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The Journal of Neuroscience, June 1, 2003, 23(11):4395-4400
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Math1 Gene Transfer Generates New Cochlear Hair Cells in Mature Guinea Pigs In Vivo
Kohei Kawamoto,1,2 Shin-Ichi Ishimoto,1,3 Ryosei Minoda,1,4 Douglas E. Brough,5 andYehoash Raphael1 1 Kresge Hearing Research Institute, Department of Otolaryngology, The University of Michigan, Ann Arbor, Michigan 48109-0648, 2 Department of Otolaryngology, Kansai Medical University, Moriguchi, Osaka, 570-8506, Japan, 3 Department of Otolaryngology, Tokyo University, Bunkyo-ku, Tokyo, 113-8655, Japan, 4 Department of Otolaryngology–Head and Neck Surgery, Kumamoto University School of Medicine, Kumamoto, 860-8556, Japan, and 5 GenVec Inc., Gaithersburg, Maryland 20878
Abstract
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Abstract
Introduction
Hair cell loss in the mammalian cochlea is irreversible and results in permanent hearing loss. Math1, the basic helix-loop-helix transcription factor homolog of the Drosophila atonal gene, is a positive regulator of hair cell differentiation during cochlear development. Developing hair cells express Math1, and nonsensory cells do not. We set out to determine the outcome of overexpression of Math1 in nonsensory cells of the cochlea on the phenotype of these cells. We demonstrate that in vivo inoculation of adenovirus with the Math1 gene insert into the endolymph of the mature guinea pig cochlea results in Math1 overexpression in nonsensory cochlear cells, as evident from the presence of Math1 protein in supporting cells of the organ of Corti and in adjacent nonsensory epithelial cells. Math1 overexpression leads to the appearance of immature hair cells in the organ of Corti and new hair cells adjacent to the organ of Corti in the interdental cell, inner sulcus, and Hensen cell regions. Axons are extended from the bundle of auditory nerve toward some of the new hair cells, suggesting that the new cells attract auditory neurons. We conclude that nonsensory cells in the mature cochlea retain the competence to generate new hair cells after overexpression of Math1 in vivo and that Math1 is necessary and sufficient to direct hair cell differentiation in these mature nonsensory cells.
Key words: hair cell; guinea pig; regeneration; Math1; gene therapy; adenovirus; supporting cell
Introduction
The auditory sensory epithelium in the inner ear, the organ of Corti, is an epithelial mosaic made of hair cells and supporting cells. Hair cell loss may result from aging, excessive exposure to loud stimuli, bacterial and viral infections, or ototoxic drugs. Cellular renewal on the basis of stem (basal) cell proliferation is a hallmark of most epithelial tissues. However, the organ of Corti lacks basal cells, and the terminally differentiated auditory hair cells are not replaced once lost (Hawkins, 1973). Thus, cochlear hair cell loss leads to permanent hearing impairment, the most common sensory disorder in humans.
On the basis of data obtained in avian inner ears, differentiated supporting cells are able to change their phenotype and become new hair cells (Corwin and Cotanche, 1988
The discovery of developmental genes that encode hair cell differentiation facilitates the design of interventions to promote generation of new hair cells in cochleae with hair cell loss. Basic helix-loop-helix (bHLH) transcription factors regulate the development of a variety of systems in vertebrates and invertebrates (Hutcheson and Vetter, 2001
Materials and Methods
Adenovirus vectors. The Math1 cDNA used for the construct was obtained from Huda Zoghbi (Baylor College of Medicine, Houston, TX). The three vectors, Ad.Math1.11D, Ad.LacZ, and adenovirus with no gene insert, were based on human adenovirus serotype 5 with E1, E3, and E4 regions deleted, as described previously (Brough et al., 1996Animals and inoculation surgery. We used young adult guinea pigs (4–5 weeks of age) weighing 350–500 gm at the beginning of the experiment. Inoculation surgery and composition of artificial endolymph were essentially as described by Ishimoto et al. (2002
-galactosidase). Animals used to assess for new hair cells were examined using scanning electron microscopy or myosin VIIa antibody. These animals were killed 30 d (n = 5 for scanning electron microscopy; n = 4 for myosin VIIa) or 60 d (n = 9 for scanning electron microscopy; n = 5 for myosin VIIa) after the inoculation. Neurofilament staining was performed on normal animals, as well as those killed 60 d after Ad.Math1.11D or control vector inoculation. At least three animals were used for each control group.
