Foxg1 is required for morphogenesis and histogenesis of the mammalian inner ear.

The forkhead genes are involved in patterning, morphogenesis, cell fate determination, and proliferation. Several Fox genes (Foxi1, Foxg1) are expressed in the developing otocyst of both zebrafish and mammals. We show that Foxg1 is expressed in most cell types of the inner ear of the adult mouse and that Foxg1 mutants have both morphological and histological defects in the inner ear. These mice have a shortened cochlea with multiple rows of hair cells and supporting cells. Additionally, they demonstrate striking abnormalities in cochlear and vestibular innervation, including loss of all crista neurons and numerous fibers that overshoot the organ of Corti. Closer examination shows that some anterior crista fibers exist in late embryos. Tracing these fibers shows that they do not project to the brain but, instead, to the cochlea. Finally, these mice completely lack a horizontal crista, although a horizontal canal forms but comes off the anterior ampulla. Anterior and posterior cristae, ampullae, and canals are reduced to varying degrees, particularly in combination with Fgf10 heterozygosity. Compounding Fgf10 heterozygotic effects suggest an additive effect of Fgf10 on Foxg1, possibly mediated through bone morphogenetic protein regulation. We show that sensory epithelia formation and canal development are linked in the anterior and posterior canal systems. Much of the Foxg1 phenotype can be explained by the participation of the protein binding domain in the delta/notch/hes signaling pathway. Additional Foxg1 effects may be mediated by the forkhead DNA binding domain.

Audiovestibular findings in patients with mitochondrial A1555G mutation.

OBJECTIVE: The aims of this study were to explore the prevalence of the A1555G mutation among a group of Japanese patients and to assess the pathophysiology of the hearing impairment associated with the mutation. STUDY DESIGN: Genetic study and retrospective chart review. METHODS: We screened for the mitochondrial DNA A1555G mutation in 138 unrelated Japanese deaf patients, including 63 sporadic cases and 75 familial cases with different patterns of inheritance. When available, patients carrying the mutation received audiovestibular examinations, including speech audiometry, distortion-product otoacoustic emission (DPOAE) testing, electrocochleography (ECochG), and electronystagmography. RESULTS: One of 63 sporadic cases (1.6%) and 6 of 75 familial cases (8.0%) carried the A1555G mutation. Patients with the mutation and a familial history included two with autosomal recessive inheritance and four with maternal inheritance. In addition, two of six patients (33.3%) presenting with aminoglycoside-induced sensorineural hearing loss (SNHL) were associated with the A1555G mutation. All but one of the patients carrying the mutation showed high-frequency SNHL. Distortion-product levels of DPOAE were reduced to the noise levels, suggesting the A1555G mutation caused cochlear deafness. Cochlear microphonics in ECochG showed elevation of the detection thresholds and corresponding audiometric thresholds. The ECochG data implied that patients with high-frequency SNHL had impairment of the cochlear hair cells that was most severe toward the basal turn. The electronystagmographic findings indicated no apparent vestibular dysfunction. CONCLUSIONS: Screening for the A1555G mutation, even in patients with idiopathic bilateral SNHL, likely would be useful for preventing further development and/or acceleration of the deafness.

Identification of Vangl2 and Scrb1 as planar polarity genes in mammals.

In mammals, an example of planar cell polarity (PCP) is the uniform orientation of the hair cell stereociliary bundles within the cochlea. The PCP pathway of Drosophila refers to a conserved signalling pathway that regulates the coordinated orientation of cells or structures within the plane of an epithelium. Here we show that a mutation in Vangl2, a mammalian homologue of the Drosophila PCP gene Strabismus/Van Gogh, results in significant disruptions in the polarization of stereociliary bundles in mouse cochlea as a result of defects in the direction of movement and/or anchoring of the kinocilium within each hair cell. Similar, but less severe, defects are observed in animals containing a mutation in the LAP protein family gene Scrb1 (homologous with Drosophila scribble). Polarization defects in animals heterozygous for Vangl2 and Scrb1 are comparable with Vangl2 homozygotes, demonstrating genetic interactions between these genes in the regulation of PCP in mammals. These results demonstrate a role for the PCP pathway in planar polarization in mammals, and identify Scrb1 as a PCP gene.

Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice.

