CD44 is a marker for the outer pillar cells in the early postnatal mouse inner ear.
ABSTRACT: Cluster of differentiation antigens (CD proteins) are classically used as immune cell markers. However, their expression within the inner ear is still largely undefined. In this study, we explored the possibility that specific CD proteins might be useful for defining inner ear cell populations. mRNA expression profiling of microdissected auditory and vestibular sensory epithelia revealed 107 CD genes as expressed in the early postnatal mouse inner ear. The expression of 68 CD genes was validated with real-time RT-PCR using RNA extracted from microdissected sensory epithelia of cochleae, utricles, saccules, and cristae of newborn mice. Specifically, CD44 was identified as preferentially expressed in the auditory sensory epithelium. Immunohistochemistry revealed that within the early postnatal organ of Corti, the expression of CD44 is restricted to outer pillar cells. In order to confirm and expand this finding, we characterized the expression of CD44 in two different strains of mice with loss- and gain-of-function mutations in Fgfr3 which encodes a receptor for FGF8 that is essential for pillar cell development. We found that the expression of CD44 is abolished from the immature pillar cells in homozygous Fgfr3 knockout mice. In contrast, both the outer pillar cells and the aberrant Deiters' cells in the Fgfr3 ( P244R/ ) (+) mice express CD44. The deafness phenotype segregating in DFNB51 families maps to a linkage interval that includes CD44. To study the potential role of CD44 in hearing, we characterized the auditory system of CD44 knockout mice and sequenced the entire open reading frame of CD44 of affected members of DFNB51 families. Our results suggest that CD44 does not underlie the deafness phenotype of the DFNB51 families. Finally, our study reveals multiple potential new cell type-specific markers in the mouse inner ear and identifies a new marker for outer pillar cells.
Project description:The heterozygous Pro250Arg substitution mutation in fibroblast growth factor receptor 3 (FGFR3), which increases ligand-dependent signalling, is the most common genetic cause of craniosynostosis in humans and defines Muenke syndrome. Since FGF signalling plays dosage-sensitive roles in the differentiation of the auditory sensory epithelium, we evaluated hearing in a large group of Muenke syndrome subjects, as well as in the corresponding mouse model (Fgfr3(P244R)). The Muenke syndrome cohort showed significant, but incompletely penetrant, predominantly low-frequency sensorineural hearing loss, and the Fgfr3(P244R) mice showed dominant, fully penetrant hearing loss that was more severe than that in Muenke syndrome individuals, but had the same pattern of relative high-frequency sparing. The mouse hearing loss correlated with an alteration in the fate of supporting cells (Deiters'-to-pillar cells) along the entire length of the cochlear duct, with the most extreme abnormalities found at the apical or low-frequency end. In addition, there was excess outer hair cell development in the apical region. We conclude that low-frequency sensorineural hearing loss is a characteristic feature of Muenke syndrome and that the genetically equivalent mouse provides an excellent model that could be useful in testing hearing loss therapies aimed at manipulating the levels of FGF signalling in the inner ear.
Project description:Adeno-associated virus (AAV) has been successfully used to deliver gene therapy to improve auditory function in mouse models of hereditary hearing loss. Many forms of hereditary hearing loss have mutations which affect the cochlear hair cells, the mechanosensory cells which allow for sound detection and processing. While most conventional AAVs infect inner hair cells (IHCs) with various efficiencies, they infect outer hair cells (OHCs) and supporting cells at lower levels in the cochlea. Here we examine the infection patterns of two synthetic AAVs (AAV2.7m8 and AAV8BP2) in the mouse inner ear. AAV2.7m8 infects both IHCs and OHCs with high efficiency. In addition, AAV2.7m8 infects inner pillar cells and inner phalangeal cells with high efficiency. Our results suggest that AAV2.7m8 is an excellent viral vector for inner ear gene therapy targeting cochlear hair cells and supporting cells, and it will likely greatly expand the potential applications for inner ear gene therapy.
