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The stria vascularis of the mammalian cochlea is composed primarily of three types of cells. Marginal cells line the lumen of the cochlear duct and are of epithelial origin. Basal cells also form a continuous layer and they may be mesodermal or derived from the neural crest. Intermediate cells are melanocyte-like cells, presumably derived from the neural crest, and are scattered between the marginal and basal cell layers. The marginal cells form extensive interdigitations with the basal and intermediate cells in the normal adult stria. The stria also contains a rich supply of blood vessels. We investigated the role of melanocytes in the stria vascularis by studying its development in a mouse mutant, viable dominant spotting, which is known to have a primary neural crest defect leading to an absence of recognisable melanocytes in the skin. Melanocytes were not found in the stria of most of the mutants examined, and from about 6 days of age onwards a reduced amount of interdigitation amongst the cells of the stria was observed. These ultrastructural anomalies were associated with strial dysfunction. In the normal adult mammal, the stria produces an endocochlear potential (EP), a resting dc potential in the endolymph in the cochlear duct, which in mice is normally about + 100 mV. In our control mice, EP rose to adult levels between 6 and 16 days after birth. In most of the mutants we studied, EP was close to zero at all ages from 6 to 20 days. Melanocyte-like cells appear to be vital for normal stria vascularis development and function. They may be necessary to facilitate the normal process of interdigitation between marginal and basal cell processes at a particular stage during development, and the lack of adequate interdigitation in the mutants may be the cause of their strial dysfunction. Alternatively, melanocytes may have some direct, essential role in the production of an EP by the stria. Melanocytes may be important both for normal strial development and for the production of the EP. We believe this is the clearest demonstration yet of a role for migratory melanocytes other than their role in pigmentation.

Both genetic background and single gene mutations may affect the development of the auditory system. A classification system is presented for those single gene mutations causing hearing impairment. The new feature of this classification is the inclusion of a category for hereditary deafness of central origin. The other categories involve peripheral abnormalities and are: morphogenetic defects, in which the overall structure of the labyrinth is deformed; neuroepithelial degeneration, in which the primary defect appears to occur in the organ of Corti; and cochleo-saccular degeneration, where the stria vascularis is abnormal and Reissner's membrane collapses, leading to further degeneration.

The deafness (dn/dn) mouse has an hereditary cochlear dysfunction throughout its development, and spiral ganglion cell density decreases progressively over the three age groups we examined. We have used this mutant to examine inferior colliculus evoked responses to modiolar electrical stimulation as a function of spiral ganglion degeneration. No differences were found between mutants and control mice or between ages in either threshold for detection of the response or latency of the response. However, peak-to-peak amplitudes of the response were larger in the mutants than in the controls in the young and intermediate age groups. There was a poor correlation between spiral ganglion degeneration and size of the evoked response: for example, mutants in the old age group had similar amplitudes of response as controls while spiral ganglion cell density was reduced to 21% of the value in young mice, and mutants in the intermediate age group with 50% spiral ganglion degeneration showed response amplitudes more than double that in controls. These data may be relevant to the significant numbers of people with hereditary deafness among the hearing-impaired human population.

The hush puppy mouse mutant has been shown previously to have skull and outer, middle, and inner ear defects, and an increase in hearing threshold. The fibroblast growth factor receptor 1 (Fgfr1) gene is located in the region of chromosome 8 containing the mutation. Sequencing of the gene in hush puppy heterozygotes revealed a missense mutation in the kinase domain of the protein (W691R). Homozygotes were found to die during development, at approximately embryonic day 8.5, and displayed a phenotype similar to null mutants. Reverse transcription PCR indicated a decrease in Fgfr1 transcript in heterozygotes and homozygotes. Generation of a construct containing the mutation allowed the function of the mutated receptor to be studied. Immunocytochemistry showed that the mutant receptor protein was present at the cell membrane, suggesting normal expression and trafficking. Measurements of changes in intracellular calcium concentration showed that the mutated receptor could not activate the IP3 pathway, in contrast to the wild-type receptor, nor could it initiate activation of the Ras/MAP kinase pathway. Thus, the hush puppy mutation in fibroblast growth factor receptor 1 appears to cause a loss of receptor function. The mutant protein appears to have a dominant negative effect, which could be due to it dimerising with the wild-type protein and inhibiting its activity, thus further reducing the levels of functional protein. A dominant modifier, Mhspy, which reduces the effect of the hush puppy mutation on pinna and stapes development, has been mapped to the distal end of chromosome 7 and may show imprinting. Electronic supplementary material The online version of this article (doi:10.1007/s00335-011-9324-8) contains supplementary material, which is available to authorized users.

