Jeffrey T. Corwin

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.

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 overstimulation 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 μm of the hair-cell lesions. These cells first entered S-phase ∼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.

Fish n' Chicks: Model Recipes for Hair-Cell Regeneration?

after hair cells have been killed, but the response is In nearly every waking moment, our brains respond to much weaker than in nonmammals. All those epithelia signals that originate from hair cells. These cells reside contain relatively undifferentiated supporting cells that in six separate epithelia in our internal ears and are cannot be reliably subdivided on thebasis of histological detectors of head rotation, gravity, and sound. Hair cells characteristics, so a relative lack of supporting cell difcan be killed by loud sounds, certain antibiotics, and ferentiation and a capacity for regenerative proliferation other drugs. Some are lost through infections and aging. may be linked (Table 1). Such a linkage is consistent Any loss is potentially significant, since hair cells are with the apparent lack of plasticity in the organ of Corti, not added to the human ear after birth, according to the the auditory epithelium of placental mammals. Its supaccepted view. “Nerve deafness,” a permanent form of porting cells are structurally specialized as five differenhearing loss, actually results from loss of hair cells in tiated subtypes, which are all effectively nonproliferative most cases, not from damage to nerves. Permanent during postembryonic life. In rare cases of damage in balance dysfunctions also result from hair-cell loss in cultures from neonates, the organ of Corti may be able many cases. These conditions affect z10% of the poputo replace hair cells after birth, and that also can occur lation and 25% of people over the age of 65, making hairthrough a cell-fate change in embryonic organs (Kelley cell loss one of the most common neurological deficits. et al., 1996). One study of organs of Corti cultured from Unfortunately, despite considerable progress in underneonatal rats has reported dramatic and complete healstanding the physiology of hair cellsand the neural basis ing after hair-cell poisoning by an antibiotic, but other of hearing and balance, most deficits that result from investigators have challenged that report. Partial healing hair-cell loss have remained irreversible. responses in this organ have recently been reported. In contrast to the situation in humans, hair cells are Hair-Cell Regeneration in Chicks produced throughout life in the ears of fish, amphibians, Each chick cochlea contains roughly 10,000 hair cells and birds. The discovery that hundreds of thousands of that form a phenotypic gradient, wherein the cells differ hair cells are added to the ears of postembryonic sharks progressively in size and shape along and across the led to the proposal that hair-cell loss might be repaired epithelium, as do the numbers, dimensions, and geovia regenerative replacement mechanisms that could metric arrangements of the stereocilia in their hair bunform the basis for regenerative treatments. Hair cell redles. There is little, if any, proliferation in the undamaged generation does indeed occur in the ears of many verteauditory epithelia, but a loss of hair cells evokes proliferbrates, even in organs such as the chick’s cochlea, ation within z200 mm of the site of loss, beginning after where cell production normally ends before birth. Treat16 hr (Warchol and Corwin, 1996). It appears that the ments that cause permanent hearing and balance defidivision of a supporting cell can give rise to one cell cits in humans also kill hair cells in birds, fish, and amthat becomes a hair cell and to another that becomes phibians, but in those species the loss of hair cells can a supporting cell (Jones and Corwin, 1996). Supporting evoke cell proliferation at the site of damage. The newly cell divisions may also give rise to pairs of supporting generated cells differentiate into supporting cells and cells and pairs of hair cells. One hypothesis proposes replacement hair cells, which make synapses with surthat new cells become hair cells by default unless they viving neurons. In nonmammalian ears, these repair prodevelop in contact with a cell already determined as a cesses can lead to rapid recovery from hearing and hair cell. Homologs of the Delta-Notch signaling system balance deficits that would be permanent in a human of Drosophila may control cell-fate choices in hair cell (references in Corwin and Warchol, 1991; Cotanche and epithelia via lateral inhibition during development (Whit-

Stereociliary bundles reorient during hair cell development and regeneration in the chick cochlea

We have examined changes in the orientation of stereociliary bundles of hair cells in the cochlear sensory epithelium that occur during normal embryonic development and during the regeneration of hair cells that follows acoustic trauma. At the time when hair cell surfaces become recognizable in the embryonic cochlea, the bundles of stereocilia exhibit a range of orientations, as indicated by the position of the kinocilium and later, by the location of the tallest row of stereocilia. With time, the orientations of bundles on neighboring hair cells become more uniform, a condition that is maintained in the adult. Changes in stereocilia orientation are also observed during the regeneration of hair cells after acoustic trauma. When new hair cells first differentiate at sites of trauma in the recovering sensory epithelium, their stereociliary bundles are not uniformly oriented. Then as the cells mature over a period of days, the bundles become aligned both with the neighboring bundles in the region of the previous lesion and with the pre-existing bundles that surround the site of regeneration. We conclude that the stereociliary bundles of hair cells are reorienting as the cells differentiate. A common mechanism may guide reorientation both during embryonic development and during regeneration. Observations in living cochleae indicate that differentiating stereociliary bundles establish asymmetric linkages to the extracellular matrix of the developing tectorial membrane. During the growth of the tectorial membrane, its progressive extension across the surface of the sensory epithelium may exert traction forces through those asymmetric linkages that pull the bundles of the hair cells into uniform alignment.

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.

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.

