Usherin is required for maintenance of retinal photoreceptors and normal development of cochlear hair cells.

Usher syndrome type IIA (USH2A), characterized by progressive photoreceptor degeneration and congenital moderate hearing loss, is the most common subtype of Usher syndrome. In this article, we show that the USH2A protein, also known as usherin, is an exceptionally large ( approximately 600-kDa) matrix protein expressed specifically in retinal photoreceptors and developing cochlear hair cells. In mammalian photoreceptors, usherin is localized to a spatially restricted membrane microdomain at the apical inner segment recess that wraps around the connecting cilia, corresponding to the periciliary ridge complex described for amphibian photoreceptors. In sensory hair cells of the cochlea, it is associated transiently with the hair bundles during postnatal development. Targeted disruption of the Ush2a gene in mice leads to progressive photoreceptor degeneration and a moderate but nonprogressive hearing impairment, mimicking the visual and hearing deficits in USH2A patients. These data suggest that usherin is required for the long-term maintenance of retinal photoreceptors and for the development of cochlear hair cells. We propose a model in which usherin in photoreceptors is tethered via its C terminus to the plasma membrane and its large extracellular domain projecting into the periciliary matrix, where they may interact with the connecting cilium to fulfill important structural or signaling roles.

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear.

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

MicroRNA gene expression in the mouse inner ear.

MicroRNAs (miRNAs) are small non-coding RNAs that function through the RNA interference (RNAi) pathway and post-transcriptionally regulate gene expression in eukaryotic organisms. While miRNAs are known to affect cellular proliferation, differentiation, and morphological development, neither their expression nor roles in mammalian inner ear development have been characterized. We have investigated the extent of miRNA expression at various time points throughout maturation of the postnatal mouse inner ear by microarray analysis. Approximately one third of known miRNAs are detected in the inner ear, and their expression persists to adulthood. Expression of such miRNAs is validated by quantitative PCR and northern blot analysis. Further analysis by in situ hybridization demonstrates that certain miRNAs exhibit cell-specific expression patterns in the mouse inner ear. Notably, we demonstrate that miRNAs previously associated with mechanosensory cells in zebrafish are also expressed in hair cells of the auditory and vestibular endorgans. Our results demonstrate that miRNA expression is abundant in the mammalian inner ear and that certain miRNAs are evolutionarily associated with mechanosensory cell development and/or function. The data suggest that miRNAs contribute substantially to genetic programs intrinsic to development and function of the mammalian inner ear and that specific miRNAs might influence formation of sensory epithelia from the primitive otic neuroepithelium.

The very large G-protein-coupled receptor VLGR1: a component of the ankle link complex required for the normal development of auditory hair bundles.

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

Atp2b2, encoding plasma membrane Ca2+-ATPase type 2, (PMCA2) exhibits tissue-specific first exon usage in hair cells, neurons, and mammary glands of mice.

Atp2b2 encodes the plasma membrane Ca(2+)-ATPase type 2 (PMCA2) expressed in various tissues, including stereocilia of cochlear and vestibular hair cells, cerebellar Purkinje cells, and lactating mammary epithelia. Mutations of the gene lead to deafness, ataxia, and reduced Ca(2+) levels in milk. Heterozygous mutants also have abnormal hearing, suggesting that precise regulation of Atp2b2 is required for normal function. In this study, we describe Atp2b2 5'-untranslated region genomic structure and transcript usage in mice. Using 5'-rapid amplification of cDNA ends, we observed four transcripts: types alpha, beta, mu and delta, each splicing into a common ATG-containing exon. Types alpha and beta correspond to previously published mammalian cDNA sequences. Types mu and delta constitute novel 5'-untranslated region sequences, and were observed at high levels only in lactating mammary gland. Using real-time reverse transcriptase polymerase chain reaction, we quantified relative transcript usage across several tissues. We show that alpha and beta are abundant throughout the CNS, as well as the cochlea. When we microdissected the cochlea into hair cell and spiral ganglion containing fractions, we found that cochlear hair cell expression is mediated through the type alpha transcript. In situ hybridization studies in cerebellum using exon-specific probes revealed that alpha dominates in Purkinje neurons, while beta is enriched in cerebellar granule neurons. We compared 5'-untranslated region sequence across multiple species, and found high conservation around the first exons for alpha and beta in mammals, but not other species. The regions around the mu and delta first exons are highly conserved between rat and mouse, but less so with other species. Our results show that expression of Atp2b2 is highly regulated, using four different transcriptional start regions, two of which are differentially expressed in neuronal tissue. This suggests that unique regulatory mechanisms are used to control Atp2b2 expression in different types of cells.

Characterization of proliferating cells from newborn mouse cochleae.

