Actin filaments, stereocilia, and hair cells of the bird cochlea. V. How the staircase pattern of stereociliary lengths is generated.

The stereocilia on each hair cell are arranged into rows of ascending height, resulting in what we refer to as a "staircase-like" profile. At the proximal end of the cochlea the length of the tallest row of stereocilia in the staircase is 1.5 micron, with the shortest row only 0.3 micron. As one proceeds towards the distal end of the cochlea the length of the stereocilia progressively increases so that at the extreme distal end the length of the tallest row of the staircase is 5.5 micron and the shortest row is 2 micron. During development hair cells form their staircases in four phases of growth separated from each other by developmental time. First, stereocilia sprout from the apical surfaces of the hair cells (8-10-d embryos). Second (10-12-d embryos), what will be the longest row of the staircase begins to elongate. As the embryo gets older successive rows of stereocilia initiate elongation. Thus the staircase is set up by the sequential initiation of elongation of stereociliary rows located at increased distances from the row that began elongation. Third (12-17-d embryos), all the stereocilia in the newly formed staircase elongate until those located on the first step of the staircase have reached the prescribed length. In the final phase (17-d embryos to hatchlings) there is a progressive cessation of elongation beginning with the shortest step and followed by taller and taller rows with the tallest step stopping last. Thus, to obtain a pattern of stereocilia in rows of increasing height what transpires are progressive go signals followed by a period when all the stereocilia grow and ending with progressive stop signals. We discuss how such a sequence could be controlled.

Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochlea.

The sensory epithelium of the chick cochlea contains only two cell types, hair cells and supporting cells. We developed methods to rapidly dissect out the sensory epithelium and to prepare a detergent-extracted cytoskeleton. High salt treatment of the cytoskeleton leaves a "hair border", containing actin filament bundles of the stereocilia still attached to the cuticular plate. On SDS-PAGE stained with silver the intact epithelium is seen to contain a large number of bands, the most prominent of which are calbindin and actin. Detergent extraction solubilizes most of the proteins including calbindin. On immunoblots antibodies prepared against fimbrin from chicken intestinal epithelial cells cross react with the 57- and 65-kD bands present in the sensory epithelium and the cytoskeleton. It is probable that the 57-kD is a proteolytic fragment of the 65-kD protein. Preparations of stereocilia attached to the overlying tectorial membrane contain the 57- and 65-kD bands. A 400-kD band is present in the cuticular plate. By immunofluorescence, fimbrin is detected in stereocilia but not in the hair borders after salt extraction. The prominent 125 A transverse stripping pattern characteristic of the actin cross-bridges in a bundle is also absent in hair borders suggesting fimbrin as the component that gives rise to the transverse stripes. Because the actin filaments in the stereocilia of hair borders still remain as compact bundles, albeit very disordered, there must be an additional uncharacterized protein besides fimbrin that cross-links the actin filaments together.

Regeneration of sensory hair cells after acoustic trauma.

Any loss of cochlear hair cells has been presumed to result in a permanent hearing deficit because the production of these cells normally ceases before birth. However, after acoustic trauma, injured sensory cells in the mature cochlea of the chicken are replaced. New cells appear to be produced by mitosis of supporting cells that survive at the lesion site and do not divide in the absence of trauma. This trauma-induced division of normally postmitotic cells may lead to recovery from profound hearing loss.

Regeneration of hair cells in the vestibulocochlear system of birds and mammals.

Regeneration of hair cells leads to a structural and functional recovery in the mature avian vestibular and auditory sensory epithelia. This regeneration replaces hair cells that have been lost as a result of noise damage, ototoxic drug poisoning, or other trauma. Recent findings suggest that it may be possible to induce a similar mechanism for repair in the vestibular and auditory epithelia of mammals, including humans.

Uptake of fluorescent gentamicin by vertebrate sensory cells in vivo.