Two large-scale phenotyping efforts, the European Mouse Disease Clinic (EUMODIC) and the Wellcome Trust Sanger Institute Mouse Genetics Project (SANGER-MGP), started during the late 2000s with the aim to deliver a comprehensive assessment of phenotypes or to screen for robust indicators of diseases in mouse mutants. They both took advantage of available mouse mutant lines but predominantly of the embryonic stem (ES) cells resources derived from the European Conditional Mouse Mutagenesis programme (EUCOMM) and the Knockout Mouse Project (KOMP) to produce and study 799 mouse models that were systematically analysed with a comprehensive set of physiological and behavioural paradigms. They captured more than 400 variables and an additional panel of metadata describing the conditions of the tests. All the data are now available through EuroPhenome database ( www.europhenome.org ) and the WTSI mouse portal ( http://www.sanger.ac.uk/mouseportal/ ), and the corresponding mouse lines are available through the European Mouse Mutant Archive (EMMA), the International Knockout Mouse Consortium (IKMC), or the Knockout Mouse Project (KOMP) Repository. Overall conclusions from both studies converged, with at least one phenotype scored in at least 80% of the mutant lines. In addition, 57% of the lines were viable, 13% subviable, 30% embryonic lethal, and 7% displayed fertility impairments. These efforts provide an important underpinning for a future global programme that will undertake the complete functional annotation of the mammalian genome in the mouse model.

SummaryWHRN (DFNB31) mutations cause diverse hearing disorders: profound deafness (DFNB31) or variable hearing loss in Usher syndrome type II. The known role of WHRN in stereocilia elongation does not explain these different pathophysiologies. Using spontaneous and targeted Whrn mutants, we show that the major long (WHRN-L) and short (WHRN-S) isoforms of WHRN have distinct localizations within stereocilia and also across hair cell types. Lack of both isoforms causes abnormally short stereocilia and profound deafness and vestibular dysfunction. WHRN-S expression, however, is sufficient to maintain stereocilia bundle morphology and function in a subset of hair cells, resulting in some auditory response and no overt vestibular dysfunction. WHRN-S interacts with EPS8, and both are required at stereocilia tips for normal length regulation. WHRN-L localizes midway along the shorter stereocilia, at the level of inter-stereociliary links. We propose that differential isoform expression underlies the variable auditory and vestibular phenotypes associated with WHRN mutations.

MicroRNAs, small non-coding RNAs which act as repressors of target genes, were discovered in 1993, and since then have been shown to play important roles in the development of numerous systems. Consistent with this role, they are also implicated in the pathogenesis of multiple diseases. Here we review the involvement of microRNAs in mouse development and disease, with particular reference to deafness as an example.

Cadherin23 has been proposed to form the upper part of the tip link, an interstereocilial link believed to control opening of transducer channels of sensory hair cells. However, we detect tip link-like links in mouse mutants with null alleles of Cdh23, suggesting the presence of other components that permit formation of a link between the tip of one stereocilium and the side of the adjacent taller stereocilium.