Loss of hair cells in mammals including human beings causes permanent hearing loss because the cochlea cannot regenerate hair cells spontaneously. Here we show that the newborn mouse cochleae contain sphere-forming cells that have the capacity for proliferation in culture, differentiating to form cells that express hair cell markers. When treated with epidermal growth factor or basic fibroblast growth factor, the number of spheres formed increases. The sphere cells express genes that are indicative of inner ear progenitor cells. After differentiation, some sphere cells grow a hair cell bundle-like structure that expresses hair cell marker myosin VIIA and espin. The sphere-forming cells being capable of differentiating into hair cell-like cells implies the possibility of using these sphere-forming cells for reconstructing the damaged cochlear hair cells.

Hair cell development: commitment through differentiation.

The perceptions of sound, balance and acceleration are mediated through the vibration of stereociliary bundles located on the lumenal surfaces of mechanosensory hair cells located within the inner ear. In mammals, virtually all hair cells are generated during a relatively brief period in embryogenesis with any subsequent hair cell loss leading to a progressive and permanent loss of sensitivity. In light of the importance of these cells, considerable effort has been focused on understanding the molecular genetic pathways that regulate their development. The results of these studies have begun to elucidate the signaling molecules that regulate several key events in hair cell development. In particular, significant progress has been made in the understanding of hair cell commitment, survival and differentiation. In addition, several aspects of the development of the stereociliary bundle, including its elongation and orientation, have recently been examined. This review will summarize results from each of these developmental events and describe the molecular signaling pathways involved.

Progression of inner ear pathology in Ames waltzer mice and the role of protocadherin 15 in hair cell development.

The Ames waltzer (av) mouse mutant exhibits auditory and vestibular abnormalities resulting from mutation of protocadherin 15 (Pcdh15). Ames waltzer has been identified as an animal model for inner ear pathology associated with Usher syndrome type 1F. Studies correlating anatomical phenotype with severity of genetic defect in various av alleles are providing better understanding of the role played by Pcdh15 in inner ear development and of sensorineural abnormalities associated with alterations in Pcdh15 protein structure as a result of gene mutation. In this work we present new findings on inner ear pathology in four alleles of av mice with differing mutations of Pcdh15 as well as varying alterations in inner ear morphology. Two alleles with in-frame deletion mutations (Pcdh15 (av-J) and Pcdh15 (av-2J)) and two presumptive functional null alleles (Pcdh15 (av-3J) and Pcdh15 (av-Tg)) were studied. Light and electron microscopic observations demonstrated that the severity of cochlear and vestibular pathology in these animals correlates positively with the extent of mutation in Pcdh15 from embryonic day 18 (E18) up to 12 months. Electron microscopic analysis of immature ears indicated early abnormalities in the arrangement of stereocilia and the inner and outer hair cell cuticular plates, stereocilia rootlets, and the actin meshwork within the cuticular plate. In severe cases, displacement of the kinocilium and alterations in the shape of the cuticular plate was also observed. Mice harboring in-frame deletion mutations showed less disorganization of stereocilia and cuticular plates in the organ of Corti than the presumptive functional null alleles at P0-P10. A slower progression of pathology was also seen via light microscopy in older animals with in-frame deletions, compared to the presumptive functional null mutations. In summary, our results demonstrate that mutation in Pcdh15 affects the initial formation of stereocilia bundles with associated changes in the actin meshwork within the cuticular plate; these effects are more pronounced in the presumed null mutation compared to mutations that only affect the extracellular domain. The positive correlation of severity of effects with extent of mutation can be seen well into adulthood.

Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear.

The espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation.

Inhibition of Notch/RBP-J signaling induces hair cell formation in neonate mouse cochleas.

Mammalian inner ear hair cells in cochleas are believed to be incapable of regeneration after birth, which hampers treatment of sensorineural hearing impairment mainly caused by hair cell loss. Sensory epithelia of cochleas are composed of hair cells and supporting cells, both of which originate from common progenitors. Notch/RBP-J signaling is an evolutionally conserved pathway involved in specification of various cell types in developmental stage and even in some of postnatal mammalian organs. The specification of hair cell fate from the progenitors is inhibited by Notch/RBP-J signaling in embryonic inner ears. However, its function in postnatal inner ears is unknown. We showed that inhibition of Notch/RBP-J signaling, by either conditional disruption of the Rbpsuh gene or treatment with a gamma-secretase inhibitor, could give rise to ectopic hair cells in the supporting cell region in organs of Corti from neonatal mouse cochleas where hair cells have not been considered to regenerate after birth. We also showed that down-regulation of Hes5 and up-regulation of Math1 were associated with ectopic hair cell induction. These results suggest that Notch/RBP-J signaling inhibits supporting cells from differentiation into hair cells even in postnatal days, implying that inhibitors of Notch/RBP-J signaling can be used to help regenerating hair cells after birth and thus serve for potential treatment of intractable sensorineural hearing impairment caused by hair cell loss without genetical manipulation.