Aminoglycoside uptake in the inner ear remains poorly understood. We subcutaneously injected a fluorescently-conjugated aminoglycoside, gentamicin-Texas Red (GTTR), to investigate the in vivo uptake of GTTR in the inner ear of several vertebrates, and in various murine sensory cells using confocal microscopy. In bullfrogs, GTTR uptake was prominent in mature hair cells, but not in immature hair cells. Avian hair cells accrued GTTR more rapidly at the base of the basilar papilla. GTTR was associated with the hair bundle; and, in guinea pigs and mice, somatic GTTR fluorescence was initially diffuse before punctate (endosomal) fluorescence could be observed. A baso-apical gradient of intracellular GTTR uptake in guinea pig cochleae could only be detected at early time points (<3h). In 21-28 day mice, cochlear GTTR uptake was greatly reduced compared to guinea pigs, 6-day-old mice, or mice treated with ethacrynic acid. In mice, GTTR was also rapidly taken up, and retained, in the kidney, dorsal root and trigeminal ganglia. In linguinal and vibrissal tissues rapid GTTR uptake cleared over a period of several days. The preferential uptake of GTTR by mature saccular, and proximal hair cells resembles the pattern of aminoglycoside-induced hair cell death in bullfrogs and chicks. Differences in the degree of GTTR uptake in hair cells of different species suggests variation in serum levels, clearance rates from serum, and/or the developmental and functional integrity of the blood-labyrinth barrier. GTTR uptake by hair cells in vivo suggests that GTTR has potential to elucidate aminoglycoside transport mechanisms into the inner ear, and as a bio-tracer for in vivo pharmacokinetic studies.

The supporting-cell antigen: a receptor-like protein tyrosine phosphatase expressed in the sensory epithelia of the avian inner ear.

After noise- or drug-induced hair-cell loss, the sensory epithelia of the avian inner ear can regenerate new hair cells. Few molecular markers are available for the supporting-cell precursors of the hair cells that regenerate, and little is known about the signaling mechanisms underlying this regenerative response. Hybridoma methodology was used to obtain a monoclonal antibody (mAb) that stains the apical surface of supporting cells in the sensory epithelia of the inner ear. The mAb recognizes the supporting-cell antigen (SCA), a protein that is also found on the apical surfaces of retinal Müller cells, renal tubule cells, and intestinal brush border cells. Expression screening and molecular cloning reveal that the SCA is a novel receptor-like protein tyrosine phosphatase (RPTP), sharing similarity with human density-enhanced phosphatase, an RPTP thought to have a role in the density-dependent arrest of cell growth. In response to hair-cell damage induced by noise in vivo or hair-cell loss caused by ototoxic drug treatment in vitro, some supporting cells show a dramatic decrease in SCA expression levels on their apical surface. This decrease occurs before supporting cells are known to first enter S-phase after trauma, indicating that it may be a primary rather than a secondary response to injury. These results indicate that the SCA is a signaling molecule that may influence the potential of nonsensory supporting cells to either proliferate or differentiate into hair cells.

Hair cell differentiation in the developing chick cochlea and in embryonic cochlear organ culture.

We have defined a method for growing chick embryonic cochleae in organ culture that preserves many aspects of hair cell differentiation. Cochlear ducts were isolated from embryonic day 8 chicks, placed in organ culture, and incubated for 48 hours (to a point equivalent to embryonic day 10). The cultured ducts were then fixed and processed for scanning electron microscopy. As controls, cochlear ducts at embryonic days 8 and 10 were dissected and immediately fixed and processed for scanning electron microscopy. We chose this period to culture cochleae because at the corresponding time in vivo hair cells undergo a dynamic phase of differentiation. During this time, the number of stereocilia in the stereociliary bundle increases, and two to three rows of stereocilia nearest the kinocilium elongate, initiating the staircase pattern of the bundle. Also, the orientation of many hair cells shifts from nonpolarized at embryonic day 8 to polarized toward the inferior edge of the basilar papilla at embryonic day 10. Many of these aspects of hair cell differentiation proceed normally in organ culture. The appropriate distal-to-proximal gradients of hair cell density, apical surface area, and stereociliary number are preserved. Elongation of the 1-2 stereociliary rows next to the kinocilium continues, and more stereociliary bundles are oriented toward the inferior edge in cultured cochleae than in embryonic day 8 chicks. It appears that cochlear organ culture can serve as an effective method with which to study how hair cell differentiation is regulated.

Identification of the timing of S phase and the patterns of cell proliferation during hair cell regeneration in the chick cochlea.

Birds respond to hair cell loss by stimulating cell division in the otherwise mitotically quiescent sensory epithelium and by generating new hair cells. We examined cell proliferation during hair cell regeneration in chick basilar papilla by using 5-bromo-2'-deoxyuridine (BrdU). Chicks were noise exposed for 4 or 24 hours and injected with BrdU, and cochleae were immunohistochemically labeled to detect BrdU. Immunoreactivity after short-term postinjection survival identified when cells entered S phase. For both 4 and 24 hour exposures, cells in S phase were first detected in the sensory epithelium after an injection at 18 hours after the onset of exposure and were also present after injections at 24, 30, 36, 42, 48, 72, 96, 120, and 144 hours. The most cells in S (or G2) phase were detected at 42 and 72 hours for 24 hour exposures and at 48 hours for 4 hour exposures. Chicks that survived for long periods after injection had BrdU-labeled hair cells, indicating that precursor cells that divided in the presence of BrdU generated new hair cells. Moreover, labeled hair cells and supporting cells were grouped into discrete clusters, suggesting that cells within each cluster are clonally related. Support for this hypothesis was provided by experiments showing that the number of labeled cells increased when chicks survived for longer periods after a single BrdU injection. These findings suggest that progenitors within the sensory epithelium may undergo several rounds of division to generate the appropriate number of new hair cells and supporting cells.

