Characterization of a new allele of Ames waltzer generated by ENU mutagenesis.

Mutation in the protocadherin 15 (Pcdh15) gene causes hair cell dysfunction and is associated with abnormal stereocilia development. We have characterized the first allele (Pcdh15(av-nmf19)) of Ames waltzer (av) obtained by N-ethyl-N-nitrosourea (ENU) mutagenesis. Pcdh15(av-nmf19) was generated in the Neuroscience Mutagenesis Facility (NMF) at The Jackson Lab (Bar Habor, USA). Pcdh15(av-nmf19) mutants display circling and abnormal swimming behavior along with lack of auditory-evoked brainstem response at the highest intensities tested. Mutation analysis shows base substitution (A--> G) in the consensus splice donor sequence linked to exon 14 resulting in the skipping of exon 14 and the splicing of exon 13-15. This results in the introduction of a stop codon in the coding sequence of exon 15 due to shift in the reading frame. The effect of nmf19 mutation is expected to be severe since the expressed Pcdh15 protein is predicted to truncate in the 5th cadherin domain. Abnormalities of cochlear hair cell stereocilia are apparent in Pcdh15(av-nmf19) mutants near the time of birth and by about P15 (15 days after birth) there is evidence of sensory cell degeneration. Disorganization of outer hair cell stereocilia is observed as early as P2. Inner hair cell stereocilia are also affected, but less severely than those of the outer hair cells. These results are consistent with characteristics of the mutation in the Pcdh15(av-nmf19) allele and they support our previous finding that Protocadherin 15 plays an important role in hair-bundle morphogenesis.

Genetic influences in individual susceptibility to noise: a review.

Individual animals and humans show differing susceptibility to noise damage even under very carefully controlled exposure conditions. This difference in susceptibility may be related to unknown genetic components. Common experimental animals (rats, guinea pigs, chinchillas, cats) are outbred-their genomes contain an admixture of many genes. Many mouse strains have been inbred over many generations reducing individual variability, making them ideal candidates for studying the genetic modulation of individual susceptibility. Erway et al. (1993) demonstrated a recessive gene associated with early presbycusis in the C57BL/6J inbred mouse. A series of studies have shown that mice homozygous for Ahl allele are more sensitive to the damaging effects of noise. Recent work has shown that mice homozygous for Ahl are not only more sensitive to noise, but also are probably damaged in a different manner by noise than mice containing the wild-type gene (Davis et al., 2001). Recent work in Noben-Trauth's lab (Di Palma et al., 2001) has shown that the wild-type Ahl gene codes for a hair cell specific cadherin. Cadherins are calcium dependent proteins that hold cells together at adherins junctions to form tissues and organs. The cadherin of interest named otocadherin or CDH23, is localized to the stereocillia of the outer hair cells. Our working hypothesis, suggests that otocadherin may form the lateral links between stereocilia described by Pickles et al (1989). Reduction of, or missing otocadherin weakens the cell and may allow stereocilia to be more easily physically damaged by loud sounds and by aging.

Suppression of the deafness and thyroid dysfunction in Thrb-null mice by an independent mutation in the Thra thyroid hormone receptor alpha gene.

Deletion of thyroid hormone receptor beta (TR beta), a ligand-dependent transcription factor encoded by the Thrb gene, causes deafness and thyroid hyperactivity in Thrb-null (Thrb(tm1/tm1)) mice and in a recessive form of the human syndrome of resistance to thyroid hormone. Here, we have determined that a targeted mutation (Thra(tm2)) in the related Thra gene, encoding thyroid hormone receptor alpha suppresses these phenotypes in mice. Thra encodes a TR alpha 1 receptor which is non-essential for hearing and a TR alpha 2 splice variant of unknown function that neither binds thyroid hormone nor transactivates. The Thra(tm2) mutation deletes TR alpha 2 and concomitantly causes overexpression of TR alpha 1 as a consequence of the exon structure of the gene. Thra(tm2/tm2) mice have normal auditory thresholds indicating that TR alpha 2 is dispensable for hearing, and have only marginally reduced thyroid activity. However, a potent function for the Thra(tm2) allele is revealed upon its introduction into Thrb(tm1/tm1) mice, where it suppresses the auditory and thyroid phenotypes caused by loss of TR beta. These findings reveal a novel modifying function for a Thra allele and suggest that increased expression of TR alpha 1 may substitute for the absence of TR beta. The TR isotypes generated by the distinct Thrb and Thra genes represent a small family of receptors that have diverged to mediate different physiological roles; however, the ability of changes in Thra expression to compensate for loss of Thrb indicates that many functions of these genes remain closely related.