In a screen for mouse mutations with dominant behavioral anomalies, we identified Wheels, a mutation associated with circling and hyperactivity in heterozygotes and embryonic lethality in homozygotes. Mutant Wheels embryos die at E10.5-E11.5 and exhibit a host of morphological anomalies which include growth retardation and anomalies in vascular and hindbrain development. The latter includes perturbation of rhombomeric boundaries as detected by Krox20 and Hoxb1. PECAM-1 staining of embryos revealed normal formation of the primary vascular plexus. However, subsequent stages of branching and remodeling do not proceed normally in the yolk sac and in the embryo proper. To obtain insights into the circling behavior, we examined development of the inner ear by paint-filling of membranous labyrinths of Whl/+ embryos. This analysis revealed smaller posterior and lateral semicircular canal primordia and a delay in the canal fusion process at E12.5. By E13.5, the lateral canal was truncated and the posterior canal was small or absent altogether. Marker analysis revealed an early molecular phenotype in heterozygous embryos characterized by perturbed expression of Bmp4 and Msx1 in prospective lateral and posterior cristae at E11.5. We have constructed a genetic and radiation hybrid map of the centromeric portion of mouse Chromosome 4 across the Wheels region and refined the position of the Wheels locus to the approximately 1.1-cM region between D4Mit104 and D4Mit181. We have placed the locus encoding Epha7, in the Wheels candidate region; however, further analysis showed no mutations in the Epha7-coding region and no detectable changes in mRNA expression pattern. In summary, our findings indicate that Wheels, a gene which is essential for the survival of the embryo, may link diverse processes involved in vascular, hindbrain, and inner ear development.

The proteins of the normal mouse tectorial membrane were analysed using SDS polyacrylamide gel electrophoresis. Three major protein bands were found with molecular weights of 145 000, 155000 and 165 000, together with a number of minor components. Abnormal tectorial membranes from thiouracil-treated mice and from Deafness, Jerker, and Varitint-Waddler mutant mice were also examined in this way, but no evidence could be found to support the hypothesis that the distortion of the membranes might be due to an abnormal protein constitution. No PAS-positive material could be demonstrated in gels of normal membranes.ZUSAMMENFASSUNGDie Proteine der normalen Membrana tectoria der Maus wurden durch SDS Polyacrylamid-Gel-EIektrophorese analysiert. Drei wichtige Proteinbander mit Molekularge-wichten von 145 000, 155000 und 165000 und eine Anzahl kleinerer Teile, wurden gefunden. Abnorme Membrana tectoria von Mausen, die man mit Thiouracil behandelte, und von Deafness-, Jerker- und Varitint-Waddler-Mutan-ten, wurden a...

• A classification scheme for inner-ear abnormalities is defined, based on animals with hereditary inner-ear defects. Three major groups are (1) morphogenetic abnormalities, involving gross structural deformities of the labyrinth; (2) neuroepithelial abnormalities, in which there appears to be a primary organ of Corti defect and Reissner's membrane remains in its normal position; and (3) cochleosaccular abnormalities, showing collapse of Reissner's membrane and restriction of vestibular abnormalities to the saccule. Recent experimental findings in hearingimpaired animal mutants are taken into account. The relevance of the classification scheme to conditions in humans is discussed. It is suggested that the Mondini and Michel deformities in humans are best considered together in the morphogenetic group. Some cases of inner-ear abnormality in humans might be considered as belonging to the neuroepithelial group. ( Arch Otolaryngol 1983;109:22-29)

Deafness mice are profoundly deaf from birth as a result of genetically determined cochlear dysfunction. Evoked potentials in response to direct electrical stimulation of the cochlear nerve can readily be recorded in the inferior colliculus ofdeafness mice, and such responses are larger in amplitude than those in control mice. These observations indicate that at least some central connections become functional in thedeafness central auditory pathway in the absence of peripheral stimulation, and are relevant to the general problem of restoring function by direct nerve stimulation in the profoundly deaf.

