Hair cell regeneration in the chick inner ear following acoustic trauma: ultrastructural and immunohistochemical studies

The regeneration of hair cells in the chick inner ear following acoustic trauma was examined using transmission electron microscopy. In addition, the localization of proliferation cell nuclear antigen (PCNA) and basic fibroblast growth factor (b-FGF) was demonstrated immunohistochemically. The auditory sensory epithelium of the normal chick consists of short and tall hair cells and supporting cells. Immediately after noise exposure to a 1500-Hz pure tone at a sound pressure level of 120 decibels for 48 h, all the short hair cells disappeared in the middle region of the auditory epithelium. Twelve hours to 1 day after exposure, mitotic cells, binucleate cells and PCNA-positive supporting cells were observed, and b-FGF immunoreactivity was shown in the supporting cells and glial cells near the habenula perforata. Spindle-shaped hair cells with immature stereocilia and a kinocilium appeared 3 days after exposure; these cells had synaptic connections with the newly developed nerve endings. The spindle-shaped hair cell is considered to be a transitional cell in the lineage of the supporting cell to the mature short hair cell. These results indicate that, after acoustic trauma, the supporting cells divide and differentiate into new short hair cells via spindle-shaped hair cells. Furthermore, it is suggested that b-FGF is related to the proliferation of the supporting cells and the extension of the nerve fibers.     

Otx1 null mutant mice show partial segregation of sensory epithelia comparable to lamprey ears

We investigated the development of inner ear innervation in Otx1 null mutants, which lack a horizontal canal, between embryonic day 12 (E12) and postnatal day 7 (P7) with DiI and immunostaining for acetylated tubulin. Comparable to control animals, horizontal crista-like fibers were found to cross over the utricle in Otx1 null mice. In mutants these fibers extend toward an area near the endolymphatic duct, not to a horizontal crista. Most Otx1 null mutants had a small patch of sensory hair cells at this position. Measurement of the area of the utricular macula suggested it to be enlarged in Otx1 null mutants. We suggest that parts of the horizontal canal crista remain incorporated in the utricular sensory epithelium in Otx1 null mutants. Other parts of the horizontal crista appear to be variably segregated to form the isolated patch of hair cells identifiable by the unique fiber trajectory as representing the horizontal canal crista. Comparison with lamprey ear innervation reveals similarities in the pattern of innervation with the dorsal macula, a sensory patch of unknown function. SEM data confirm that all foramina are less constricted in Otx1 null mutants. We propose that Otx1 is not directly involved in sensory hair cell formation of the horizontal canal but affects the segregation of the horizontal canal crista from the utricle. It also affects constriction of the two main foramina in the ear, but not their initial formation. Otx1 is thus causally related to horizontal canal morphogenesis as well as morphogenesis of these foramina.     

Dark hair cells in the inner ear of the rainbow trout. A study of the influence of different fixation methods

The occurrence of dark staining cells in different tissues has been suggested to be artefactual and caused during the fixation process. In inner ear sensory epithelia, dark hair cells (DHC) have been suggested to be apoptotic cells. We have examined whether dark cells represent dying cells or whether they are the results of fixation artefacts. The effects of buffer osmolarity and different fixation methods on the incidence of dark hair cells in the inner ear macula sacculi of the rainbow trout (Oncorhynchus mykiss) were investigated by light and electron microscopy. Glutaraldehyde in phosphate buffer with osmolarities of 0, 135, 225, 425, and 560 mosmol were used for fixation by immersion. For comparison, fixation by vascular perfusion as well as the effects of mechanical injury and delayed fixation were studied. DHC were found in all examined saccular maculae except for the delayed fixation protocol where almost all the sensory cells were lost. The number of DHC accounted for 2.5–12.9‰ of the sensory cells. Neither the buffer osmolarity nor the fixation method had significant effects on the frequencies of DHC. Mitotic cell division events were seen exclusively in the apical cell strata of the sensory epithelium. The DHC are suggested to be associated with apoptosis rather than fixation artefacts.     