Genetic basis for susceptibility to noise-induced hearing loss in mice.

The C57BL/6J (B6) and DBA/2J (D2) inbred strains of mice exhibit an age-related hearing loss (AHL) due to a recessive gene (Ahl) that maps to Chromosome 10. The Ahl gene is also implicated in the susceptibility to noise-induced hearing loss (NIHL). The B6 mice (Ahl/Ahl) are more susceptible to NIHL than the CBA/CaJ (CB) mice (+(Ahl)). The B6xD2.F(1) hybrid mice (Ahl/Ahl) are more susceptible to NIHL than the CBxB6.F(1) mice (+/Ahl) [Erway et al., 1996. Hear. Res. 93, 181-187]. These genetic effects implicate the Ahl gene as contributing to NIHL susceptibility. The present study demonstrates segregation for the putative Ahl gene and mapping of such a gene to Chromosome 10, consistent with other independent mapping of Ahl for AHL in 10 strains of mice [Johnson et al., 2000. Genomics 70, 171-180]. The present study was based on a conventional cross between two inbred strains, CBxB6.F(1) backcrossed to B6 with segregation for the putative +/Ahl:Ahl/Ahl. These backcross progeny were exposed to 110 dB SPL noise for 8 h. All of the progeny were tested for auditory evoked brainstem responses and analyzed for any significant permanent threshold shift of NIHL. Cluster analyses were used to distinguish the two putative genotypes, the least affected with NIHL (+/Ahl) and most affected with PTS (Ahl/Ahl). Approximately 1/2 of the backcross progeny exhibited PTS, particularly at 16 kHz. These mice were genotyped for two D10Mit markers. Quantitative trait loci analyses (log of the odds=15) indicated association of the genetic factor within a few centiMorgan of the best evidence for Ahl [Johnson et al., 2000. Genomics 70, 171-180]. All of the available evidence supports a role for the Ahl gene in both AHL and NIHL among these strains of mice.

A major gene affecting age-related hearing loss is common to at least ten inbred strains of mice.

Inbred strains of mice offer promising models for understanding the genetic basis of human presbycusis or age-related hearing loss (AHL). We previously mapped a major gene affecting AHL in C57BL/6J mice. Here, we show that the same Chromosome 10 gene (Ahl) is a major contributor to AHL in nine other inbred mouse strains-129P1/ReJ, A/J, BALB/cByJ, BUB/BnJ, C57BR/cdJ, DBA/2J, NOD/LtJ, SKH2/J, and STOCK760. F1 hybrids between each of these inbred strains and the normal-hearing inbred strain CAST/Ei retain good hearing, indicating that inheritance of AHL is recessive. To follow segregation of hearing loss, F1 hybrids were backcrossed to the parental strains with AHL. Auditory-evoked brain-stem response thresholds were used to assess hearing in more than 1500 N2 mice and analyzed as quantitative traits for linkage associations with Chromosome 10 markers. Highly significant linkage was found in all nine strain backcrosses, with the highest probability (LOD > 70) near the marker D10Mit112. This map position for Ahl is near the waltzer mutation (v) and the modifier of deaf waddler locus (mdfw), suggesting the possibility of allelism. Results from an intercross of C57BL/6J and NOD/LtJ mice indicate that the 6- to 10-month difference in AHL onset between these two strains is not due to allelic heterogeneity of the Ahl gene.

Neuroepithelial defects of the inner ear in a new allele of the mouse mutation Ames waltzer.