Deafness in spontaneously occurring mouse mutants is often associated with defects in cochlea sensory hair cells, opening an avenue to systematically identify genes critical for hair cell structure and function. The classical semidominant mouse mutant varitint-waddler (Va) exhibits early-onset hearing loss, vestibular defects, pigmentation abnormalities, and perinatal lethality. A second allele, Va(J), which arose in a cross segregating for Va, shows a less severe phenotype. By using a positional cloning strategy, we identify two additional members of the mucolipin gene family (Mcoln2 and Mcoln3) in the 350-kb Va(J) minimal interval and provide evidence for Mcoln3 as the gene mutated in varitint-waddler. Mcoln3 encodes a putative six-transmembrane-domain protein with sequence and motif similarities to the family of nonselective transient-receptor-potential (TRP) ion channels. In the Va allele an Ala419Pro substitution occurs in the fifth transmembrane domain of Mcoln3, and in Va(J), a second sequence alteration (Ile362Thr) occurring in cis partially rescues the Va allele. Mcoln3 localizes to cytoplasmic compartments of hair cells and plasma membrane of stereocilia. Hair cell defects are apparent by embryonic day 17.5, assigning Mcoln3 an essential role during early hair cell maturation. Our data suggest that Mcoln3 is involved in ion homeostasis and acts cell-autonomously. Hence, we identify a molecular link between hair cell physiology and melanocyte function. Last, MCOLN2 and MCOLN3 are candidate genes for hereditary and/or sporadic forms of neurosensory disorders in humans.

A novel stereocilia defect in sensory hair cells of the deaf mouse mutant Tasmanian devil.

Stereocilia are specialized actin-filled, finger-like processes arrayed in rows of graded heights to form a crescent or W-shape on the apical surface of sensory hair cells. The stereocilia are deflected by the vibration of sound, which opens transduction channels and allows an influx of ions to depolarize the hair cell, in turn triggering synaptic activity. The specialized morphology and organization of the stereocilia bundle is crucial in the process of sensory transduction in the inner ear. However, we know little about the development of stereocilia in the mouse and few molecules that are involved in stereocilia maturation are known. We describe here a new mouse mutant with abnormal stereocilia development. The Tasmanian devil (tde) mouse mutation arose by insertional mutagenesis and has been mapped to the middle of chromosome 5. Homozygotes show head-tossing and circling and have raised thresholds for cochlear nerve responses to sound. The gross morphology of the inner ear was normal, but the stereocilia of cochlear and vestibular hair cells are abnormally thin, and they become progressively disorganized with increasing age. Ultimately, the hair cells die. This is the first report of a mutant showing thin stereocilia. The association of thin stereocilia with cochlear dysfunction emphasizes the critical role of stereocilia in auditory transduction, and the discovery of the Tasmanian devil mutant provides a resource for the identification of an essential molecule in hair cell function.

FGFR1 is required for the development of the auditory sensory epithelium.

The mammalian auditory sensory epithelium, the organ of Corti, comprises the hair cells and supporting cells that are pivotal for hearing function. The origin and development of their precursors are poorly understood. Here we show that loss-of-function mutations in mouse fibroblast growth factor receptor 1 (Fgfr1) cause a dose-dependent disruption of the organ of Corti. Full inactivation of Fgfr1 in the inner ear epithelium by Foxg1-Cre-mediated deletion leads to an 85% reduction in the number of auditory hair cells. The primary cause appears to be reduced precursor cell proliferation in the early cochlear duct. Thus, during development, FGFR1 is required for the generation of the precursor pool, which gives rise to the auditory sensory epithelium. Our data also suggest that FGFR1 might have a distinct later role in intercellular signaling within the differentiating auditory sensory epithelium.

Mutation of the novel gene Tmie results in sensory cell defects in the inner ear of spinner, a mouse model of human hearing loss DFNB6.