Development of location-specific hair cell stereocilia in denervated embryonic ears.

The developmental mechanisms that allow physiological coding of acoustic pitch have remained unexplained. Cochlear hair cells that have different structures respond to different sound frequencies and synapse with neurons that project to different locations in the brain. How do these hair cells develop appropriate structures, and how are the connections between specific hair cells and the neurons that code for their pitch sensitivities matched? We have investigated one aspect of this by denervating embryonic chicken ears, before the time of hair cell production, and then transplanting them to the aneural chorioallantoic membrane of host embryos where they have continued to develop. We report that vestibular and auditory hair cell phenotypes differentiate appropriately and that correct gradients of hair cell structural phenotypes, as expressed in stereocilia bundles, develop in the cochleae of these denervated ears. Therefore, the normal development of gradients in hair cell stereocilia properties must be controlled by location-specific cues originating in the ear itself. Neuronally directed modification of target cell phenotypes is not required for the quite specific phenotype development represented by the stereocilia bundles of individual hair cells and the connectional matching in the numerous distinct peripheral information lines of the auditory system.

Distribution of nerve fibers in the basilar papilla of normal and sound-damaged chick cochleae.

Epifluorescent light microscopy and confocal laser scanning microscopy were employed to visualize the distribution of nerve fibers in whole-mount preparations of normal and sound-damaged chick basilar papillae (BP). In normal cochleae, we identified a consistent pattern of nerve processes that ran transversely across the BP. The transverse processes increase in number from the proximal to the distal ends of the epithelium. However, when the processes are separated into populations of thin fibers and thick bundles, the thin fibers are more prevalent in distal regions whereas thick bundles are more extensive in proximal regions. Furthermore, the thick bundles form an elaborate longitudinal network in the border cell and hyaline cell region. Based on these data and no other previous studies, the thin fibers appear to be afferent nerves and the thick bundles represent efferent nerves. When birds are exposed to acoustic trauma, the normal pattern and number of nerve processes is not altered by levels of sound that produce moderate levels of damage, i.e., damage that leads to hair cell loss and regeneration. However, the nerve pattern is disrupted by severe levels of damage that destroy both hair cells and supporting cells. These findings indicate that the level of sound exposure that induces hair cell regeneration may damage the synaptic endings associated with the lost hair cells, but that the nerve processes that give rise to these endings remain intact within the sensory epithelium. In contrast, severe damage destroys both the hair cells and their associated nerve fibers.

Contractile proteins in the hyaline cells of the chicken cochlea.

Hyaline cells are a single layer of epithelial cells found at the inferior edge of the sensory epithelium in the chick cochlea. They rest directly above a specialized region of the basilar membrane at a point where it connects to the fibrocartilaginous skeleton of the cochlear duct. The basal cytoplasm of the hyaline cells contains a bundle of linearly aligned actin filaments that resemble stress fibers in their organization. The actin filaments are anchored in the basal plasma membranes of the cells, which are, in turn, associated with the underlying basal lamina and the extracellular matrix of the basilar membrane. We have used a combination of transmission electron microscopy, differential-interference-contrast and epifluorescence light microscopy, and confocal laser scanning microscopy to study the composition and organization of these actin bundles within the hyaline cells. The bundles are arranged into triangular wedges that are oriented radially across the basilar membrane. Each cell contains one or two actin wedges. Adjacent cells can have them aligned in opposite directions so that in a whole-mount surface preparation they appear as interdigitations. Immunofluorescent staining of the hyaline cells has shown that smooth muscle myosin and alpha-actinin are co-localized to the actin bundles. Smooth muscle myosin is also found throughout the cytoplasm of the cells. The fact that hyaline cells in the chick cochlea are contacted by efferent nerve fibers suggests that these cells may regulate tension on the basilar membrane via the specialized bundle of actin filaments.

The development of stereociliary bundles in the cochlear duct of chick embryos.