This report presents new findings regarding a recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice homozygous for the mutation. The mutant locus was mapped to a region on mouse chromosome 10 close to three spontaneous recessive mutations causing deafness: Ames waltzer (av), Waltzer (v), and Jackson circler (jc). Complementation testing revealed that the TgN2742Rpw mutation is allelic with av. Histological and auditory brainstem response (ABR) evaluation of animals that have the new allele balanced with the av(J) allele (called compound heterozygotes, TgN2742Rpw/av(J)) supports our genetic analysis. ABR evaluation shows complete absence of auditory response throughout the life span of TgN2742Rpw/av(J) compound heterozygotes. Scanning electron microscopy revealed abnormalities of inner and outer hair cell stereocilia in the cochleae of TgN2742Rpw mutants at 10 days after birth (DAB). The organ of Corti subsequently undergoes degeneration, leading to nearly complete loss of the cochlear neuroepithelium in older mutants by about 50 DAB. The vestibular neuroepithelia remain morphologically normal until at least 30 DAB. However, by 50 days, degenerative changes are evident in the saccular macula, which progresses to total loss of the saccular neuroepithelium in older animals. The new allele of av reported here will be designated av(TgN2742Rpw).

Mice lacking the basolateral Na-K-2Cl cotransporter have impaired epithelial chloride secretion and are profoundly deaf.

In chloride-secretory epithelia, the basolateral Na-K-2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl(-) and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K(+) that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1(-/-)) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl(-) channel, were reduced in jejunum, cecum, and trachea of Nkcc1(-/-) mice, indicating that NKCC1 contributes to cAMP-induced Cl(-) secretion. In contrast, secretion of gastric acid in adult Nkcc1(-/-) stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1(-/-) mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K(+) movements involved in generation of the K(+)-rich endolymph and the endocochlear potential.

Vestibular responses to linear acceleration are absent in otoconia-deficient C57BL/6JEi-het mice.

UNLABELLED: Vestibular evoked potentials (VsEPs) were measured in normal mice and in mice homozygous for the head tilt mutation (het/het, abbr. het). The het mice lack otoconia, the inertial mass critical for natural stimulation of inner ear gravity receptors. Our findings demonstrate that vestibular neural responses to pulsed linear acceleration are absent in het mice. THE RESULTS: (1) confirm that adequate sensory stimuli fail to activate gravity receptors in the het model; and (2) serve as definitive evidence that far-field vestibular responses to pulsed linear acceleration depend critically on otolith end organs. The C57BL/6JEi-het mouse may be an excellent model of gravity receptor sensory deprivation.

Quantitative measure of genetic differences in susceptibility to noise-induced hearing loss in two strains of mice.

The CBA/CaJ (CB) and C57BL/6J (B6) inbred strains of mice were exposed for 1 h to noise intensities between 98 and 119 dB SPL. Previous studies indicated that the B6 mice exhibited permanent threshold shifts (PTS) after 1h exposure to 110 dB, whereas the CB mice did not exhibit any PTS. These differences in susceptibility to noise-induced hearing loss (NIHL) appear to be due to a gene for age-related hearing loss (AHL). The current study was designed to determine dose-response curves for NIHL over the ranges of intensities of noise that would characterize the B6 and CB inbred strains of mice. Because of the considerable differences in sensitivity to NIHL, the noise exposures for the two strains overlapped only at 110 and 113 dB. Nevertheless, the two strains exhibited two different dose-response curves, offset and with different slopes. We postulate that the B6 strain of mice exhibits a more linear increase for PTS from 98-113 dB, consistent with incremental effects on some metabolic physiological mechanism(s); the abrupt transition in NIHL between 113 and 116 dB for the CB mice is consistent with an ototraumatic structural injury.

A new mouse insertional mutation that causes sensorineural deafness and vestibular defects.