The recessive mutation at the mouse spinner (sr) locus results in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified the mutant locus responsible for this pathology. The affected gene (Tmie) lies within a 40 kb deletion in the original sr allele. In a newly identified allele, Tmie contains a nonsense mutation expected to truncate the C-terminal end of its product. The 153 amino acid protein encoded by the gene shows no similarity to other known proteins, and is predicted to contain a signal peptide and at least one transmembrane domain. Tmie transcripts were identified in several tissues, including the cochlea. Loss of function of Tmie results in postnatal alterations of sensory hair cells in the cochlea, including defects in stereocilia, the apical projections of hair cells that are important in mechanotransduction of sound. These morphological defects are associated with a profound failure to develop normal auditory function. Consistent with a conserved role for this gene in the cochlea, the genetic mapping data presented here support human TMIE as the gene affected at DFNB6, a non-syndromic hearing loss locus. The spinner mutant is thus a valuable model for insight into mechanisms of human deafness and development of sensory cell function.

Breed differences in cochlear integrity in adult, commercially raised chickens.

Two types of chickens are commercially available. Broiler birds are bred to develop quickly for meat production, while egg layers are bred to attain a smaller adult size. Because we have observed breed differences in the response of central auditory neurons to cochlear ablation in adult birds [Edmonds et al. (1999) Hear. Res. 127, 62-76], we examined cochleae from the two breeds for differences in integrity. We evaluated cochlear hair cell structure using scanning electron microscopy and cochlear hair cell function using distortion product otoacoustic emissions (DPOAEs) and the auditory brainstem response. We observed striking breed differences in cochlear integrity in adult but not hatchling birds. In adult broiler birds, all cochleae showed damage, encompassing at least the basal 29% of the cochlea. In 15 of 18 broiler ears, damage was observed throughout the basal 60% of the cochlea. In contrast, cochleae from egg layer adults were largely normal. Two thirds of egg layer ears showed no anatomical abnormalities, while in the remainder cochlear damage was seen within the basal 48% of the cochlea. DPOAEs recorded from egg layer birds showed loss of high frequency emissions in every ear for which the cochlea displayed anatomical damage. Average sound pressure levels in both commercial facilities were 90 dB, suggesting these two breeds may exhibit differential susceptibility to noise damage.

Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome.

Following the positional cloning of PDS, the gene mutated in the deafness/goitre disorder Pendred syndrome (PS), numerous studies have focused on defining the role of PDS in deafness and PS as well as elucidating the function of the PDS-encoded protein (pendrin). To facilitate these efforts and to provide a system for more detailed study of the inner-ear defects that occur in the absence of pendrin, we have generated a Pds-knockout mouse. Pds(-/-) mice are completely deaf and also display signs of vestibular dysfunction. The inner ears of these mice appear to develop normally until embryonic day 15, after which time severe endolymphatic dilatation occurs, reminiscent of that seen radiologically in deaf individuals with PDS mutations. Additionally, in the second postnatal week, severe degeneration of sensory cells and malformation of otoconia and otoconial membranes occur, as revealed by scanning electron and fluorescence confocal microscopy. The ultrastructural defects seen in the Pds(-/-) mice provide important clues about the mechanisms responsible for the inner-ear pathology associated with PDS mutations.

Stereocilia defects in the sensory hair cells of the inner ear in mice deficient in integrin alpha8beta1.

The mammalian inner ear contains organs for the detection of sound and acceleration, the cochlea and the vestibule, respectively. Mechanosensory hair cells within the neuroepithelia of these organs transduce mechanical force generated by sound waves or head movements into neuronal signals. Defects in hair cells lead to deafness and balance defects. Hair cells have stereocilia that are indispensable for mechanosensation, but the molecular mechanisms regulating stereocilia formation are poorly understood. We show here that integrin alpha8beta1, its ligand fibronectin and the integrin-regulated focal adhesion kinase (FAK) co-localize to the apical hair-cell surface where stereocilia are forming. In mice homozygous for a targeted mutation of Itga8 (encoding the alphabeta8 subunit), this co-localization is perturbed and hair cells in the utricle, a vestibular subcompartment, lack stereocilia or contain malformed stereocilia. Most integrin alpha-8beta1-deficient mice die soon after birth due to kidney defects. Many of the survivors have difficulty balancing, consistent with the structural defects of the inner ear. Our data suggest that integrin alpha8beta1, and potentially other integrins, regulates hair-cell differentiation and stereocilia maturation. Mutations affecting matrix molecules cause inherited forms of inner ear disease and integrins may mediate some effects of matrix molecules in the ear; thus, mutations in integrin genes may lead to inner-ear diseases as well.

Hair cells in the inner ear of the pirouette and shaker 2 mutant mice.