Mice homozygous for the recessive quivering gene are largely unresponsive to sound, although their cochleas appear normal by light microscopy and cochlear hair cells do not degenerate with age. Cochlear potentials and inferior colliculus evoked responses were recorded in quivering mice (qv/qv) and in littermate controls (+/qv or +/+). There were no significant differences between mutants and controls in cochlear microphonic amplitudes or compound action potential thresholds, amplitudes and latencies. However, inferior colliculus evoked responses in mutants were small in amplitude and abnormal in waveform. Latencies were long and thresholds were elevated by at least 50 dB at all frequencies tested. These data suggest that the genetic hearing impairment in quivering mice is retrocochlear.

A distinctive cochlear pathology was found in deafness mutant mice. There was a delay in the formation of the fluid-filled Nuel and tunnel spaces in the organ of Corti, the hair cells were distorted and degenerate, and there was poor maintenance of synapses. No hair cells appeared normal by TEM, but SEM revealed some areas where stereocilia appeared relatively normal, suggesting that SEM of the surface of the organ of Corti is not necessarily a good indicator of hair cell pathology in hereditary hearing impairment. Mutant mice show normal development of endocochlear potential, but have no measurable cochlear microphonics or compound action potential. The data suggest that the deafness gene affects the organ of Corti and that cochlear hair cells in deafness mice are never functional.

Many mouse mutants have an apparent deficiency in their responsiveness to sound1. Most of these mutants have other abnormalities in addition to their hearing deficit, and the only two which have been subjected to a detailed anatomical and physiological study, shaker-1 and Ames waltzer, also have motor abnormalities2,3. The existence of such motor abnormalities throws some doubt on the usefulness of these two mutants as possible models for hereditary deafness in man, which is most frequently uncomplicated4. Deol and Kocher have described the deafness mutation in which mice homozygous for the recessive deafness gene (dn/dn) were unresponsive to sound and had no significant behavioural abnormality5. Cochlear hair cells in deafness mice develop normally and then degenerate, and the adult animals are completely deaf6. We have now studied deafness mice in order to determine the nature of their inherited deafness. Our data indicate that stimulus-related cochlear potentials do not develop even though hair cells are present in the young animal. The endocochlear potential is present in the scala media, but behaves abnormally during anoxia.

Most viable dominant spotting (W v /W v ) mutant mice, which show cochleo-saccular degeneration, were found to have an endocochlear potential (EP) around zero together with a structurally abnormal stria vascularis. Inner hair cells were well preserved, but outer hair cells in the basal half of the cochlea were degenerating, possibly as a result of primary striai dysfunction. Thresholds for the detection of a compound action potential were raised to around 100 dB SPL in the mutants with no EP, and there was little if any cochlear microphonic at the round window. Of the 20 W v /W v mice studied, five partially escaped the effects of the mutation and had measurable positive potentials (15–86 mV) in scala media in the basal turn; responses in these animals were intermediate between control responses and those of mutants with no EP. These findings confirm that the pathological processes in this mutant, with cochleo-saccular abnormalities, are fundamentally different from the pathological processes in animals with neuroepithelial abnormalities reported previously [see Steel and Bock (1983) Arch. Otolaryngol. 109, 22–29, for references].

The Light mutation (Blt) is a dominant allele of the b-locus on mouse chromosome 4 which causes progressive dilution of coat colour. Melanocytes within the hair follicles of mutant mice develop normally but later degenerate, due to the accumulation of a toxic product, so that the hair becomes lighter with age. Previous studies on W-locus spotting mutants, from which melanocytes are absent, have shown that melanocytes in the stria vascularis of the inner ear are essential for the development and/or maintenance of the endocochlear potential (EP) which is normally around 100 mV. In this study, physiological recordings from the ears of Light mutants were correlated with strial ultrastructure. EPs recorded from all b/b controls and young homozygous and heterozygous mutants (20-22 days old) were normal (77 to 113 mV), but were reduced (19 to 59 mV) in about 30% of ears from older mutants (Blt/Blt and Blt/b). Strial function therefore appears to develop normally but later degenerates in some mutants. This suggests that strial melanocytes are affected by the Light allele and that the continued presence of melanocytes is necessary for strial function. There was no obvious association between the recorded EP value and the ultrastructural appearance of the stria. No structural abnormalities of the stria were noted in control or mutant mice aged 20 days to 4 months including those which had a reduced EP. Strial atrophy was common in old controls and mutants (1-2 years), and appeared to be an age-related process rather than an effect of the Light mutation. Similarly, pigment build-up was common in all strial cells of old mice. However, the accumulations of lipofuscin-like pigment were much larger and more abundant in aged brown non-agouti mice than those observed in old agouti mice, which suggests that this age-related process also has a genetic component.

