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35 Yamauchi, T. et al. (2001) The mechanisms by which both heterozygous peroxisome proliferatoractivated receptor γ (PPARγ) deficiency and PPARγ agonist improve insulin resistance. J. Biol. Chem. 276, 41245–41254 36 Weyer, C. et al. (2000) Enlarged subcutaneous abdominal adipocyte size, but not obesity itself, predicts type II diabetes independent of insulin resistance. Diabetologia 43, 1498–1506 37 Mori, Y. et al. (1999) Effect of troglitazone on body fat distribution in type 2 diabetic patients. Diabetes Care 22, 908–912 38 Akazawa, S. et al. (2000) Efficacy of troglitazone on body fat distribution in type 2 diabetes. Diabetes Care 23, 1067–1071 39 Bolinder, J. et al. (1982) Postreceptor defects causing insulin resistance in normoinsulinemic non-insulindependent diabetes mellitus. Diabetes 31, 911–916 40 Boyko, E.J. et al. (2000) Visceral adiposity and risk of type 2 diabetes: a prospective study among Japanese Americans. Diabetes Care 23, 465–471 41 Reed, J. and Leff, T. (2002) The antidiabetic PPARγ ligand: update on compunds in development. Curr. Med. Chem. Immunol. Endocrine Metab. Agents 2, 33–47 42 Cao, H. and Hegele, R.A. (2000) Nuclear lamin A/C R482Q mutation in canadian kindreds with Dunnigan-type familial partial lipodystrophy. Hum. Mol. Genet. 9, 109–112 43 Agarwal, A.K. and Garg, A. (2002) A novel heterozygous mutation in peroxisome proliferator-activated receptor γ gene in a patient with familial partial lipodystrophy. J. Clin. Endocrinol. Metab. 87, 408–411 44 Hegele, R.A. (2000) Insulin resistance in human partial lipodystrophy. Curr. Atheroscler. Rep. 2, 397–404 45 Zhang, Y. et al. (1994) Positional cloning of the mouse obese gene and its human homologue. Nature 372, 425–432

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Mouse models are one of the major tools used for discovery and characterization of genes for non-syndromic deafness in humans. The similarities between the mouse and human genomes, and between the physiology and morphology of their auditory systems, are striking. This article describes the latest mouse models, including spontaneous, ‘knockout’ and ENU (N-ethyl-N-nitrosourea)-induced mutants, and the recent discovery of modifier genes that are involved in mouse deafness; this discovery is leading the search for genetic modifiers for human disorders. Published online: 8 August 2002

The auditory system is highly complex and includes many components that must work in harmony. The inner-ear sensory epithelia are responsible for the http://tmm.trends.com

M

Nadav Ahituv and Karen B. Avraham

ISEAS

E

D

Mouse models for human deafness: current tools for new fashions

O DEL S

mechano–chemical transduction of sound and vestibular information [1]. The cells that sense these stimuli are called ‘hair cells’ and are characterized by cytoskeletal projections on their apical surface called ‘stereocilia’ (Fig. 1a). These projections have an actin-rich core and are thought to have transduction channels that open upon deflection of the stereocilia bundle in response to a change in position or a sound wave. This channel opening causes an ion influx, resulting in depolarization of the hair cells and further propagation of the stimulus to the brain. This tightly coordinated system can be impaired by many factors, making hearing loss the most common form of sensory loss known to man. Over

1471-4914/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S1471-4914(02)02388-2

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(a)

Scc

Stria vascularis

Tectorial membrane Spiral limbus

Cochlea

Saccule

(b)

(c)

Stereocillia

Ihc

Utricle

(d) Ihc Ohc

Ohc

Supporting cells

+/+

(e)