The differentiation of hair cell stereociliary bundles was investigated during early stages of embryonic development in the chick cochlear duct. The ultrastructural characteristics of the differentiating stereocilia and the position of the hair cells at the time of their differentiation were determined with scanning (SEM) and transmission (TEM) electron microscopy. Stereocilia were first identifiable with SEM as early as embryonic day 6 (stage 29) in only the distal region of the basilar papilla. By embryonic day 7.5 (stage 32) stereocilia were detected with both SEM and TEM on hair cells located in the distal two-thirds of the basilar papilla. Stereociliary bundles were recognizable throughout the entire basilar papilla by embryonic day 9 (stage 35). At this stage the hair cells exhibited a distal-to-proximal gradient in the cell surface area and the number of stereocilia on each hair cell. These results suggest that there is a distal-to-proximal wave of hair cell differentiation which occurs at a very early time period in the development of the chick cochlea. Both the timing and the direction of the stereociliary differentiation contrast with previous ultrastructural reports of avian hair cell development, yet compare favorably with the patterns of functional development in the auditory system.

Regeneration of hair cell stereociliary bundles in the chick cochlea following severe acoustic trauma.

Examination of pure-tone acoustic damage in the chick cochlea revealed a significant amount of hair cell recovery over a 10 day period following the exposure. The recovery included both a regeneration of stereociliary bundles to replace those that were lost and a reshuffling of the mosaic pattern of the hair cell surfaces that survived. Ten-day-old chicks were exposed to a 1500 Hz pure tone at 120 dB SPL for 48 h and their cochleae were processed for scanning, transmission and light microscopy at 0 h, 24 h, 48 h, 4 d, 6 d and 10 d after exposure. Immediately after exposure the damaged region exhibited two types of hair cell trauma. The first was a defined area of complete hair cell loss and the second was an area where the hair cells survived but exhibited varying amounts of stereocilia injury. After 48 h of recovery, new hair cells were identifiable in the region of hair cell loss and with time they underwent a progressive maturation of their stereociliary bundles. The surviving hair cells showed a dramatic rearrangement and expansion of their surfaces but exhibited no repair of the damaged stereociliary bundles. These results suggest that the chick cochlea is capable of a significant amount of recovery and regeneration following acoustic trauma.

Regeneration of the tectorial membrane in the chick cochlea following severe acoustic trauma.

Damage to the tectorial membrane caused by acoustic trauma was examined with scanning and transmission electron microscopy immediately after exposure and at selected time points over a 10 day recovery period. At 0 h of recovery the structure of the tectorial membrane overlying the region of hair cell damage was severely disrupted and connections between the membrane and the basilar papilla were lost. By 24 h of recovery, regeneration of the tectorial membrane was evident in the secretion of new matrix materials by the supporting cells of the basilar papilla. By 10 days of recovery a new honeycomb-like matrix had replaced the segment of damaged tectorial membrane, re-established connections with hair cell stereocilia and become fused with adjacent regions of undamaged tectorial membrane. However, the regenerated segment included only the honeycomb-like structure of the lower layer of the normal tectorial membrane. The laterally-oriented fibers which form the upper layer of the membrane were not regenerated over the damaged region. These findings indicate that the tectorial membrane is regenerated in parallel with the hair cells during recovery from acoustic trauma but the full extent of this recovery and its effect on cochlear function are not yet clear.

Development of hair cell stereocilia in the avian cochlea.

The establishment of an embryonic hair cell's stereociliary bundle involves the coordinated regulation of several morphogenetic events. The developing hair cell organizes the assembly of individual stereocilia, regulates the growth of the stereociliary bundle, and aligns the orientation of the bundle. During development, individual stereocilia exhibit three phases of growth: (1) an initial assembly and elongation of a small number of actin filaments; (2) the development of the stereocilia rootlet and the addition of more filaments to each stereocilium; (3) a second growth phase where elongation of the actin filaments resumes. These three phases involve different biochemical conditions for actin assembly and, thus, are temporally separated during development. Each hair cell also regulates the size of its stereociliary bundle so that it fits into the precise basal-to-apical gradient in stereocilia length and width seen in the mature cochlea. Orientation of the stereociliary bundles also changes during development. Very young hair cells exhibit a non-uniform orientation. Early in development, neighboring groups of cells rapidly acquire a uniform orientation. A more gradual shift in orientation continues throughout development, so that by maturity most of the hair cells are oriented toward the abneural edge of the sensory epithelium.

SEM analysis of the developing tectorial membrane in the chick cochlea.