Morphological and molecular changes in the inner hair cell region of the rat cochlea after amikacin treatment

This study investigates the morphological and molecular changes that occur in the inner hair cell area of the rat cochlea following aminoglycoside treatment. Rats were injected daily with 500 mg/kg of amikacin between postnatal day 9 (PND9) and PND16. Cochleae were examined at PND16 to PND120 using both scanning and transmission electron microscopy and molecular fluorescent labeling. The inner hair cells showed obvious signs of apoptosis in response to amikacin treatment and most of them were missing by one week after the end of the aminoglycoside exposure period. Concomitantly, the epithelium became scarred as the surrounding supporting cells expanded and filled the space vacated by the missing IHCs. The mid-basolateral region of these modified supporting cells was surrounded by many afferent and efferent terminals. However, these cells expressed neither calbindin nor SNAP25, proteins that are both expressed by IHCs in the normal, untreated organ of Corti in the rat. In addition, these supporting cells remained attached to the basal lamina by a thin cytoplasmic process. The supporting cells surrounding the inner hair cells therefore appear unable to convert directly into inner hair cells following aminoglycoside induced hair-cell loss but may be able to provide trophic support for the remaining afferent and efferent neurites.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.     

Hes1 is a negative regulator of inner ear hair cell differentiation

Hair cell fate determination in the inner ear has been shown to be controlled by specific genes. Recent loss-of-function and gain-of-function experiments have demonstrated that Math1, a mouse homolog of the Drosophila gene atonal, is essential for the production of hair cells. To identify genes that may interact with Math1 and inhibit hair cell differentiation, we have focused on Hes1, a mammalian hairy and enhancer of split homolog, which is a negative regulator of neurogenesis. We report here that targeted deletion of Hes1 leads to formation of supernumerary hair cells in the cochlea and utricle of the inner ear. RT-PCR analysis shows that Hes1 is expressed in inner ear during hair cell differentiation and its expression is maintained in adulthood. In situ hybridization with late embryonic inner ear tissue reveals that Hes1 is expressed in supporting cells, but not hair cells, of the vestibular sensory epithelium. In the cochlea, Hes1 is selectively expressed in the greater epithelial ridge and lesser epithelial ridge regions which are adjacent to inner and outer hair cells. Co-transfection experiments in postnatal rat explant cultures show that overexpression of Hes1 prevents hair cell differentiation induced by Math1. Therefore Hes1 can negatively regulate hair cell differentiation by antagonizing Math1. These results suggest that a balance between Math1 and negative regulators such as Hes1 is crucial for the production of an appropriate number of inner ear hair cells.     

Ultrastructural differentiation of glandular stomach (proventriculus) in chick embryo

Cytochemical characterization of mucosubstances of chick glanular stomach (proventriculus) changes from 15 days of development to postnatal and adult stages was studied. To corroborate these data cytochemical, ultrastructural and ultracytochemical study of chick embryo proventriculus from 7 to 20 days of development was performed. At the 7th day several layers of undifferentiated cells formed an epithelium which covered the walls of the glandular stomach. Mocosubstances were not found. Between the 9th and 5th day a single layer of cylindrical cells was encountered forming invaginations which originated deep glands. Three types of cells were separated from the above mentioned layer, dark, clear and undifferentiated. The dark cells had organelles which are involved in protein synthesis and the clear ones were rich in mitochondria. Argentaffine cells appeared at 15th day instead mucosubstances formed a thin coat on the epithelium at 9th day which increased at the end of development in the apical cytoplasm and gland cells. These observations demonstrate that proventriculus of chick embryo has ultrastructurally differentiated cells involved with enzymatic and hydrochloric acid secretion after the 9th day. These progressive events are correlated with the digestion process of yolk during embryogenesis. At the end of development the proventriculus has completely organized the glandular layer.     