Biotinidase reveals the morphogenetic sequence in cochlea and cochlear nucleus of mice.

Hearing loss affects children with biotinidase deficiency, an inherited metabolic disorder in the recycling of biotin. The deficit appears shortly after birth during development of the auditory system. Using a mouse model, we sought to discover where and when biotinidase is expressed in the normal development of the cochlea and cochlear nucleus. In the process, we reconstructed the normal morphogenetic sequences of the constituent cells. Immunolabeling for biotinidase was localized to neurons and other cells of the adult and immature mouse, including the embryonic precursors of these regions dating from the stage of the otocyst. Its distribution was compared to the particular morphological changes occurring at each developmental stage. Biotinidase was localized in cells and their processes at the critical stages in their proliferation, migration, structural differentiation, and innervation, covering the entire span of their development. The prevalence of immunostaining peaked in the adult animal, including hair cells and ganglion cells of the cochlea and neurons of the cochlear nucleus. The findings suggest that biotinidase plays a role in the normal development of the auditory system. Besides the pattern of localization of biotinidase, this study provides the first systematic account of each developmental stage in a mammalian auditory system.

The membranous labyrinth during larval development in lamprey (Lampetra planeri, Bloch, 1784).

SEM and CLSM studies were performed on the membranous labyrinth of Lampetra planeri, a threatened species of brook lamprey, spanning from the 1st to the 4th year of ammocoetes larval stages and on the adults. In all the examined stages, the entire membranous labyrinth does not show any morphologic differences, but only a progressive increase in size. SEM and CLSM observations show that the ciliated chamber is lined with numerous unsensorial multiciliated cells. In the early stages, the ciliary bundles were approximately 15 microm long, while in the late stages they reached 30 microm. In the crista sensory area, we observed two populations of hair cells. "Type II" cells are peculiar for this species and show both long stereocilia decreasing in length and a long kinocilium (10-12 microm). Two other types of ciliary bundles have been found on the sensory hair cells of the Macula communis: the first one has both kinocilium and stereocilia about 4-5 microm long; the second shows a long kinocilium (7-10 microm in length) and short stereocilia bundles with a gradual increase in length. In the early stages of development, the three macular areas show few and sparsely distributed hair cells. In the late developmental stages, hair cells become more numerous and densely populated.

Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals.

In the mammalian auditory system, sensory cell loss resulting from aging, ototoxic drugs, infections, overstimulation and other causes is irreversible and leads to permanent sensorineural hearing loss. To restore hearing, it is necessary to generate new functional hair cells. One potential way to regenerate hair cells is to induce a phenotypic transdifferentiation of nonsensory cells that remain in the deaf cochlea. Here we report that Atoh1, a gene also known as Math1 encoding a basic helix-loop-helix transcription factor and key regulator of hair cell development, induces regeneration of hair cells and substantially improves hearing thresholds in the mature deaf inner ear after delivery to nonsensory cells through adenovectors. This is the first demonstration of cellular and functional repair in the organ of Corti of a mature deaf mammal. The data suggest a new therapeutic approach based on expressing crucial developmental genes for cellular and functional restoration in the damaged auditory epithelium and other sensory systems.

Age effects and size effects in the ears of gekkonomorph lizards: inner ear.

Audiograms have indicated greater auditory sensitivity in larger than in smaller geckos; part of this difference, interspecifically and intraspecifically, is explained by middle-ear proportions. To investigate the contribution of the inner ear to the variation in sensitivity, we examined it in museum specimens representing 11 species and three subfamilies. We measured papilla basilaris length, and, when intact, the saccular otoconial mass. Papilla length approximated 1% of rostrum-anus length in large geckos but 2% in small geckos; in some species some inter-aural difference was indicated. Over the lumped material, relative papilla length varied as a function of body length, with highly significant correlation. Similar relations prevailed within each subfamily. However, intraspecifically the correlation of papilla basilaris length with animal size was usually nonsignificant. Hair cell populations assessed from SEM photographs were larger in the larger species but intraspecifically did not relate to an individual's size. Hence interspecifically, the dependence of auditory sensitivity on animal size seems supported by inner-ear differences but intraspecifically this relation derives only from the middle ear. Otoconial mass, as measured by its volume, was correlated with animal length both interspecifically and intraspecifically.

Survival of adult spiral ganglion neurons requires erbB receptor signaling in the inner ear.