The development of the tectorial membrane in the embryonic chick cochlea was studied using scanning electron microscopy. Chick embryos ranged in age from embryonic day 7 (E7) to post-hatching day 15. Our studies revealed that a fine filamentous matrix arose on the apical surface of the basilar papilla at approximately E7. This matrix was secreted by the supporting cells which encircled the hair cells. By E9, the early matrix had increased in volume but remained filamentous in structure, except at the inferior edge of the basilar papilla where it was condensed into a layer of laterally-oriented columns. At E9 the TM exhibited an additional layer of matrix, called the amorphous component. It appeared to originate from the homogene cell population, and attached to the early columnar matrix at the inferior edge of the basilar papilla. The two components of the TM were separated by a longitudinal ridge, called the 'track', which marked the inferior edge of the amorphous component. As the cochlea developed, the basilar papilla increased in width, the columnar component elongated and the track appeared to recede. These morphological findings point to separate developmental origins for the two components of the tectorial membrane.

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.

Hair cell and supporting cell response to acoustic trauma in the chick cochlea.

Damage to the chick cochlea in response to progressively increased periods of noise exposure was studied with scanning electron microscopy. Ten day-old chick hatchlings were exposed to a 1500 Hz pure tone at 120 dB SPL for 4, 8, 12, 24, and 48 h. Measurements of hair cell and supporting cell surface areas within a defined region of the cochlea showed that the average hair cell surface area decreased over the first 12 h of exposure. Between 12 h and 48 h there was no significant change in hair cell surface area. Supporting cells showed a corresponding increase in surface area over the same period. Noise damage first appeared after 4 h of exposure as a localized expansion of supporting cell surfaces near the inferior edge of the basilar papilla (BP). Between 8 and 12 h of exposure the supporting cell surface area increased dramatically and was visible throughout the noise damaged region. Hair cell expulsion was first seen after 12 h. Exposure to noise for 24-48 h resulted in further expansion of supporting cells, extensive expulsion of hair cells from the BP, and the appearance of a strip of noise damage along the superior, edge of the BP.

Potential role of bFGF and retinoic acid in the regeneration of chicken cochlear hair cells.

Messenger RNAs (mRNA) of several growth factor receptors and relate genes were examined with reverse transcriptase polymerase chain reaction (RT-PCR) in normal and noise-damaged chicken basilar papillae (BP). Analysis of the amplification products indicated the presence of mRNAs for epidermal growth factor receptor (EGFR), fibroblast factor receptor (FGFR), insulin-like growth factor receptor (IGFR), insulin receptor (IR), retinoic acid receptor beta (RAR beta), retinoic acid receptor gamma (RXR gamma), and basic fibroblast growth factor (BFGF) in both normal and noise-damaged BP. The RT-PCR products generated were characterized by size and sequencing analysis to confirm the identities of the target molecules. The subcellular localization of the mature protein analogs for EGFR, FGFR, IGFR, RAR beta, and BFGF were identified using fluorescence immunocytochemistry and confocal laser scanning microscopy. These experiments indicated that EGFR is present in the stereociliary bundles in the hair cells, IGFR is not present in the cells of the BP, BFGF localizes in the nuclei of supporting cells in the BP, but not hair cells or hyaline cells, and that RAR beta localizes in the perinuclear regions of hair cells. The subcellular distributions of these proteins were consistent in both noise-damaged and control BP. FGFR, in contrast, changed its distribution in the tissue after noise damage. In normal BP, FGFR is concentrated in the stereocilia of hair cells. However, in damaged regions of noise-exposed chick cochleae, FGFR is heavily expressed in the expanded apical regions of the supporting cells. These findings suggest that BFGF and retinoic acid may potentially play a role in the mechanisms which regulate the regeneration of chicken cochlear hair cells.

Secretion of a new basal layer of tectorial membrane following gentamicin-induced hair cell loss.

Scanning electron microscopy (SEM) and video-enhanced DIC light microscopy were used to assess morphological changes in the chick tectorial membrane (TM) following gentamicin-induced hair cell loss. Gentamicin was administered (100 mg/kg/day for 3 days) and isolated and in-situ TMs were examined in both fixed and unfixed preparations at days 5 and 10 after the initial injection. Although this protocol induced hair cell damage extending up to 75% of the length of the basilar papilla, there was no apparent damage to the TM itself. However, the ejection of damaged hair cells appeared to sever the filamentous attachments between the TM and the apical surface of the basilar papilla. In SEM preparations this detachment caused the TM to shrink back toward the superior edge. Interestingly, despite the lack of TM damage, gentamicin treatment did reveal the secretion of a new basal layer of TM. Secretion of this new basal layer had begun by day 5 and it was well organized by day 10. This new layer formed attachments to both the recovering basilar papilla and the overlying original TM, a step thought to be necessary for the restoration of auditory function in the regenerating cochlea.