We have used a probe derived from TRP-2/DT to detect migratory melanoblasts shortly after they emerge from the neural crest, as early as 10 days post coitum (dpc). TRP-2/DT expression is otherwise restricted to the pre sumptive pigmented retinal epithelium, the developing telencephalon and the endolymphatic duct. The pattern of steel and c-kit hybridisation in the developing brain differed from that of TRP-2. TRP-1 and tyrosinase probes also detected melanoblasts but were both expressed later in development than TRP-2. We used the TRP-2/DT probe to investigate the way that the Steel-dickie (Sld) mutation interferes with melanocyte development, and found that the membrane-bound steel growth factor which is missing in Sld/Sld mutants is necessary for the survival of melanoblasts but not for their early migration and initial differentiation.

Nonsyndromic hearing impairment (NSHI) is a highly heterogeneous condition with more than eighty known causative genes. However, in the clinical setting, a large number of NSHI families have unexplained etiology, suggesting that there are many more genes to be identified. In this study we used SNP-based linkage analysis and follow up microsatellite markers to identify a novel locus (DFNA66) on chromosome 6q15-21 (LOD 5.1) in a large Danish family with dominantly inherited NSHI. By locus specific capture and next-generation sequencing, we identified a c.574C>T heterozygous nonsense mutation (p.R192*) in CD164. This gene encodes a 197 amino acid transmembrane sialomucin (known as endolyn, MUC-24 or CD164), which is widely expressed and involved in cell adhesion and migration. The mutation segregated with the phenotype and was absent in 1200 Danish control individuals and in databases with whole-genome and exome sequence data. The predicted effect of the mutation was a truncation of the last six C-terminal residues of the cytoplasmic tail of CD164, including a highly conserved canonical sorting motif (YXXФ). In whole blood from an affected individual, we found by RT-PCR both the wild-type and the mutated transcript suggesting that the mutant transcript escapes nonsense mediated decay. Functional studies in HEK cells demonstrated that the truncated protein was almost completely retained on the plasma cell membrane in contrast to the wild-type protein, which targeted primarily to the endo-lysosomal compartments, implicating failed endocytosis as a possible disease mechanism. In the mouse ear, we found CD164 expressed in the inner and outer hair cells of the organ of Corti, as well as in other locations in the cochlear duct. In conclusion, we have identified a new DFNA locus located on chromosome 6q15-21 and implicated CD164 as a novel gene for hearing impairment., Author Summary It is known that hearing impairment running in families can be caused by mutations in more than eighty different genes. However, there are still families where the responsible gene is unknown. By studying a large Danish family with dominant inherited hearing impairment, we found that the disorder cosegregates with genetic markers on chromosome 6, suggesting that the responsible mutation lies within this chromosomal region. By sequencing this genetic locus, we discovered a mutation in the CD164 gene that is passed on to all the affected individuals. In the mouse ear, we demonstrated that the CD164 protein is expressed in hair cells and other sites known to be important for correct hearing. The identified mutation is predicted to result in shortening of the protein, leading to loss of an evolutionary conserved sequence important for cellular trafficking of CD164. Using cell lines, we show that the truncated protein is trapped on the cell surface while the normal protein is internalized. This finding is important because it implicates for the first time a role for CD164 in the complex physiological processes of hearing and suggests that failed endocytosis may be a possible disease mechanism for some types of hearing impairment.

