonic organ of Corti retain response mechanisms that can lead to self-repair.

Supporting cells in avian vestibular organs proliferate in serum-free culture.

Explants of saccules and utricles taken from hatchling chicks were cultured in medium that contained fetal bovine serum and in serum-free medium. The mitotic tracers [3H]thymidine and bromo-deoxyuridine were added to the media to label proliferating cells. High numbers of labeled supporting cells were found in cultures that were maintained in both serum-containing and serum-free media. After seven days in culture, some of the labeled cells had begun to differentiate as hair cells. The results suggest that any mitogenic factors necessary for supporting cell proliferation and the factors required for the initial stages of hair cell differentiation are produced by cells contained within explants of the vestibular sensory epithelia.

The developing organ of Corti contains retinoic acid and forms supernumerary hair cells in response to exogenous retinoic acid in culture.

The mammalian organ of Corti has one of the most highly ordered patterns of cells in any vertebrate sensory epithelium. A single row of inner hair cells and three or four rows of outer hair cells extend along its length. The factors that regulate the formation of this strict pattern are unknown. In order to determine whether retinoic acid plays a role during the development of the organ of Corti, exogenous retinoic acid was added to embryonic mouse cochleae in vitro. Exogenous retinoic acid significantly increased the number of cells that developed as hair cells and resulted in large regions of supernumerary hair cells and supporting cells containing two rows of inner hair cells and up to 11 rows of outer hair cells. The effects of retinoic acid were dependent on concentration and on the timing of its addition. Western blot analysis indicated that cellular retinoic acid binding protein (CRABP) was present in the sensory epithelium of the embryonic cochlea. The amount of CRABP apparently increased between embryonic day 14 and postnatal day 1, but CRABP was not detectable in sensory epithelia from adults. A retinoic acid reporter cell line was used to demonstrate that retinoic acid was also present in the developing organ of Corti between embryonic day 14 and postnatal day 1, and was also present in adult cochleae at least in the vicinity of the modiolus. These results suggest that retinoic acid is involved in the normal development of the organ of Corti and that the effect of retinoic acid may be to induce a population of prosensory cells to become competent to differentiate as hair cells and supporting cells.

Cochlear cytogenesis visualized through pulse labeling of chick embryos in culture.

Cytogenesis in the basilar papilla sensory epithelium of the chicken was investigated through pulse labeling of proliferative cells. Tritiated-thymidine was injected intravenously in chick embryos cultured in petri dishes. All embryos received the injection on the seventh day of incubation (E7), when the progenitors of hair cells and supporting cells are replicating deoxyribonucleic acid (DNA). Cells that were in the synthesis phase of the cell cycle, either at the time of the 3H-thymidine pulse or within 2 hours, incorporated detectable levels of the radioactive DNA precursor. Labeled cells were identified in cochleae from embryos fixed at 0.5, 1, 2, 4, 6, 12 hours, 6 and 8 days after the pulse. One hour after the injection the majority of labeled nuclei were in the basal and middle strata of the sensory epithelium. Four to 6 hours after the injection, a greater number of labeled cells appeared in the lumenal stratum. The patterns of labeled cells in embryos fixed immediately after the injection of 3H-thymidine and in others fixed 6 to 8 days after the injection were unchanged, suggesting that the progenitor cells divide and their progeny differentiate in the sensory epithelium without appreciable transverse migration. Mitotic figures were usually observed only in the lumenal stratum. Analysis of DNA content in the populations of Feulgen-stained nuclei at three levels of depth through the epithelium also produced results consistent with the conclusion that vertical nuclear migration occurs during development of the cells in this sensory epithelium.

Regenerative proliferation in inner ear sensory epithelia from adult guinea pigs and humans.

Supporting cells in the vestibular sensory epithelia from the ears of mature guinea pigs and adult humans proliferate in vitro after treatments with aminoglycoside antibiotics that cause sensory hair cells to die. After 4 weeks in culture, the epithelia contained new cells with some characteristics of immature hair cells. These findings are in contrast to expectations based on previous studies, which had suggested that hair cell loss is irreversible in mammals. The loss of hair cells is responsible for hearing and balance deficits that affect millions of people.

Ultrastructural evidence for hair cell regeneration in the mammalian inner ear.

It has long been thought that hair cell loss from the inner ears of mammals is irreversible. This report presents scanning electron micrographs and thin sections of the utricles from the inner ears of guinea pigs that show that, after hair cell loss caused by treatment with the aminoglycoside gentamicin, hair cells reappeared. Four weeks after the end of treatment, a large number of cells with immature hair bundles in multiple stages of development could be identified in the utricle. Thin sections showed that lost type 1 hair cells were replaced by cells with a morphology similar to that of type 2 hair cells. These results indicate an unexpected capacity for hair cell regeneration in vivo in the mature mammalian inner ear.

Replacement of lateral line sensory organs during tail regeneration in salamanders: identification of progenitor cells and analysis of leukocyte activity.

It has been proposed that supporting cells may be the progenitors of regenerated hair cells that contribute to recovery of hearing in birds, but regeneration is difficult to visualize in the ear, because it occurs deep in the skull. Hair cells and supporting cells that are comparable to those in the ear are present in lateral line neuromasts, and in axolotl salamanders these cells are accessible to microscopic observation in vivo. After amputation of a segment of the tail that contains neuromasts, cells from the posteriormost neuromast on the tail stump divide rapidly and form a migratory regenerative placode. The cells of the regenerative placode represent a lineage that eventually produces both hair cells and supporting cells in replacement neuromasts. We sought to identify the progenitors of the regenerative placode by using differential interference contrast microscopy combined with time-lapse video recording in living axolotl salamanders. In response to amputation, the mantle-type supporting cells at the posteroventral edge of the neuromast that is nearest to the wound increased their frequency of cell division, and gave rise to the first cells of the placode. The increase in mitotic activity of mantle-type supporting cells was accompanied by an unexplained decrease in the frequency of divisions in the same neuromast's population of internal supporting cells. The time-lapse records suggested that the changes in the mitotic activity of supporting cells might have been linked to the presence of phagocytic leukocytes in the vicinity of the neuromast that was nearest to the wound. Leukocytes were evenly distributed around control neuromasts, but during regeneration leukocyte activity increased significantly in the vicinity of the posterior half of the posteriormost neuromast. The redistribution of leukocytes occurred early in the regenerative response, but a causal role for the leukocytes has not been conclusively established. It is possible that the leukocytes could contribute to the formation of the regenerative placode at that location by breaking down the glycocalyx that ensheaths the outermost cells of the neuromast, or through the secretion of mitogenic growth factors.