Bth/+

Ohc

Ohc Ohc Ihc

Ihc

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Fig. 1. Various tools are available for studying the morphology, physiology and function of the inner ear in mice, some of which are shown here. (a) The inner ear. The detail shows a cross-section of the organ of Corti. (b,c) Organ of Corti explant cultures can be used to study the development of the sensory epithelium of the inner ear and the expression of inner-ear proteins. Organs of Corti from newborn mice were cultured, fixed and labeled with an affinity-purified antibody. (b) Portion of an organ of Corti labeled with an antibody specific for the cytoplasmic myosin VIIa protein, using fluorochrome-labeled antibodies for detection (green). Colocalization of actin and myosin VIIa is shown in yellow. (c) Entire organ of Corti labeled with an antibody specific for the transcription factor Pou4f3 (which is expressed exclusively in the nuclei of the hair cells), using an enzyme-conjugated antibody for detection. (d) The patch-clamp technique is used to record transducer currents in the mouse cochlea. The hair bundle of an outer hair cell is stimulated by a fluid jet (left). The fluid stimulus provides a force that can be varied sinusoidally (as shown) or stepwise. Bundle movements (top) are monitored using a laser interferometer (red dot), and transducer currents are measured with a patch pipette (right). Modified by C. Kros from Ref. [6]. (e) Scanning electron microscopy of cochleas derived from wild-type (+/+) and Beethoven mutant (Bth/+) mice. The Bth/+ sample shows hair-cell degeneration (indicated by arrows) in the basal region of the cochlea at post-natal day 30. Scale bar = 5 µm. Modified, with permission, from [27]. Abbreviations: Ihc, inner hair cells; Ohc, outer hair cells; Ssc, semicircular canals.

Nadav Ahituv Karen B. Avraham* Dept of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. *e-mail: karena@ post.tau.ac.il

half of prelingual hearing loss is caused by genetic factors (reviewed in [2]). Genetic non-syndromic hearing loss (NSHL) is inherited in a recessive mode in ~80% of cases (DFNB loci), in a dominant mode in ~18% (DFNA), and is either X-linked (DFN loci) or mitochondrial in 2–3% of this group. Approximately 36% of people over the age of 75 suffer from presbycusis, a high-tone hearing impairment that presents with advanced age and is thought to be largely caused by genetic factors [2]. The rapid advancement in the discovery of genes responsible for NSHL has been outstanding. The first human nuclear gene, X-linked POU3F4, was identified only in 1995 [3], whereas, to date, 28 human genes have been cloned, and, indeed, the number rises almost monthly (see the Hereditary Hearing Loss Homepage at http://dnalab-www.uia.ac.be/dnalab/hhh/). With the cloning of each new human gene, more light is shed on the molecular basis of the auditory pathway, although the information revealed is limited http://tmm.trends.com

because of the confines of human experiments. Mice are proving to be an invaluable tool for study of the morphology and physiology of the inner ear, using techniques such as scanning electron microscopy (SEM) [4], patch clamping on individual hair cells [5], immunohistochemistry, and culturing of the organ of Corti [6] (Fig. 1). Mouse models for human deafness include those that have arisen spontaneously, and those created by random mutagenesis techniques and gene-targeting technology for the formation of ‘knockouts’ (Table 1). From mice to men

Mutations in a large repertoire of genes, encoding many different types of proteins, have been implicated in human hereditary NSHL. Detection of some of these genes in humans was facilitated by the earlier cloning of their orthologous genes in mice. There are a large number of deaf mouse mutants, most of which also exhibit vestibular dysfunction, manifested as ‘circling’ or head tossing. These phenotypes are consequences of either defects in cochlear and vestibular hair cells or extracellular membranes, or defects in inner-ear development or endolymph homeostasis (see the Hereditary Hearing Impairment in Mice website at http://www.jax.org/research/hhim/ and the Mouse Mutants with Hearing or Balance Defects website at http://www.ihr.mrc.ac.uk/ hereditary/mousemutants.htm). One group of proteins that is frequently associated with NSHL is the myosins, three of which were discovered owing to their corresponding mouse mutants [7–9]. Myosins are molecular motors that move along actin filaments. They have been implicated in various cellular functions, such as cell movement,

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Table 1. Mouse models for human hearing loss

449

a

Mouse model

Mouse gene

Protein function

Corresponding human phenotype

Refs

waltzer (v)

Cadherin 23 (Cdh23)

DFNB12 and Usher 1D syndrome

[35]

Col11a2 knockout Gjb2 conditional knockout Snell's waltzer (sv) shaker 1 (sh1) shaker 2 (sh2) Pou3f4 knockouts, sex-linked fidget (Slf) b Pou4f3 knockouts, dreidel (ddl) Slc26a4 knockout

Collagen type XI α 2 (Col11a2) Connexin 26 (Gjb2) Myosin VI (Myo6) Myosin VIIa (Myo7a) Myosin XV (Myo15) Pou3f4

Calcium-dependent cell–cell adhesion Extracellular matrix Gap junction Motor molecule Motor molecule Motor molecule Transcription factor

DFNA13 and Stickler syndrome type 2 DFNA3, DFNB1 DFNA22 DFNA11, DFNB2 and Usher 1B syndrome DFNB3 DFN3