Scanning electron microscopy. Guinea pigs were anesthetized and transcardially perfused with saline, followed by 2% glutaraldehyde in cacodylate buffer (0.15 M). Cochleae were processed for scanning electron microscopy using the osmium thiocarbohydrazide method (Osborne and Comis, 1991
Immunocytochemistry. Whole mounts of the auditory sensory epithelium and surrounding tissues were used to localize Math1, myosin VIIa, and neurofilament. We fixed cochleae in 4% paraformaldehyde in phosphate buffer, pH 7.4, removed the spiral ligament, stria vascularis, and tectorial membrane, and then permeabilized the tissue with 0.3% Triton X-100 in PBS for 10 min. Nonspecific binding of secondary antibodies was blocked with 5% BSA in PBS for 20 min. Tissues were reacted with primary antibody, rinsed, and incubated with the secondary antibody. To perform double staining of neurofilaments and myosin VIIa, we used FITC secondary antibody for neurofilaments and tetramethylrhodamine isothiocyanate (TRITC) fluorescence for myosin VIIa. To double stain for F-actin, we used FITC-conjugated phalloidin (1:400; Molecular Probes, Junction City, OR). Specimens were mounted on glass slides using CrystalMount (Biomeda, Foster City, CA). Cryosections of the organ of Corti and surrounding cochlear epithelium were used to localize
Primary antibodies were a rabbit polyclonal anti-myosin VIIa antibody (a gift from Tama Hasson, University of California San Diego, San Diego, CA) diluted 1:200 in PBS with 0.1% BSA for 1 hr, a rabbit polyclonal anti-Math1 (a gift from Jane Johnson, The University of Texas Southwestern Medical Center at Dallas, Dallas, TX) diluted 1:200, a monoclonal antibody against neurofilament 200 kDa (Sigma, St. Louis, MO) diluted 1:200 in PBS for 1 hr, and a rabbit polyclonal against
Specimens were examined and recorded using a Leica DMRB epifluorescence microscope (Leica, Eaton, PA) using 40 and 100x oil objectives and a CCD Cooled SPOT-RT digital camera (Diagnostic Instruments, Sterling Heights, MI).
Results
Nonsensory cochlear cells express transgenes
To insert genes into nonsensory cells in the cochlea, we used adenovirus vectors. We constructed an adenoviral vector designated Ad.Math1.11D, with the Math1 cDNA insert, as described previously (Brough et al., 1996
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Figure 1. Epifluorescence of
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Figure 2. Myosin VIIa and neurofilaments in Ad.Math1.11D-treated and normal cochleae. a, Myosin VIIa-positive ectopic hair cells (arrows) among interdental cells. Inner hair cells (arrowheads) mark the medial border of the organ of Corti. b, A myosin VIIa-positive hair cell (arrow) among Hensen cells. c, An ectopic myosin VIIa-positive hair cell (red) in the interdental cell region. An axon (green) extends from the organ of Corti (arrowhead) to ectopic hair cell. d, An axon (green) extends laterally into the Hensen cell region (arrowheads, lateral border of organ of Corti). e, Myosin VIIa and axons in the organ of Corti (cochlear area similar to that shown in Fig. 1b, adjacent to the inoculation site) 60 d after artificial endolymph inoculation. Inner (arrowhead) and outer (arrows) hair cells are myosin VIIa positive. Several outer hair cells are missing. f, Neurofilament staining is restricted to the organ of Corti in normal (noninoculated) cochlea. Myosin VIIa (red) is in inner (arrowhead) and outer (arrows) hair cells. Micrographs are oriented with medial (modiolar) side down. Scale bars, 25 µm.
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Figure 3. Scanning electron microscopy of cochleae after Ad.Math1.11D treatment. a, An interdental cell area (boxed) with several ectopic hair cells medial to the organ of Corti (arrowhead). b, Box in a enlarged to show ectopic hair cells (arrows). c, Higher magnification of ectopic hair cell in interdental cell area (a, arrow) with a well developed stereocilia bundle. d, Inoculation-lesioned organ of Corti exhibits cells with short stereocilia (arrow) and small hair cells (arrowhead). Micrographs are oriented with medial (modiolar) side down. e, The site of inoculation showing the injured organ of Corti (arrowhead) and Hensen cells (bar). f, An ectopic hair cell with short stereocilia in the inner sulcus (e, arrow). g, Hair cells in the organ of Corti distant from the inoculation site are missing or injured. h, An ectopic hair cell in the inner sulcus (g, arrow). i, Lateral to the organ of Corti (arrowhead), Hensen cells (bar) exhibit an ectopic hair cell (arrow). j, Higher magnification of ectopic hair cell depicted in i. Micrographs are oriented with medial (modiolar) side down. Scale bars: a, e, i, 50 µm; b, d, g, 20 µm; c, f, h, j, 2 µm.
To detect reporter gene expression, we killed the animals 4 d after Ad.LacZ inoculation and analyzed cochlear cryosections. At the site of inoculation, Ad.LacZ transgene expression was found in several cell types in the epithelium, including supporting cells of the organ of Corti and adjacent epithelial cells that reside lateral or medial to the organ of Corti (Fig. 1a). These epithelial cells included Hensen cells and cells in the inner sulcus and interdental cell regions (Figs. 1a, 4).