This article describes a new recessive insertional mutation in the transgenic line TgN2742Rpw that causes deafness and circling behavior in mice. Histologic analysis revealed virtually complete loss of the cochlear neuroepithelium (the organ of Corti) in adult mutant mice. In association with the neuroepithelial changes, there is a dramatic reduction of the cochlear nerve supply. Adult mutants also show morphological defects of the vestibular apparatus, including degeneration of the saccular neuroepithelium and occasional malformation of utricular otoconia. Audiometric evaluations demonstrated that the mice displaying the circling phenotype are completely deaf. Molecular analysis of this mutant line revealed that the transgenic insertion occurred without creating a large deletion of the host DNA sequences. The mutant locus was mapped to a region on mouse chromosome 10, where other spontaneous, recessive mutations causing deafness in mice have been mapped.

Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses.

The common occurrence of hearing loss in both humans and mice, and the anatomical and functional similarities of their inner ears, attest to the potential of mice being used as models to study inherited hearing loss. A large-scale, auditory screening project is being undertaken at The Jackson Laboratory (TJL) to identify mice with inherited hearing disorders. To assess hearing sensitivity, at least five mice from each inbred strain had auditory brainstem response (ABR) thresholds determined. Thus far, we have screened 80 inbred strains of mice; 60 of them exhibited homogeneous ABR threshold values not significantly different from those of the control strain CBA/CaJ. This large database establishes a reliable reference for normal hearing mouse strains. The following 16 inbred strains exhibited significantly elevated ABR thresholds before the age of 3 months: 129/J, 129/ReJ, 129/SvJ, A/J, ALR/LtJ, ALS/LtJ, BUB/BnJ, C57BLKS/J, C57BR/cdJ, C57L/J, DBA/2J, I/LnJ, MA/MyJ, NOD/LtJ, NOR/LtJ, and SKH2/J. These hearing impaired strains may serve as models for some forms of human non-syndromic hearing loss and aid in the identification of the underlying genes.

Inner ear and kidney anomalies caused by IAP insertion in an intron of the Eya1 gene in a mouse model of BOR syndrome.

A spontaneous mutation causing deafness and circling behavior was discovered in a C3H/HeJ colony of mice at the Jackson Laboratory. Pathological analysis of mutant mice revealed gross morphological abnormalities of the inner ear, and also dysmorphic or missing kidneys. The deafness and abnormal behavior were shown to be inherited as an autosomal recessive trait and mapped to mouse chromosome 1 near the position of the Eya1 gene. The human homolog of this gene, EYA1, has been shown to underly branchio-oto-renal (BOR) syndrome, an autosomal dominant disorder characterized by hearing loss with associated branchial and renal anomalies. Molecular analysis of the Eya1 gene in mutant mice revealed the insertion of an intracisternal A particle (IAP) element in intron 7. The presence of the IAP insertion was associated with reduced expression of the normal Eya1 message and formation of additional aberrant transcripts. The hypomorphic nature of the mutation may explain its recessive inheritance, if protein levels in homozygotes, but not heterozygotes, are below a critical threshold needed for normal developmental function. The new mouse mutation is designated Eya1(bor) to denote its similarity to human BOR syndrome, and will provide a valuable model for studying mutant gene expression and etiology.

Thyroid hormone receptor beta-dependent expression of a potassium conductance in inner hair cells at the onset of hearing.

To elucidate the role of thyroid hormone receptors (TRs) alpha1 and beta in the development of hearing, cochlear functions have been investigated in mice lacking TRalpha1 or TRbeta. TRs are ligand-dependent transcription factors expressed in the developing organ of Corti, and loss of TRbeta is known to impair hearing in mice and in humans. Here, TRalpha1-deficient (TRalpha1(-/-)) mice are shown to display a normal auditory-evoked brainstem response, indicating that only TRbeta, and not TRalpha1, is essential for hearing. Because cochlear morphology was normal in TRbeta-/- mice, we postulated that TRbeta regulates functional rather than morphological development of the cochlea. At the onset of hearing, inner hair cells (IHCs) in wild-type mice express a fast-activating potassium conductance, IK,f, that transforms the immature IHC from a regenerative, spiking pacemaker to a high-frequency signal transmitter. Expression of IK,f was significantly retarded in TRbeta-/- mice, whereas the development of the endocochlear potential and other cochlear functions, including mechanoelectrical transduction in hair cells, progressed normally. TRalpha1(-/-) mice expressed IK,f normally, in accord with their normal auditory-evoked brainstem response. These results establish that the physiological differentiation of IHCs depends on a TRbeta-mediated pathway. When defective, this may contribute to deafness in congenital thyroid diseases.