Electron microscope observations on in vitro cultures of the isolated fowl embryo otocyst

In vitro cultures of isolated fowl embryo otocysts were studied with the electron microscope. Hair cells of the developing organ of Corti and crista ampullaris have been examined with particular reference to the structure of the cilia and of the cell membrane. Two types of hair cells could be distinguished on the basis whether or not they possessed a "kinocilium" and "stereocilia," or "stereocilia" only. The cytoplasmic membranes were simple and there were no multiple vesicular layers in any of the hair cells. The supporting elements consisted of supporting cells flanking the hair cells, fibroblasts, and the cartilaginous otic capsule. Both the cochlear and vestibular sensory area showed rich innervation by mainly non-myelinated fibers with partial myelinization in others. There were well developed ganglion cells present. Bare axons penetrated the basement membrane and spread, amongst the supporting cells sheltering them, to the base of the hair cells where they formed bud-shaped nerve endings but, at the stage of development examined, no calyces. These in vitro cultures of the isolated fowl embryo otocyst provided convenient and suitable material for the electron microscope study of the sensory epithelium of the ear and revealed further that the isolated fowl embryo otocyst possesses great powers of self-differentiation also at the ultrastructural level.     

Inner hair cell Cre-expressing transgenic mouse

Cochlear hair cells of the inner ear are mechanosensory transducers critical for sound reception in mammals. A mouse with a specific expression of Cre recombinase activity in hair cells is essential for hair cell-specific gene targeting. Here we report a transgenic mouse in which Cre activity is detected in inner hair cells, not in supporting cells, in the cochlea. The Cre activity was visualized with both X-gal staining and beta-galactosidase immunostaining in progeny of a cross between our Cre line and the reporter ROSA26R line. In inner hair cells, the Cre activity started at postnatal day 14 and was maintained throughout adulthood. Starting at postnatal day 50, a few outer hair cells in the outermost row of cochlear apical and middle turns displayed the Cre activity. In vestibular hair cells and spiral ganglia, the Cre activity was also detected. Cre activity was present in cells widely distributed throughout brain, testis, and retina, but was absent in many other tissues such as kidney, heart, liver, and intestine. This Cre mouse line can thus be used for conditional gene targeting in mature inner hair cells of the cochlea. genesis 39:173-177, 2004. Copyright 2004 Wiley-Liss, Inc.     

Electron Microscope Observations on in Vitro Cultures of the Isolated Fowl Embryo Otocyst

In vitro cultures of isolated fowl embryo otocysts were studied with the electron microscope. Hair cells of the developing organ of Corti and crista ampullaris have been examined with particular reference to the structure of the cilia and of the cell membrane. Two types of hair cells could be distinguished on the basis whether or not they possessed a "kinocilium" and "stereocilia," or "stereocilia" only. The cytoplasmic membranes were simple and there were no multiple vesicular layers in any of the hair cells. The supporting elements consisted of supporting cells flanking the hair cells, fibroblasts, and the cartilaginous otic capsule. Both the cochlear and vestibular sensory area showed rich innervation by mainly non-myelinated fibers with partial myelinization in others. There were well developed ganglion cells present. Bare axons penetrated the basement membrane and spread, amongst the supporting cells sheltering them, to the base of the hair cells where they formed bud-shaped nerve endings but, at the stage of development examined, no calyces. These in vitro cultures of the isolated fowl embryo otocyst provided convenient and suitable material for the electron microscope study of the sensory epithelium of the ear and revealed further that the isolated fowl embryo otocyst possesses great powers of self-differentiation also at the ultrastructural level.     

Inner ear gene transfection in neonatal mice using adeno-associated viral vector: a comparison of two approaches

Local gene transfection is a promising technique for the prevention and/or correction of inner ear diseases, particularly those resulting from genetic defects. Adeno-associated virus (AAV) is an ideal viral vector for inner ear gene transfection because of its safety, stability, long-lasting expression, and its high tropism for many different cell types. Recently, a new generation of AAV vectors with a tyrosine mutation (mut-AAV) has demonstrated significant improvement in transfection efficiency. A method for inner ear gene transfection via the intact round window membrane (RWM) has been developed in our laboratory. This method has not been tested in neonatal mice, an important species for the study of inherited hearing loss. Following a preliminary study to optimize the experimental protocol in order to reduce mortality, the present study investigated inner ear gene transfection in mice at postnatal day 7. We compared transfection efficiency, the safety of the scala tympani injection via RWM puncture, and the trans-RWM diffusion following partial digestion with an enzyme technique. The results revealed that approximately 47% of inner hair cells (IHCs) and 17% of outer hair cells (OHCs) were transfected via the trans-RWM approach. Transfection efficiency via RWM puncture (58% and 19% for IHCs and OHCs, respectively) was slightly higher, but the difference was not significant.     

Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death

Mutations in the gene encoding the gap junction protein connexin26 (Cx26) are responsible for the autosomal recessive isolated deafness, DFNB1, which accounts for half of the cases of prelingual profound hereditary deafness in Caucasian populations. To date, in vivo approaches to decipher the role of Cx26 in the inner ear have been hampered by the embryonic lethality of the Cx26 knockout mice. To overcome this difficulty, we performed targeted ablation of Cx26 specifically in one of the two cellular networks that it underlies in the inner ear, namely, the epithelial network. We show that homozygous mutant mice, Cx26(OtogCre), have hearing impairment, but no vestibular dysfunction. The inner ear developed normally. However, on postnatal day 14 (P14), i.e., soon after the onset of hearing, cell death appeared and eventually extended to the cochlear epithelial network and sensory hair cells. Cell death initially affected only the supporting cells of the genuine sensory cell (inner hair cell, IHC), thus suggesting that it could be triggered by the IHC response to sound stimulation. Altogether, our results demonstrate that the Cx26-containing epithelial gap junction network is essential for cochlear function and cell survival. We conclude that prevention of cell death in the sensory epithelium is essential for any attempt to restore the auditory function in DFNB1 patients.     

Two contrasting roles for Notch activity in chick inner ear development: specification of prosensory patches and lateral inhibition of hair-cell differentiation

Lateral inhibition mediated by Notch is thought to generate the mosaic of hair cells and supporting cells in the inner ear, but the effects of the activated Notch protein itself have never been directly tested. We have explored the role of Notch signalling by transiently overexpressing activated Notch (NICD) in the chick otocyst. We saw two contrasting consequences, depending on the time and site of gene misexpression: (1) inhibition of hair-cell differentiation within a sensory patch; and (2) induction of ectopic sensory patches. We infer that Notch signalling has at least two functions during inner ear development. Initially, Notch activity can drive cells to adopt a prosensory character, defining future sensory patches. Subsequently, Notch signalling within each such patch mediates lateral inhibition, restricting the proportion of cells that differentiate as hair cells so as to generate the fine-grained mixture of hair cells and supporting cells.     

The role of Math1 in inner ear development: Uncoupling the establishment of the sensory primordium from hair cell fate determination

During embryonic development of the inner ear, the sensory primordium that gives rise to the organ of Corti from within the cochlear epithelium is patterned into a stereotyped array of inner and outer sensory hair cells separated from each other by non-sensory supporting cells. Math1, a close homolog of the Drosophila proneural gene atonal, has been found to be both necessary and sufficient for the production of hair cells in the mouse inner ear. Our results indicate that Math1 is not required to establish the postmitotic sensory primordium from which the cells of the organ of Corti arise, but instead is limited to a role in the selection and/or differentiation of sensory hair cells from within the established primordium. This is based on the observation that Math1 is only expressed after the appearance of a zone of non-proliferating cells that delineates the sensory primordium within the cochlear anlage. The expression of Math1 is limited to a subpopulation of cells within the sensory primordium that appear to differentiate exclusively into hair cells as the sensory epithelium matures and elongates through a process that probably involves radial intercalation of cells. Furthermore, mutation of Math1 does not affect the establishment of this postmitotic sensory primordium, even though the subsequent generation of hair cells is blocked in these mutants. Finally, in Math1 mutant embryos, a subpopulation of the cells within the sensory epithelium undergo apoptosis in a temporal gradient similar to the basal-to-apical gradient of hair cell differentiation that occurs in the cochlea of wild-type animals.     