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Figure 4. Math1-positive nuclei and ectopic hair cells. The schematic of the organ of Corti is oriented similar to the mid-modiolar cross sections in Figure 1, a and d–g, in which medial is on left and lateral is on right. The epithelial regions that exhibit Math1-positive nuclei include the organ of Corti (yellow) and ectopic areas adjacent to the organ of Corti (red). Ectopic new hair cells were identified in the interdental cell, inner sulcus, and Hensen cell regions.
We assessed the extent of Math1 transgene expression using a Math1-specific antibody in cochleae processed 4 d after Ad.Math1.11D inoculation. Numerous cells in the third turn of Ad.Math1.11D-inoculated animals were Math1 positive in the organ of Corti and in adjacent regions, including Hensen cells, inner sulcus areas (Fig. 1b), and the interdental cell area (data not shown). Most Math1-positive cells were within the normal boundaries of the organ of Corti (Fig. 1b,d). To better localize Math1-positive cells and distinguish hair cells from supporting cells or scars (sites of missing hair cells), cochleae were double stained with FITC phalloidin (Fig. 1b,c). We determined that most Math1-positive cells were nonsensory cells (Fig. 1c).
We used cryosections to localize Math1-positive cells in the fourth (apical) and second cochlear turns, flanking the site of inoculation. We determined that the extent of lesion decreased in areas distant from the inoculation site, with most hair cells surviving (Fig. 1d,e). Many nonsensory epithelial cells were Math1 positive, whereas most hair cells were Math1 negative (Fig. 1d,e). Control-inoculated cochleae were Math1 negative (Fig. 1f,g), demonstrating the absence of Math1 expression in the mature cochlea. These data demonstrate robust and efficient expression of Math1 in nonsensory cells of the auditory epithelium after Ad.Math1.11D inoculation into the third-turn endolymph.
New and immature hair cells in the cochlea
To assess the surface morphology of the cochlear epithelium, we performed scanning electron microscopy analysis in the inner ears obtained from animals killed 30 or 60 d after the inoculation. After Ad.Math1.11D inoculation, we observed hair cells adjacent to the organ of Corti, in which hair cells are typically absent (Fig. 3) (for schematic, see Fig. 4). The most remote area that contained ectopic hair cells was the interdental cell region (Fig. 3a–c). The morphology of some ectopic hair cells appeared similar to normal mature hair cells (Fig. 3c). Typically, approximately one-third of the ectopic hair cells exhibited a well differentiated surface morphology. Within the organ of Corti, we detected hair cells with an immature appearance (Fig. 3d). No immature-looking hair cells were observed in any of the control-inoculated cochleae (data not shown).In most Ad.Math1.11D-inoculated cochleae, the number of hair cells with an immature appearance was between 25 and 50. However, we could not reliably distinguish between old (preexisting) and new hair cells within the boundaries of the organ of Corti. In the inner sulcus area, which resides immediately medial to the organ of Corti (Fig. 4), we observed some immature hair cells with short stereocilia (Fig. 3e,f) and some hair cells with longer stereocilia (Fig. 3g,h). Ectopic hair cells were also found in the Hensen cell area, immediately lateral to the organ of Corti (Figs. 3i,j, 4).
All Ad.Math1.11D-inoculated animals assessed with scanning electron microscopy exhibited new hair cells (n = 14). In five animals killed 60 d after the inoculation, the number of ectopic cells varied from 2 to 10 per cochlea. In four animals killed 30 d after Ad.Math1.11D inoculation, we observed two to nine ectopic cells per cochlea. Cells observed to have features typical of hair cells in ectopic locations were not counted if there was any doubt as to their phenotypic identification. Cochleae receiving control inoculations did not exhibit any ectopic hair cells (data not shown; n = 12). These data demonstrate that nonsensory cells in the mature mammalian cochlea retain the competence to generate hair cells after viral-mediated overexpression of Math1.