[36] [24] [8] [7] [9] [37–39]

Transcription factor Anion transporter

DFNA15 DFNB4 and Pendred syndrome

[40–42] [43]

Extracellular matrix Predicted channel or transporter

DFNA8, DFNA12 and DFNB21 DFNA36, DFNB7 and DFNB11

[44] [25,27]

Tecta knockout deafness (dn), Beethoven (Bth)

Pou4f3 Solute carrier family 26, member 4 (Slc26a4) Tectorin α (Tecta) Transmembrane cochlearexpressed gene 1 (Tmc1)

a

b

Relevant genes have been cloned in both mice and humans. See http://www.informatics.jax.org/

Acknowledgements K.B.A.’s work on mouse models for human deafness is supported by grants from the European Commission (QLG2-CT1999–00988), the Israel Ministry of Health and the Israel Science Foundation. We thank Corne Kros, David Gurwitz, Martine CohenSalmon, Ari Elson and Yehoash Raphael for valuable comments; Tama Hasson for myosin VIIa antibodies; and Ronna Hertzano and Varda Wexler for figures. This review is dedicated to the memory of George D. Snell, a pioneer in mouse genetics, whose ‘namesake’ – Snell’s waltzer – introduced us to the world of mouse models for deafness.

membrane traffic and signal transduction [10]. Several Myo7a mutations have been found to cause recessive hearing loss in shaker 1 (sh1) mice [7] and, in humans, MYO7A mutations (DFNB2 and DFNA11) cause both recessive and dominant hearing loss [11,12], as well as Usher 1B syndrome (characterized by profound hearing loss, retinitis pigmentosa and vestibular dysfunction) [13]. An elegant combination of organotypic cultures of organs of Corti and patch-clamp techniques has been adapted to study mouse hair bundles [5] (Fig. 1c,d). Recently, this technique has provided key insights into the function of myosin VIIa in mechano–electrical transduction of cochlear hair cells, suggesting that myosin VIIa helps fasten membrane-bound elements to the actin core of the stereocilium, thereby playing a role in auditory adaptation (i.e. a mechanism that allows channels to be open even in the absence of stimuli) [14]. Another myosin, myosin 1c, is thought to be the main motor molecule involved in auditory adaptation, and a recent report used a chemical–genetic approach and transgenic mice to demonstrate the role of myosin 1c in this mechanism [15]. However, as yet, no human deaf families are known to have mutations in the gene for myosin 1c. Mutations in the genes for myosin VI (MYO6) [16] and myosin XV (MYO15) [17] occur in both humans and mice, and electron microscopy of Snell’s waltzer (Myo6sv) [18] and shaker2 (Myo15sh2) [9] mice has revealed fused stereocilia and shortened stereocilia, respectively. Through these mouse models, we see that the damage caused by loss-of-function of each gene is unique, although they all ultimately lead to the same phenotypic outcome. From men to mice

When a gene is known to cause NSHL in humans, and a spontaneous mouse model does not exist, the race is on to generate a knockout model. But, for connexin 26 (Cx26, locus designation GJB2), a knockout mouse had a phenotype that was completely different to that in humans. In most cases, mutations in GJB2 lead to http://tmm.trends.com

severe to profound congenital deafness in humans [19]. In fact, this might be the most prevalent form of genetic deafness as it accounts for 30–50% of autosomal recessive deafness in many parts of the world [20,21]. Surprisingly, Cx26 homozygous knockout mice show a lethal phenotype at embryonic day 11 [22]. Cx26 is a gap-junction protein that contributes to two independent cellular networks in the cochlea: an epithelial network connecting the supporting cells of hair cells and adjacent epithelial cells, and a connective tissue network connecting mesenchymal cells and fibrocytes (as well as basal and intermediate cells of the stria vascularis) (reviewed in Ref.[23]). The early death of Cx26 knockout mice is thought to reflect defective transplacental uptake of glucose, and the difference in phenotype between mice and humans might be explained by the different morphology of the mouse and human placenta. To study Cx26 function in the inner ear, Cx26 was recently inactivated in the inner-ear epithelial gap-junction network using Cre-lox targeting technology [24]. Tissue-specific knockouts show a moderate to profound hearing impairment with no balance dysfunction caused by the degeneration of the cochlear neuroepithelium. Mutations in the TMC1 gene were recently identified in both recessive and dominant forms of hearing loss in humans: DFNB7/B11 and DFNA36, respectively [25]. The only two mouse mutants shown, as yet, to suffer from isolated hearing loss with no balance defect – deafness (dn) and Beethoven (Bth) – are models for these forms of human deafness. Their loci have been mapped to mouse chromosome 19, to a region homologous to human chromosome 9 where TMC1 is localized [26,27]. Bth was generated in a large-scale N-ethyl-N-nitrosourea (ENU) screen [28]. Bth mutants show dominant progressive hearing loss and their hair cells appear to have normal function before they begin to degenerate around post-natal day 20 (Fig. 1e). Once mutations in TMC1 were found in the human forms of deafness, the mouse ortholog, Tmc1, became an excellent