Degeneration of cochlear sensory neurons is an important cause of hearing loss, but the mechanisms that maintain the survival of adult cochlear sensory neurons are not clearly defined. We now provide evidence implicating the neuregulin (NRG)-erbB receptor signaling pathway in this process. We found that NRG1 is expressed by spiral ganglion neurons (SGNs), whereas erbB2 and erbB3 are expressed by supporting cells of the organ of Corti, suggesting that these molecules mediate interactions between these cells. Transgenic mice in which erbB signaling in adult supporting cells is disrupted by expression of a dominant-negative erbB receptor show severe hearing loss and 80% postnatal loss of type-I SGNs without concomitant loss of the sensory cells that they contact. Quantitative RT-PCR analysis of neurotrophic factor expression shows a specific downregulation in expression of neurotrophin-3 (NT3) in the transgenic cochleas before the onset of neuronal death. Because NT3 is critical for survival of type I SGNs during development, these results suggest that it plays similar roles in the adult. Together, the data indicate that adult cochlear supporting cells provide critical trophic support to the neurons, that survival of postnatal cochlear sensory neurons depends on reciprocal interactions between neurons and supporting cells, and that these interactions are mediated by NRG and neurotrophins.

Development of f2/f1 ratio functions in humans.

Otoacoustic emissions (OAEs) presumably represent active processes within the cochlea fundamental to frequency-selectivity in peripheral auditory function. Maturation of the cochlear amplifier, vis-a-vis frequency encoding or selectivity, has yet to be fully characterized in humans. The purpose of this study was to further investigate the maturation of features of the f2/f1 frequency ratio (Distortion Product OAE amplitude X f2/f1 ratio) presumed to reflect cochlear frequency selectivity. A cross-sectional, multivariate study was completed comparing three age groups: pre-term infants, term infants and young adult subjects. Frequency ratio functions were analyzed at three f2 frequencies--2000, 4000 and 6000 Hz. An analysis included an estimation of the optimal ratio (OR) and a bandwidth-like measure (Q3). Analysis revealed significant interactions of age x frequency x gender for optimal ratio and a significant interaction of age x frequency for Q3. Consistent and statistically significant differences for both OR and Q3 were found in female subjects and when f2 = 2 or 6 kHz. This supports research by others [Abdala, J. Acoust. Soc. Am. 114, 3239-3250 (2003)] suggesting that the development of cochlear active mechanisms may still be somewhat in flux at least through term birth. Furthermore, OAEs appear to demonstrate gender differences in the course of such maturational changes.

Expression of beta-catenin in developing auditory epithelia of mice.

This study investigated the role of beta-catenin in the development of mouse auditory epithelia. Inner ears obtained from embryonic and newborn mice were used. Expression of beta-catenin was examined together with the expression of Ki-67, a marker for proliferating cells, or myosin VIIa, a marker for differentiated hair cells. In the early phase of development, intense expression of beta-catenin was found in auditory epithelia in which a number of Ki-67-positive cells were identified. Together with a decrease in proliferating cells, the intensity and area of beta-catenin expression were reduced. In addition, during differentiation and maturation of hair cells, the area of beta-catenin expression was further limited. These findings suggest that patterns of expression of beta-catenin are closely linked with the status of auditory epithelia development.

Correlation of expression of the actin filament-bundling protein espin with stereociliary bundle formation in the developing inner ear.

The vertebrate hair cell is named for its stereociliary bundle or hair bundle that protrudes from the cell's apical surface. Hair bundles mediate mechanosensitivity, and their highly organized structure plays a critical role in mechanoelectrical transduction and amplification. The prototypical hair bundle is composed of individual stereocilia, 50-300 in number, depending on the animal species and on the type of hair cell. The assembly of stereocilia, in particular, the formation during development of individual rows of stereocilia with descending length, has been analyzed in great morphological detail. Electron microscopic studies have demonstrated that stereocilia are filled with actin filaments that are rigidly cross-linked. The growth of individual rows of stereocilia is associated with the addition of actin filaments and with progressively increasing numbers of cross-bridges between actin filaments. Recently, a mutation in the actin filament-bundling protein espin has been shown to underlie hair bundle degeneration in the deaf jerker mouse, subsequently leading to deafness. Our study was undertaken to investigate the appearance and developmental expression of espin in chicken inner ear sensory epithelia. We found that the onset of espin expression correlates with the initiation and growth of stereocilia bundles in vestibular and cochlear hair cells. Intense espin immunolabeling of stereocilia was colocalized with actin filament staining in all types of hair cells at all developmental stages and in adult animals. Our analysis of espin as a molecular marker for actin filament cross-links in stereocilia is in full accordance with previous morphological studies and implicates espin as an important structural component of hair bundles from initiation of bundle assembly to mature chicken hair cells.