Project description:The organ of Corti, the auditory organ of the inner ear, contains two types of sensory hair cells and at least seven types of supporting cells. Most of these supporting cell types rely on Notch-dependent expression of Hes/Hey transcription factors to maintain the supporting cell fate. Here, we show that Notch signaling is not necessary for the differentiation and maintenance of pillar cell fate, that pillar cells are distinguished by Hey2 expression, and that-unlike other Hes/Hey factors-Hey2 expression is Notch independent. Hey2 is activated by FGF and blocks hair cell differentiation, whereas mutation of Hey2 leaves pillar cells sensitive to the loss of Notch signaling and allows them to differentiate as hair cells. We speculate that co-option of FGF signaling to render Hey2 Notch independent also liberated pillar cells from the need for direct contact with surrounding hair cells, and enabled evolutionary remodeling of the complex cellular mosaic of the inner ear.
Project description:Fifty percent of inner ear disorders are caused by genetic mutations. To develop treatments for genetic inner ear disorders, we designed gene replacement therapies using synthetic adeno-associated viral vectors to deliver the coding sequence for Transmembrane Channel-Like (Tmc) 1 or 2 into sensory hair cells of mice with hearing and balance deficits due to mutations in Tmc1 and closely related Tmc2. Here we report restoration of function in inner and outer hair cells, enhanced hair cell survival, restoration of cochlear and vestibular function, restoration of neural responses in auditory cortex and recovery of behavioral responses to auditory and vestibular stimulation. Secondarily, we find that inner ear Tmc gene therapy restores breeding efficiency, litter survival and normal growth rates in mouse models of genetic inner ear dysfunction. Although challenges remain, the data suggest that Tmc gene therapy may be well suited for further development and perhaps translation to clinical application.
Project description:Acetylcholine is the major neurotransmitter of the olivocochlear efferent system, which provides feedback to cochlear hair cells and sensory neurons. To study the role of cochlear muscarinic receptors, we studied receptor localization with immunohistochemistry and reverse transcription-PCR and measured olivocochlear function, cochlear responses, and histopathology in mice with targeted deletion of each of the five receptor subtypes. M2, M4, and M5 were detected in microdissected immature (postnatal days 10-13) inner hair cells and spiral ganglion cells but not outer hair cells. In the adult (6 weeks), the same transcripts were found in microdissected organ of Corti and spiral ganglion samples. M2 protein was found, by immunohistochemistry, in olivocochlear fibers in both outer and inner hair cell areas. M3 mRNA was amplified only from whole cochleas, and M1 message was never seen in wild-type ears. Auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were unaffected by loss of Gq-coupled receptors (M1, M3, or M5), as were shock-evoked olivocochlear effects and vulnerability to acoustic injury. In contrast, loss of Gi-coupled receptors (M2 and/or M4) decreased neural responses without affecting DPOAEs (at low frequencies). This phenotype and the expression pattern are consistent with excitatory muscarinic signaling in cochlear sensory neurons. At high frequencies, both ABRs and DPOAEs were attenuated by loss of M2 and/or M4, and the vulnerability to acoustic injury was dramatically decreased. This aspect of the phenotype and the expression pattern are consistent with a presynaptic role for muscarinic autoreceptors in decreasing ACh release from olivocochlear terminals during high-level acoustic stimulation and suggest that muscarinic antagonists could enhance the resistance of the inner ear to noise-induced hearing loss.
Project description:The distinctive planar polarity of auditory hair cells is evident in the polarized organization of the stereociliary bundle. Mutations in the core planar cell polarity gene Van Gogh-like 2 (Vangl2) result in hair cells that fail to properly orient their stereociliary bundles along the mediolateral axis of the cochlea. The severity of this phenotype is graded along the length of the cochlea, similar to the hair cell differentiation gradient, suggesting that an active refinement process corrects planar polarity phenotypes in Vangl2 knock-out (KO) mice. Because Vangl2 gene deletions are lethal, Vangl2 conditional knock-outs (CKOs) were generated to test this hypothesis. When crossed with Pax2-Cre, Vangl2 is deleted from the inner ear, yielding planar polarity phenotypes similar to Vangl2 KOs at late embryonic stages except that Vangl2 CKO mice are viable and do not have craniorachischisis like Vangl2 KOs. Quantification of planar polarity deficits through postnatal development demonstrates the activity of a Vangl2-independent refinement process that rescues the planar polarity phenotype within 10 d of birth. In contrast, the Pax2-Cre;Vangl2 CKO has profound changes in the shape and distribution of outer pillar cell and Deiters' cell phalangeal processes that are not corrected during the period of planar polarity refinement. Auditory brainstem response analyses of adult mice show a 10-15 dB shift in auditory threshold, and distortion product otoacoustic emission measurements indicate that this mild hearing deficit is of cochlear origin. Together, these data demonstrate a Vangl2-independent refinement mechanism that actively reorients auditory stereociliary bundles and reveals an unexpected role of Vangl2 during supporting cell morphogenesis.