Lineage Analysis of the Late Otocyst Stage Mouse Inner Ear by Transuterine Microinjection of A Retroviral Vector Encoding Alkaline Phosphatase and an Oligonucleotide Library

The mammalian inner ear subserves the special senses of hearing and balance. The auditory and vestibular sensory epithelia consist of mechanically sensitive hair cells and associated supporting cells. Hearing loss and balance dysfunction are most frequently caused by compromise of hair cells and/or their innervating neurons. The development of gene- and cell-based therapeutics will benefit from a thorough understanding of the molecular basis of patterning and cell fate specification in the mammalian inner ear. This includes analyses of cell lineages and cell dispersals across anatomical boundaries (such as sensory versus nonsensory territories). The goal of this study was to conduct retroviral lineage analysis of the embryonic day 11.5(E11.5) mouse otic vesicle. A replication-defective retrovirus encoding human placental alkaline phosphatase (PLAP) and a variable 24-bp oligonucleotide tag was microinjected into the E11.5 mouse otocyst. PLAP-positive cells were microdissected from cryostat sections of the postnatal inner ear and subjected to nested PCR. PLAP-positive cells sharing the same sequence tag were assumed to have arisen from a common progenitor and are clonally related. Thirty five multicellular clones consisting of an average of 3.4 cells per clone were identified in the auditory and vestibular sensory epithelia, ganglia, spiral limbus, and stria vascularis. Vestibular hair cells in the posterior crista were related to one another, their supporting cells, and nonsensory epithelial cells lining the ampulla. In the organ of Corti, outer hair cells were related to a supporting cell type and were tightly clustered. By contrast, spiral ganglion neurons, interdental cells, and Claudius'' cells were related to cells of the same type and could be dispersed over hundreds of microns. These data contribute new information about the developmental potential of mammalian otic precursors in vivo.     

Cell Cycle,Differentiation and Regeneration: Where to Begin?

Hair cells, the sensory cells of inner ear, perform essential functions in hearing and balance. However, mammalian hair cells, like most of the CNS neurons, lack the capacity to regenerate. This is in sharp contrast to lower vertebrates in which hair cell regeneration occurs spontaneously through cell division of supporting cells, which leads to hearing restoration. It is believed that the lack of regeneration in mammals is, to a large degree, due to the block of cell cycle re-entry imposed by negative cell growth genes in the inner ear. Recent studies have identified retinoblastoma gene, a well-known tumor suppressor, as the key gene involved in cell cycle exit of inner ear sensory cells. In the inner ear of pRb conditional knockout mice, hair cells undergo continuous cell division, and at the same time differentiate and become functional. Cell division continues in early postnatal cochlea and adult vestibule. Remarkably, the vestibular hair cells without pRb survive, and function at both the cellular and system levels. The time course and effects of pRb inhibition shows that there is a separation between the roles of pRb in cell cycle exit, and subsequent maturation and apoptosis. Those studies reveal distinctly different roles of pRb in the cochlear and vestibular sensory epithelia. The review discusses additional areas to be studied for regeneration of mature hair cells, and highlights the importance of transient and reversible block of pRb function as one of the routes to be explored for regeneration.     

Disappearance of afferent and efferent nerve terminals in the inner ear of the chick embryo after chronic treatment with beta-bungarotoxin

Beta-Bungarotoxin(beta-BT) was applied to chick embryos at 3-day intervals beginning on the 4th day of incubation to see the effect of chronically and massively applied beta-BT, and to investigate the hair cell-nerve relationship in the developing inner ear by electron microscopy. On the 10th day of incubation, nerve terminals had achieved contact with differentiating hair cells, but the acoustico-vestibular ganglion cells of treated animals were decreased in number to one-third of those of the control. By the 14th day, most of the ganglion cells degenerated and disappeared, and only a few nerve terminals were seen in the neuroepithelium. At this time, most of the hair cells lacked synaptic contacts with nerve terminals; but their presynaptic specialization remained intact and they showed evidence of continuing differentiation. On the 17th day, the acoustico-vestibular ganglion cells were completely absent. All the hair cells were devoid of afferent and efferent innervation but were fully differentiated on the 21st day. Beta-BT was found to have a similar destructive effect on cultured spinal ganglion cells. The present study shows that beta-BT kills acoustico-vestibular and spinal nerve cells when applied chronically and massively during development. Furthermore, the differentiation of hair cells proceeds normally, and their presynaptic specializations are maintained when nerve terminals are absent during later developmental stages.     