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candidate for mouse forms of deafness. Subsequently, mutations in this gene were found in both Bth and dn TMC1 is predicted to encode a channel or transporter protein, and further analysis using the Bth and dn mouse models will help assess the questions regarding the role of this protein in the inner ear and how this function is associated with hearing loss. The above example is the first of its kind demonstrating both recessive and dominant hearing loss in both mice and humans. Modifiers in mice: implications for humans

Searching for modifier genes in humans is, at times, akin to searching for a needle in a haystack, mainly because of the complexity of performing modifier linkage analysis on outbred populations. In the case of hearing loss, one such dominant modifer locus, which suppresses the recessive deafness locus DFNB26, has been mapped to chromosome 1q24 in a large consanguineous family [29]. By contrast, the genetic diversity between different inbred mouse strains has been a powerful tool for the discovery of modifier genes. One such case is the discovery that microtubule-associated protein 1A (Mtap1a) is the modifier of hearing loss in tubby mice [30]. Tubby mice have a phenotype of obesity, retinal degeneration and hearing loss in a C57BL/6J genetic background. The hearing loss is lost when crossed with AKR/J, CAST/Ei and 129P2/OlaHsd backgrounds. Chromosomal mapping, as well as the subsequent finding of sequence alterations between the Mtap1a of C57BL/6J and the other three strains, led to the hypothesis that Mtap1a is the modifier of the tubby hearing gene moth1. A transgenic approach – introduction of a 129P2/OlaHsd-derived Mtap1a gene into a C57BL/6J tubby mouse, followed by rescue of the hearing-loss phenotype – led to the final proof that Mtap1a is moth1. The vulnerability to presbycusis within the human population varies, and association studies to identify a plausible causative gene are highly complicated. Different inbred mice show a variety of age-related References 1 Gillespie, P.G. and Walker, R.G. (2001) Molecular basis of mechanosensory transduction. Nature 413, 194–202 2 Nadol, J.B., Jr and Merchant, S.N. (2001) Histopathology and molecular genetics of hearing loss in the human. Int. J. Pediatr. Otorhinolaryngol. 61, 1–15 3 de Kok, Y.J. et al. (1995) Association between X-linked mixed deafness and mutations in the POU domain gene POU3F4. Science 267, 685–688 4 Lim, D.J. (1969) Three dimensional observation of the inner ear with the scanning electron microscope. Acta Otolaryngol. 255 (Suppl.), 1–38 5 Kros, C.J. et al. (1992) Mechano–electrical transducer currents in hair cells of the cultured neonatal mouse cochlea. Proc. R. Soc. Lond. B Biol. Sci. 249, 185–193 6 Sobkowicz, H.M. et al. (1993) Tissue culture of the organ of Corti. Acta Otolaryngol. 502 (Suppl.), 3–36

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and noise-induced hearing loss throughout their life span. An immense contribution to our ability to study the diversity of hearing loss among different inbred mouse strains was made when the auditory brainstem response (ABR) was used to assess the hearing of 80 different inbred strains throughout their lifetime [31]. Some strains lose their hearing as early as three months, whereas others maintain normal hearing until the latest age examined, 15 months (these data are available at the Hereditary Hearing Impairment in Mice website). With the earlier finding of an age-related hearing-loss locus on mouse chromosome 10 (Ahl) in the C57BL/6J strain [32], this research helped demonstrate that this is the same locus that modifies hearing in 11 other inbred mouse strains. There is now genetic evidence that Ahl is allelic with the modifier of deaf waddler (mdfw) [33]. It also led to the finding of an interaction between mitochondrial DNA and Ahl in an A/J inbred mouse mitochondrial background. Mice that are homozygous for the Ahl allele and have an A/J adenine insertion in the mitochondrial gene encoding tRNA-Arg, show elevated ABR thresholds compared with those that have a different mitochondrial background [34]. Most of this research has, as yet, only provided discoveries in the mouse. However, there is great hope that modifier research in mice will provide clues regarding human age-related hearing loss and variability. Concluding remarks