New hair cells express myosin VIIa
We also characterized the phenotype of new hair cells with antibodies against myosin VIIa, a hair cell-specific marker (Hasson et al., 1995New hair cells attract neurons
To assess for the presence of axons in the vicinity of new ectopic hair cells, we double stained control-inoculated and Ad.Math1.11D-treated cochleae (time point, 60 d) with antibodies to myosin VIIa and neurofilament. Assessment of double-stained cochlear whole mounts revealed that neurofilament staining within the cochlear epithelium was restricted to the organ of Corti in noninoculated cochleae (Fig. 2f) and control-inoculated cochleae (Fig. 2e), with no axons in the regions of Hensen cells, inner sulcus, and interdental cells. In contrast, in Ad.Math1.11D-treated tissues, long and slender neurofilament-stained fibers extended over 50 µm from the organ of Corti toward some myosin VIIa-labeled ectopic hair cells in the interdental cell area (Fig. 2c) and toward the Hensen cell area (Fig. 2d), suggesting that axons grow toward newly formed ectopic hair cells in the cochlea. Nerve processes have been shown to remain in the area of the traumatized organ of Corti long after hair cells are lost (Strominger et al., 1995
Discussion
Plasticity and the potential for repair are commonly found in developing tissues. In explants of developing rat cochleae, Math1 was sufficient to produce extra hair cells via phenotypic conversion of nonsensory cells (Zheng and Gao, 2000Using immunocytochemistry with Math1-specific antibodies, we demonstrate that mature hair cells downregulate Math1 expression. This finding is in agreement with reverse transcription-PCR data showing downregulation of Math1 in the mature rat cochlea (Zheng et al., 2000
Regenerated hair cells within the organ of Corti are likely to contribute more than ectopic cells toward recovery of hearing. Nevertheless, the potential functional contribution of ectopic hair cells should not be overlooked. Ectopic hair cells in the inner sulcus are adjacent to inner hair cells. Similar to inner hair cells, the neighboring ectopic cells are situated on a part of the basilar membrane that is not free to vibrate (Slepecky, 1996
Most Math1-positive cells that were identified in cochlea 4 d after Math1 inoculation were within the normal boundaries of the organ of Corti. Surface analysis 2 months later revealed numerous cells with surface morphology resembling immature hair cells within the organ of Corti. Although we cannot unequivocally identify these cells as new hair cells, the absence of such immature cells in control-treated cochleae suggest that they are regenerated hair cells induced by Math1 overexpression. Future experiments using Math1 overexpression in cochleae that are completely depleted of their original hair cells may help identify new hair cells within the organ of Corti.
In Math1 null mice, the auditory sensory primodrium and supporting cells develop normally, but hair cells are not generated (Bermingham et al., 1999
The bundles of stereocilia on most ectopic hair cells did not reach a level of maturity seen on normal hair cells 2 months after Ad.Math1.11D inoculation. It is possible that a longer period of time is required for bundle maturation. However, it is likely that the extracellular environment and cell–cell communication in ectopic locations cannot support the formation of completely normal bundles. As such, ectopic hair cells may be experimentally useful for elucidating the requirements for normal hair cell differentiation.
In birds, nonsensory cells of the auditory epithelium spontaneously generate new hair cells after experimentally induced trauma (Corwin and Cotanche, 1988
Our data raise several issues regarding the potential for use of Math1 gene therapy for restoring hearing. First, the inoculation into the endolymph damages the organ of Corti. Candidates for inner ear gene therapy in the future are likely to have preexisting severe hair cell lesions, making the adverse effects of this procedure less troubling. It is also likely that vector inoculation into the larger human cochleae would elicit less mechanical trauma compared with that seen in guinea pigs. Second, in severely traumatized cochleae, supporting cells often become dedifferentiated and appear like cells in the inner sulcus (Leake and Hradek, 1988
Innervation of the new hair cells would be a prerequisite for restoring hearing. The ability of new hair cells to receive new nerve terminals has been demonstrated in the regenerating avian basilar papilla (Ofsie and Cotanche, 1996
In conclusion, we show that new hair cells are generated after Math1 overexpression via an adenovirus vector in the mature mammalian cochlea. The new hair cells exhibit the typical surface morphology of hair cells and stain for the hair cell-specific protein myosin VIIa. The new hair cells are ectopically positioned and able to attract auditory nerve fibers, raising the possibility that they may be functional. This is the first in vivo induction of new hair cell generation in the mammalian cochlea and the first success in inducing regeneration in any tissue in which spontaneous cell replacement does not occur. The ability to generate hair cells in the mammalian organ of Corti may lead to treatments for sensorineural deafness and the development of methods for inducing regeneration and innervation in other organs.
Footnotes
Received Feb. 21, 2003; revised Feb. 21, 2003; accepted Mar. 13, 2003.This work was supported by GenVec and National Institutes of Health/National Institute on Deafness and Other Communication Disorders Grant R01DC01634. We thank Tama Hasson, Jane Johnson, and Huda Zoghbi for reagents. We thank Christopher Zurenko and James Beals for their help in preparation of the figures. We thank Sally Camper, Tom Glaser, Peter Hitchcock, Donna Martin, and John Middlebrooks for valuable discussions and helpful comments on this manuscript.
Correspondence should be addressed to Dr. Yehoash Raphael, Kresge Hearing Research Institute, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0648. E-mail: yoash{at}umich.edu.
Copyright © 2003 Society for Neuroscience 0270-6474/03/234395-06$15.00/0
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