The W locus encodes a tyrosine kinase receptor, c-kit, which affects survival of melanoblasts from the neural crest. The primary cochlear defect in Viable Dominant Spotting (Wv/Wv) mutants is a lack of melanocytes within the stria vascularis (SV) associated with an endocochlear potential (EP) close to zero and hearing impairment. In this study, we compare inner ear pigmentation with cochlear potentials in three other W alleles (Wx, Wsh, and W41) and reveal an unequivocal correlation between presence of strial melanocytes and presence of an EP. Asymmetry was common, and 8.3% of Wsh/Wx, 25% of Wsh/Wsh, 60% of W41/Wx, and 69.2% of W41/W41 ears had a pigmented stria and an EP, while the remainder had no strial melanocytes and no EP. In those mutants that partially escaped the effects of the mutation, strial melanocytes rarely extended the entire length of the stria, but were confined to the middle and/or basal turns of the cochlea. The extent of strial pigmentation was unrelated to the EP value, which was measured from the basal turn only. Compound action potential (CAP) responses recorded from ears with an EP were variable and they showed greatly raised thresholds or were absent in all ears where the EP was close to zero. In controls, melanocytes in the vestibular part of the ear were found in the utricle, crus commune, and ampullae, whereas in many mutants only one or two of these regions were pigmented. There was a broad correlation between pigmentation of the stria and pigmentation of the vestibular region but this was not absolute. All W41/Wx, Wsh/Wsh, and W41/W41 mutants had some pigment on the pinna but, in contrast to controls where melanocytes were found in the epidermis and dermis of the pinna, pigment cells were reduced in number and generally restricted to the dermis. Injection of normal neural crest cells into 9.5-day-old mutant embryos increased the extent of skin pigmentation on the head and coat of adult chimeras and was associated with a small increase in the proportion of pigmented strias.

Anatomical studies using light microscopy have shown that mice affected by the deafness or jerker genes develop cochlear hair cells which at first appear to be normal. The hair cells then begin to degenerate, and by about three months of age most of the organ of Corti has degenerated. The object of the present study was to determine whether the cochleas of affected animals are functionally normal during the period when they appear structurally normal. Mice from the two strains aged between 12 and 50 days were anaesthetized and audiograms were determined by measuring thresholds for detecting the compound action potential recorded at the round window. No action potentials or cochlear microphonics could be recorded from affected animals at any age but littermates unaffected by the respective genes had normal thresholds. These results indicate that the apparent structural integrity of the cochleas of young deafness and jerker mice is not accompanied by normal function. Mice affected by these genes appear to b...

Inflammation of the middle ear (otitis media) is very common and can lead to serious complications if not resolved. Genetic studies suggest an inherited component, but few of the genes that contribute to this condition are known. Mouse mutants have contributed significantly to the identification of genes predisposing to otitis media The dearisch mouse mutant is an ENU-induced mutant detected by its impaired Preyer reflex (ear flick in response to sound). Auditory brainstem responses revealed raised thresholds from as early as three weeks old. Pedigree analysis suggested a dominant but partially penetrant mode of inheritance. The middle ear of dearisch mutants shows a thickened mucosa and cellular effusion suggesting chronic otitis media with effusion with superimposed acute infection. The inner ear, including the sensory hair cells, appears normal. Due to the low penetrance of the phenotype, normal backcross mapping of the mutation was not possible. Exome sequencing was therefore employed to identify a non-conservative tyrosine to cysteine (Y71C) missense mutation in the Islet1 gene, Isl1 Drsh . Isl1 is expressed in the normal middle ear mucosa. The findings suggest the Isl1 Drsh mutation is likely to predispose carriers to otitis media. Dearisch, Isl1 Drsh , represents the first point mutation in the mouse Isl1 gene and suggests a previously unrecognized role for this gene. It is also the first recorded exome sequencing of the C3HeB/FeJ background relevant to many ENU-induced mutants. Most importantly, the power of exome resequencing to identify ENU-induced mutations without a mapped gene locus is illustrated.