In vivo proliferative regeneration of balance hair cells in newborn mice

The regeneration of mechanoreceptive hair cells occurs throughout life in non-mammalian vertebrates and allows them to recover from hearing and balance deficits that affect humans and other mammals permanently. The irreversibility of comparable deficits in mammals remains unexplained, but often has been attributed to steep embryonic declines in cellular production. However, recent results suggest that gravity-sensing hair cells in murine utricles may increase in number during neonatal development, raising the possibility that young mice might retain sufficient cellular plasticity for mitotic hair cell regeneration. To test for this we used neomycin to kill hair cells in utricles cultured from mice of different ages and found that proliferation increased tenfold in damaged utricles from the youngest neonates. To kill hair cells in vivo, we generated a novel mouse model that uses an inducible, hair cell-specific CreER allele to drive expression of diphtheria toxin fragment A (DTA). In newborns, induction of DTA expression killed hair cells and resulted in significant, mitotic hair cell replacement in vivo, which occurred days after the normal cessation of developmental mitoses that produce hair cells. DTA expression induced in 5-d-old mice also caused hair cell loss, but no longer evoked mitotic hair cell replacement. These findings show that regeneration limits arise in vivo during the postnatal period when the mammalian balance epitheliums supporting cells differentiate unique cytological characteristics and lose plasticity, and they support the notion that the differentiation of those cells may directly inhibit regeneration or eliminate an essential, but as yet unidentified pool of stem cells.

Inner ear hair cells produced in vitro by a mesenchymal-to-epithelial transition

Sensory hair cell loss is a major contributor to disabling hearing and balance deficits that affect >250 million people worldwide. Sound exposures, infections, drug toxicity, genetic disorders, and aging all can cause hair cell loss and lead to permanent sensory deficits. Progress toward treatments for these deficits has been limited, in part because hair cells have only been obtainable via microdissection of the anatomically complex internal ear. Attempts to produce hair cells in vitro have resulted in reports of some success but have required transplantation into embryonic ears or coculturing with other tissues. Here, we show that avian inner ear cells can be cultured and passaged for months, frozen, and expanded to large numbers without other tissues. At any point from passage 6 up to at least passage 23, these cultures can be fully dissociated and then aggregated in suspension to induce a mesenchymal-to-epithelial transition that reliably yields new polarized sensory epithelia. Those epithelia develop numerous hair cells that are crowned by hair bundles, composed of a single kinocilium and an asymmetric array of stereocilia. These hair cells exhibit rapid permeance to FM1-43, a dye that passes through open mechanotransducing channels. Because a vial of frozen cells can now provide the capacity to produce bona fide hair cells completely in vitro, these discoveries should open new avenues of research that may ultimately contribute to better treatments for hearing loss and other inner ear disorders.

Reinforcement of cell junctions correlates with the absence of hair cell regeneration in mammals and its occurrence in birds.

Debilitating hearing and balance deficits often arise through damage to the inner ears hair cells. For humans and other mammals, such deficits are permanent, but nonmammalian vertebrates can quickly recover hearing and balance through their innate capacity to regenerate hair cells. The biological basis for this difference has remained unknown, but recent investigations in wounded balance epithelia have shown that proliferation follows cellular spreading at sites of injury. As mammalian ears mature during the first weeks after birth, the capacity for spreading and proliferation declines sharply. In seeking the basis for those declines, we investigated the circumferential bands of F‐actin that bracket the apical junctions between supporting cells in the gravity‐sensitive utricle. We found that those bands grow much thicker as mice and humans mature postnatally, whereas their counterparts in chickens remain thin from hatching through adulthood. When we cultured utricular epithelia from chickens, we found that cellular spreading and proliferation both continued at high levels, even in the epithelia from adults. In contrast, the substantial reinforcement of the circumferential F‐actin bands in mammals coincides with the steep declines in cell spreading and production established in earlier experiments. We propose that the presence of thin F‐actin bands at the junctions between avian supporting cells may contribute to the lifelong persistence of their capacity for shape change, cell proliferation, and hair cell replacement and that the postnatal reinforcement of the F‐actin bands in maturing humans and other mammals may have an important role in limiting hair cell regeneration. J. Comp. Neurol. 511:396–414, 2008.

Proliferative responses to growth factors decline rapidly during postnatal maturation of mammalian hair cell epithelia

Millions of lives are affected by hearing and balance deficits that arise as a consequence of sensory hair cell loss. Those deficits affect mammals permanently, but hearing and balance recover in nonmammals after epithelial supporting cells divide and produce replacement hair cells. Hair cells are not effectively replaced in mammals, but balance epithelia cultured from the ears of rodents and adult humans can respond to hair cell loss with low levels of supporting cell proliferation. We have sought to stimulate vestibular proliferation; and we report here that treatment with glial growth factor 2 (rhGGF2) yields a 20‐fold increase in cell proliferation within sheets of pure utricular hair cell epithelium explanted from adult rats into long‐term culture. In epithelia from neonates, substantially greater proliferation responses are evoked by rhGGF2 alone, insulin alone and to a lesser degree by serum even during short‐term cultures, but all these responses progressively decline during the first 2 weeks of postnatal maturation. Thus, sheets of utricular epithelium from newborn rats average > 40% labelling when cultured for 72 h with bromo‐deoxyuridine (BrdU) and either rhGGF2 or insulin. Those from 5‐ and 6‐day‐olds average 8–15%, 12‐day‐olds average < 1% and after 72 h there is little or no labelling in epithelia from 27‐ and 35‐day‐olds. These cells are the mammalian counterparts of the progenitors that produce replacement hair cells in nonmammals, so the postnatal quiescence described here is likely to be responsible for at least part of the mammalian ears unique vulnerability to permanent sensory deficits.