The shaker 2 (sh2) and pirouette (pi) mouse mutants display severe inner ear dysfunction that involves both auditory and vestibular manifestation. Pathology of the stereocilia of hair cells has been found in both mutants. This study was designed to further our knowledge of the pathological characteristics of the inner ear sensory epithelia in both the sh2 and pi strains. Measurements of auditory brainstem responses indicated that both mutants were profoundly deaf. The morphological assays were specifically designed to characterize a pathological actin bundle that is found in both the inner hair cells and the vestibular hair cells in all five vestibular organs in these two mutants. Using light microscope analysis of phalloidin-stained specimens, these actin bundles could first be detected on postnatal day 3. As the cochleae matured, each inner hair cell and type I vestibular hair cell contained a bundle that spans from the region of the cuticular plate to the basal end of the cell, then extends along with cytoplasm and membrane, towards the basement membrane. Abnormal contact with the basement membrane was found in vestibular hair cells. Based on the shape of the cellular extension and the actin bundle that supports it, we propose to name these extensions "cytocauds." The data suggest that the cytocauds in type I vestibular hair cells and inner hair cells are associated with a failure to differentiate and detach from the basement membrane.

Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development.

The neurally expressed genes Brn-3.1 and Brn-3.2 (refs 1-6) are mammalian orthologues of the Caenorhabditis elegans unc-86 gene that constitute, with Brn-3.0 (refs 1-3,8,9), the class IV POU-domain transcription factors. Brn-3.1 and Brn-3.2 provide a means of exploring the potentially distinct biological functions of expanded gene families in neural development. The highly related members of the Brn-3 family have similar DNA-binding preferences and overlapping expression patterns in the sensory nervous system, midbrain and hindbrain, suggesting functional redundancy. Here we report that Brn-3.1 and Brn-3.2 critically modulate the terminal differentiation of distinct sensorineural cells in which they exhibit selective spatial and temporal expression patterns. Deletion of the Brn-3.2 gene causes the loss of most retinal ganglion cells, defining distinct ganglion cell populations. Mutation of Brn-3.1 results in complete deafness, owing to a failure of hair cells to appear in the inner ear, with subsequent loss of cochlear and vestibular ganglia.

Shape deformation of the organ of Corti associated with length changes of outer hair cell.

Cochlear outer hair cells (OHC) are commonly assumed to function as mechanical effectors as well as sensory receptors in the organ of Corti (OC) of the inner ear. OHC in vitro and in organ explants exhibit mechanical responses to electrical, chemical or mechanical stimulation which may represent an aspect of their effector process that is expected in vivo. A detailed description, however, of an OHC effector operation in situ is still missing. Specifically, little is known as to how OHC movements influence the geometry of the OC in situ. Previous work has demonstrated that the motility of isolated OHCs in response to electrical stimulation and to K(+)-gluconate is probably under voltage control and causes depolarisation (shortening) and hyperpolarization (elongation). This work was undertaken to investigate if the movements that were observed in isolated OHC, and which are induced by ionic stimulation, could change the geometry of the OC. A synchronized depolarization of OHC was induced in guinea pig cochleae by exposing the entire OC to artificial endolymph (K+). Subsequent morphometry of mid-modiolar sections from these cochleae revealed that the distance between the basilar membrane (BM) and the reticular lamina (RL) had decreased considerably. Furthermore, in the three upper turns OHC had significantly shortened in all rows. The results suggest that OHC can change their length in the organ of Corti (OC) thus deforming the geometry of the OC. The experiments reveal a tonic force generation within the OC that may change the position of RL and/or BM, contribute to damping, modulate the BM-RL-distance and control the operating points of RL and sensory hair bundles. Thus, the results suggest active self-adjustments of cochlear mechanics by slow OHC length changes. Such mechanical adjustments have recently been postulated to correspond to timing elements of animal communication, speech or music.

[Variations of the stereociliary bundles of the outer hair cells in guinea pigs. A scanning electron microscopic study].

Variations of the stereociliary bundles of the outer hair cells in guinea pigs observed under scanning electron-microscope were reported. Changes including transposition and distorted outlines of the stereociliary bundles were distributed in all turns of the cochlear duct and all rows of the outer hair cells, but not in inner hair cells. The possible pathogenetic significance of the changes was discussed.

Morphological changes of cochlea in a strain of new-mutant mice.