Absence of thyroid hormone receptor beta-retinoid X receptor interactions in auditory function and in the pituitary-thyroid axis.

THYROID hormone receptor beta-deficient (TRbeta-/-) mice have defective auditory-evoked brain stem responses (ABR). Since in vitro, TRbeta binds to DNA as homodimers or as heterodimers with retinoid X receptors (RXRs), we investigated whether the TRbeta-/- phenotype may reflect loss of RXR-TRbeta heterodimer or TRbeta homodimer function. Normal ABR thresholds were recorded in RXRbeta-/-, RXRgamma-/-, RXRalpha-/+ and RXR compound mutant mice. When RXR mutations were introduced onto TRbeta-/+ or TRbeta-/- backgrounds, thresholds were dictated solely by TRbeta and not RXR genotype. TRbeta-/-mice also over-produce thyroid hormones and thyroid stimulating hormone; however, levels of these hormones were unaltered by RXR mutations. This suggests that, contrary to in vitro models, RXRs may be dispensable and that TRbeta may function in vivo by an RXR-independent mechanism in the auditory system and pituitary-thyroid axis.

Deletion mapping of the head tilt (het) gene in mice: a vestibular mutation causing specific absence of otoliths.

Head tilt (het) is a recessive mutation in mice causing vestibular dysfunction. Homozygotes display abnormal responses to position change and linear acceleration and cannot swim. However, they are not deaf. het was mapped to the proximal region of mouse chromosome 17, near the T locus. Here we report anatomical characterization of het mutants and high resolution mapping using a set of chromosome deletions. The defect in het mutants is limited to the utricle and saccule of the inner ear, which completely lack otoliths. The unique specificity of the het mutation provides an opportunity to better understand the development of the vestibular system. Complementation analyses with a collection of embryonic stem (ES)- and germ cell-induced deletions localized het to an interval near the centromere of chromosome 17 that was indivisible by recombination mapping. This approach demonstrates the utility of chromosome deletions as reagents for mapping and characterizing mutations, particularly in situations where recombinational mapping is inadequate.

Balance and hearing deficits in mice with a null mutation in the gene encoding plasma membrane Ca2+-ATPase isoform 2.

Plasma membrane Ca2+-ATPase isoform 2 (PMCA2) exhibits a highly restricted tissue distribution, suggesting that it serves more specialized physiological functions than some of the other isoforms. A unique role in hearing is indicated by the high levels of PMCA2 expression in cochlear outer hair cells and spiral ganglion cells. To analyze the physiological role of PMCA2 we used gene targeting to produce PMCA2-deficient mice. Breeding of heterozygous mice yielded live homozygous mutant offspring. PMCA2-null mice grow more slowly than heterozygous and wild-type mice and exhibit an unsteady gait and difficulties in maintaining balance. Histological analysis of the cerebellum and inner ear of mutant and wild-type mice revealed that null mutants had slightly increased numbers of Purkinje neurons (in which PMCA2 is highly expressed), a decreased thickness of the molecular layer, an absence of otoconia in the vestibular system, and a range of abnormalities of the organ of Corti. Analysis of auditory evoked brainstem responses revealed that homozygous mutants were deaf and that heterozygous mice had a significant hearing loss. These data demonstrate that PMCA2 is required for both balance and hearing and suggest that it may be a major source of the calcium used in the formation and maintenance of otoconia.

Genetics of age-related hearing loss in mice. IV. Cochlear pathology and hearing loss in 25 BXD recombinant inbred mouse strains.