Notch ligands with contrasting functions: Jagged1 and Delta1 in the mouse inner ear

Each of the sensory patches in the epithelium of the inner ear is a mosaic of hair cells and supporting cells. Notch signalling is thought to govern this pattern of differentiation through lateral inhibition. Recent experiments in the chick suggest, however, that Notch signalling also has a prior function - inductive rather than inhibitory - in defining the prosensory patches from which the differentiated cells arise. Several Notch ligands are expressed in each patch, but their individual roles in relation to the two functions of Notch signalling are unclear. We have used a Cre-LoxP approach to knock out two of these ligands, Delta1 (Dll1) and Jagged1 (Jag1), in the mouse ear. In the absence of Dll1, auditory hair cells develop early and in excess, in agreement with the lateral inhibition hypothesis. In the absence of Jag1, by contrast, the total number of these cells is strongly reduced, with complete loss of cochlear outer hair cells and some groups of vestibular hair cells, indicating that Jag1 is required for the prosensory inductive function of Notch. The number of cochlear inner hair cells, however, is almost doubled. This correlates with loss of expression of the cell cycle inhibitor p27(Kip1) (Cdkn1b), suggesting that signalling by Jag1 is also needed to limit proliferation of prosensory cells, and that there is a core part of this population whose prosensory character is established independently of Jag1-Notch signalling. Our findings confirm that Notch signalling in the ear has distinct prosensory and lateral-inhibitory functions, for which different ligands are primarily responsible.     

Localized morphological differentiation in the bipolar cells of the chick retina

On a morphological and ultrastructural level, we studied a thickening which appears on the ascending prolongation of bipolar cells in the chick retina. We first observed this thickening on day 10 of incubation and it remains unchanged throughout the postnatal life of the chick. Its presence seems to be related to the synaptic activity at a dendritic level in certain bipolar cells.     

Hair cell regeneration in the avian auditory epithelium

Regeneration of sensory hair cells in the mature avian inner ear was first described just over 20 years ago. Since then, it has been shown that many other non-mammalian species either continually produce new hair cells or regenerate them in response to trauma. However, mammals exhibit limited hair cell regeneration, particularly in the auditory epithelium. In birds and other non-mammals, regenerated hair cells arise from adjacent non-sensory (supporting) cells. Hair cell regeneration was initially described as a proliferative response whereby supporting cells re-enter the mitotic cycle, forming daughter cells that differentiate into either hair cells or supporting cells and thereby restore cytoarchitecture and function in the sensory epithelium. However, further analyses of the avian auditory epithelium (and amphibian vestibular epithelium) revealed a second regenerative mechanism, direct transdifferentiation, during which supporting cells change their gene expression and convert into hair cells without dividing. In the chicken auditory epithelium, these two distinct mechanisms show unique spatial and temporal patterns, suggesting they are differentially regulated. Current efforts are aimed at identifying signals that maintain supporting cells in a quiescent state or direct them to undergo direct transdifferentiation or cell division. Here, we review current knowledge about supporting cell properties and discuss candidate signaling molecules for regulating supporting cell behavior, in quiescence and after damage. While significant advances have been made in understanding regeneration in non-mammals over the last 20 years, we have yet to determine why the mammalian auditory epithelium lacks the ability to regenerate hair cells spontaneously and whether it is even capable of significant regeneration under additional circumstances. The continued study of mechanisms controlling regeneration in the avian auditory epithelium may lead to strategies for inducing significant and functional regeneration in mammals.