Project description:We previously mapped a novel autosomal dominant deafness locus, DFNA44, by studying a family with postlingual, progressive, nonsyndromic hearing loss. We report here on the identification of a mutation in CCDC50 as the cause of hearing loss in the family. CCDC50 encodes Ymer, an effector of epidermal growth factor (EGF)-mediated cell signaling that is ubiquitously expressed in different organs and has been suggested to inhibit down-regulation of the EGF receptor. We have examined its expression pattern in mouse inner ear. Western blotting and cell transfection results indicate that Ymer is a soluble, cytoplasmic protein, and immunostaining shows that Ymer is expressed in a complex spatiotemporal pattern during inner ear development. In adult inner ear, the expression of Ymer is restricted to the pillar cells of the cochlea, the stria vascularis, and the vestibular sensory epithelia, where it shows spatial overlap with the microtubule-based cytoskeleton. In dividing cells, Ymer colocalizes with microtubules of the mitotic apparatus. We suggest that DFNA44 hearing loss may result from a time-dependent disorganization of the microtubule-based cytoskeleton in the pillar cells and stria vascularis of the adult auditory system.
Project description:Inner ear auditory and vestibular tissues differ in their responses to mechanical stimuli. Chick cochlea and utricle sensory epithelia were microdissected at E20-E21. RNA was extracted and cRNA hybridized to Affymetrix microarrays.
Project description:Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to "self-terminate" its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.
Project description:KEY POINTS:The physiological maturation of auditory hair cells and their innervation requires precise temporal and spatial control of cell differentiation. The transcription factor gata3 is essential for the earliest stages of auditory system development and for survival and synaptogenesis in auditory sensory afferent neurons. We show that during postnatal development in the mouse inner ear gata3 is required for the biophysical maturation, growth and innervation of inner hair cells; in contrast, it is required only for the survival of outer hair cells. Loss of gata3 in inner hair cells causes progressive hearing loss and accounts for at least some of the deafness associated with the human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome. The results show that gata3 is critical for later stages of mammalian auditory system development where it plays distinct, complementary roles in the coordinated maturation of sensory hair cells and their innervation. ABSTRACT:The zinc finger transcription factor gata3 regulates inner ear development from the formation of the embryonic otic placode. Throughout development, gata3 is expressed dynamically in all the major cochlear cell types. Its role in afferent formation is well established but its possible involvement in hair cell maturation remains unknown. Here, we find that in heterozygous gata3 null mice (gata3+/- ) outer hair cells (OHCs) differentiate normally but their numbers are significantly lower. In contrast, inner hair cells (IHCs) survive normally but they fail to acquire adult basolateral membrane currents, retain pre-hearing current and efferent innervation profiles and have fewer ribbon synapses. Targeted deletion of gata3 driven by otoferlin-cre recombinase (gata3fl/fl otof-cre+/- ) in IHCs does not affect OHCs or the number of IHC afferent synapses but it leads to a failure in IHC maturation comparable to that observed in gata3+/- mice. Auditory brainstem responses in gata3fl/fl otof-cre+/- mice reveal progressive hearing loss that becomes profound by 6-7 months, whilst distortion product otoacoustic emissions are no different to control animals up to this age. Our results, alongside existing data, indicate that gata3 has specific, complementary functions in different cell types during inner ear development and that its continued expression in the sensory epithelium orchestrates critical aspects of physiological development and neural connectivity. Furthermore, our work indicates that hearing loss in human hypoparathyroidism, deafness and renal anomaly (HDR) syndrome arises from functional deficits in IHCs as well as loss of function from OHCs and both afferent and efferent neurons.