The effects of three putative genes which contribute to age-related hearing loss (AHL genes) were evaluated using auditory brainstem response (ABR) thresholds and post-mortem cochlear histopathology in 25 recombinant BXD inbred mouse strains, originally derived from C57BL/6J (B6) and DBA/2J (D2) progenitor strains. All BXD strains showed substantial elevation of ABR thresholds and loss of spiral ganglion cells (SGCs) during the first year of life. The findings are consistent with our genetic model in which D2 and B6 inbred strains both possess the Ahl (age-related hearing loss) gene, whereas D2 possesses two additional chromosomal loci with AHL genes (Ahl2 and Ahl3). The between-strain distribution in the severity of SGC loss and ABR threshold elevations suggests that the severity of hearing loss is determined in large part by the number of AH L genes an animal possesses and by additional genetic background effects. The present findings also demonstrate that, because BXD strains vary substantially in the rate and severity of progressive hearing loss (but are genetically closely related), they can provide powerful animal models for developmental studies of AHL.

Enzyme replacement therapy for murine mucopolysaccharidosis type VII leads to improvements in behavior and auditory function.

Mucopolysaccharidosis type VII (MPS VII; Sly syndrome) is one of a group of lysosomal storage diseases that share many clinical features, including mental retardation and hearing loss. Lysosomal storage in neurons of the brain and the associated behavioral abnormalities characteristic of a murine model of MPS VII have not been shown to be corrected by either bone marrow transplantation or gene therapy. However, intravenous injections of recombinant beta-glucuronidase initiated at birth reduce the pathological evidence of disease in MPS VII mice. In this study we present evidence that enzyme replacement initiated at birth improved the behavioral performance and reduced hearing loss in MPS VII mice. Enzyme-treated MPS VII mice performed similarly to normal mice and significantly better than mock- treated MPS VII mice in every phase of the Morris Water Maze test. In addition, the auditory function of treated MPS VII mice was dramatically improved, and was indistinguishable from normal mice. These data indicate that some of the learning, memory, and hearing deficits can be prevented in MPS VII mice if enzyme replacement therapy is initiated early in life. These data also provide functional correlates to the biochemical and histopathological improvements observed after enzyme replacement therapy.

A major gene affecting age-related hearing loss in C57BL/6J mice.

A major gene responsible for age-related hearing loss (AHL) in C57BL/6J mice was mapped by analyses of a (C57BL/6J x CAST/Ei) x C57BL/6J backcross. AHL, as measured by elevated auditory-evoked brainstem response (ABR) thresholds, segregated among backcross mice as expected for a recessive, primarily single-gene trait. Both qualitative and quantitative linkage analyses gave the same genetic map position for the AHL gene (Ahl on chromosome 10, near D10Mit5. Marker assisted selection was then used to produce congenic lines of C57BL/6J that contain different CAST-derived segments of chromosome 10. ABR test results and cochlear histopathology of aged progenitors of these congenic lines are presented. Ahl is the first gene causing late-onset, non-syndromic hearing loss that has been reported in the mouse.

Thyroid hormone receptor beta is essential for development of auditory function.

Congenital thyroid disorders are often associated with profound deafness, indicating a requirement for thyroid hormone (T3) and its receptors in the development of hearing. Two T3 receptor genes, Tr alpha and Tr beta are differentially expressed, although in overlapping patterns, during development. Thus, the extent to which they mediate unique or redundant functions is unclear. We demonstrate that Tr beta-deficient (Thrb-/-) mice exhibit a permanent deficit in auditory function across a wide range of frequencies, although they show no other overt neurological defects. The auditory-evoked brainstem response (ABR) in Thrb-/- mice, although greatly diminished, displayed normal waveforms, which suggested that the primary defect resides in the cochlea. Although hypothyroidism causes cochlear malformation, there was no evidence of this in Thrb-/- mice. These findings suggest that Tr beta controls the maturation of auditory function but not morphogenesis of the cochlea. Thrb-/- mice provide a model for the human endocrine disorder of resistance to thyroid hormone (RTH), which is typically associated with dominant mutations in Tr beta. However, deafness is generally absent in RTH, indicating that dominant and recessive mutations in Tr beta have different consequences on the auditory system. Our results identify Tr beta as an essential transcription factor for auditory development and indicate that distinct Tr genes serve certain unique functions.