The hearing ability and histological characteristics of the cochlea of a strain of new-mutant mice were analyzed. This new mutant arose as a spontaneous mutation in the C3H/He stock. The genetic mode is autosomal recessive and the animals show abnormal behavior such as circling, head-tossing and hyperactivity. The audiological findings exhibited no recordable auditory brain stem response (ABR) in any homozygotes at ages ranging from 11 days to 117 days. For morphological examination, we used 36 homozygote with ages ranging from 10 days to 18 months. The primary morphological abnormalities were observed in the organ of Corti. The stereocilia of the outer hair cells showed disarray throughout the whole cochlea, although outer hair cell cytoplasm became fully developed, including the nerve terminals. Age-dependent degeneration of the outer hair cells subsequently occurred from the basal to the apical part of the cochlea. The earliest change demonstrated in the outer hair cells was cuticular degeneration. Although the abnormalities of the inner hair cells occurred late, a complete loss of inner and outer hair cells was demonstrated. The stria vascularis was well preserved at a later age as were spiral ganglion cells. These histological findings confirm that this mouse is classified as a neuroepithelial-type mutant. As this animal was expected to have a single gene abnormality, molecular genetic studies on this animal can provide important information on the nature of histological changes of the hair cell from a mode of gene action.

Mutant golden hamsters with an abnormal outer hair cell stereociliary arrangement.

A new malformation of the inner ear was found in golden hamster reared at our institute. It was studied using electron microscope and auditory electrophysiological measurements including auditory brainstem response (ABR), whole nerve action potential (AP) cochlear microphonic (CM) potential, and the summating potential (SP). The stereocilia on individual first row of outer hair cells in the hamsters with malformed inner ears (F-K hamsters) were arranged in a triple W form, but the entire bundle of stereocilia was irregular in orientation. These anomalies were seen in approximately 70 to 85% of sensory hairs, in all rotations, with no difference between the right and left sides. The cuticles of the first row of outer hair cells were displaced, but lower portions did not appear to be affected. ABR and SP revealed no differences from normal hamsters and it is believed that the F-K hamsters' hearing ability was normal. The CM potential and the amplitude of AP in the F-K hamsters were significantly lower, at 50 to 80 dB sound pressure level (SPL). The linear portions of the CM input-output relation curve were separated by 4 to 6 dBSPL and the saturating voltage levels differed by 2.5 to 3.0 microV. Based on these results, the actually-measured CM potentials were shown to represent a summation of the reaction of the three individual rows of outer hair cells.

An inner ear anomaly in golden hamsters.

A new mutation of the inner ear was discovered in golden hamsters raised in our laboratories. Although scanning electron microscopy showed a normal arrangement of individual stereocilia on the first row of outer hair cells, the entire bundle of stereocilia were irregular in orientation and scattered in several directions. Seventy per cent to 85% of the stereociliary bundles were found to be abnormal throughout the cochlea, with no apparent difference between the right and left sides. Transmission electron microscopy showed that the cuticles of the first row of the outer hair cells were dislocated, but no dislocation due to this mutation was apparent in the lower portions. This mutation of the inner ear was already present in the basal turn four days after birth. The kinocilium was located outside of the stereocilia in the first row of outer hair cells, but sensory hairs were scattered in every direction, as in the adult animals. A comparison of auditory brainstem response tests revealed no difference between the abnormal and normal hamsters.

A scanning electron microscopic study of vestibular organ malformation following prenatal gamma irradiation.

Pregnant CBA/CBA mice were exposed to 1 and 2 Gy whole-body gamma irradiation on the 13th and 16th gestational days, respectively. The litters were born on the 21st day of gestation and were tested for vestibular function at the age of 1 month. The animals were then sacrificed and their inner ears were analyzed by scanning electron microscopy. No disturbances of vestibular function were noted in the animals studied. However, the cristae ampullares showed severe malformations as regards their gross shape, with irregularities of their outer contours. Type I hair cells seemed to be more severely changed than Type II hair cells, with fusion of sensory hairs, giant hair formation and bulging of the cuticular plate. In certain sites the hair cells were totally missing. These derangements were usually located in the central areas of the cristae ampullares and in the striolar portion of the maculae utriculi. The morphological damage found showed a dose-dependent, time-related pattern.