As more and more NSHL genes are found, it is intriguing to speculate on what future research will hold for the field of hearing loss. We have already learned, and will continue to learn, a great deal about ion homeostasis, auditory transduction, inner-ear development and other aspects of auditory function. Deciphering the function of the proteins encoded by these genes could lead to therapeutic advances, and the mouse models should prove particularly useful for this endeavor.

7 Gibson, F. et al. (1995) A type VII myosin encoded by the mouse deafness gene shaker-1. Nature 374, 62–64 8 Avraham, K.B. et al. (1995) The mouse Snell’s waltzer deafness gene encodes an unconventional myosin required for the structural integrity of inner ear hair cells. Nat. Genet. 11, 369–375 9 Probst, F.J. et al. (1998) Correction of deafness in shaker-2 mice by an unconventional myosin in a BAC transgene. Science 280, 1444–1447 10 Mermall, V. et al. (1998) Unconventional myosins in cell movement, membrane traffic, and signal transduction. Science 279, 527–533 11 Liu, X.Z. et al. (1997) Mutations in the myosin VIIA gene cause non-syndromic recessive deafness. Nat. Genet. 16, 188–190 12 Liu, X.Z. et al. (1997) Autosomal dominant non-syndromic deafness caused by a mutation in the myosin VIIA gene. Nat. Genet. 17, 268–269

13 Weil, D. et al. (1995) Defective myosin VIIA gene responsible for Usher syndrome type 1B. Nature 374, 60–61 14 Kros, C.J. et al. (2002) Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations. Nat. Neurosci. 5, 41–47 15 Holt, J.R. et al. (2002) A chemical–genetic strategy implicates myosin-1c in adaptation by hair cells. Cell 108, 371–381 16 Melchionda, S. et al. (2001) MYO6, the human homologue of the gene responsible for deafness in Snell’s waltzer mice, is mutated in autosomal dominant nonsyndromic hearing loss. Am. J. Hum. Genet. 69, 635–640 17 Wang, A. et al. (1998) Association of unconventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3. Science 280, 1447–1451 18 Self, T. et al. (1999) Role of myosin VI in the differentiation of cochlear hair cells. Dev. Biol. 214, 331–341

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19 Kelsell, D. et al. (1997) Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387, 80–83 20 Kelley, P.M. et al. (1998) Novel mutations in the connexin 26 gene (GJB2) that cause autosomal recessive (DFNB1) hearing loss. Am. J. Hum. Genet. 62, 792–799 21 Denoyelle, F. et al. (1997) Prelingual deafness: high prevalence of a 30delG mutation in the connexin 26 gene. Hum. Mol. Genet. 6, 2173–2177 22 Gabriel, H. et al. (1998) Transplacental uptake of glucose is decreased in embryonic lethal connexin26-deficient mice. J. Cell Biol. 140, 1453–1461 23 Petit, C. et al. (2001) Molecular genetics of hearing loss. Annu. Rev. Genet. 35, 589–646 24 Cohen-Salmon, M. et al. (2002) Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death. Curr. Biol. 12, 1106–1111 25 Kurima, K. et al. (2002) Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function. Nat. Genet. 30, 277–284 26 Keats, B.J. et al. (1995) The deafness locus (dn) maps to mouse chromosome 19. Mamm. Genome 6, 8–10 27 Vreugde, S. et al. (2002) Beethoven, a mouse model for dominant, progressive hearing loss DFNA36. Nat. Genet. 30, 257–258

28 Hrabe de Angelis, M.H. et al. (2000) Genomewide, large-scale production of mutant mice by ENU mutagenesis. Nat. Genet. 25, 444–447 29 Riazuddin, S. et al. (2000) Dominant modifier DFNM1 suppresses recessive deafness DFNB26. Nat. Genet. 26, 431–434 30 Ikeda, A. et al. (2002) Microtubule-associated protein 1A is a modifier of tubby hearing (moth1). Nat. Genet. 30, 401–405 31 Zheng, Q.Y. et al. (1999) Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear. Res. 130, 94–107 32 Johnson, K.R. et al. (1997) A major gene affecting age-related hearing loss in C57BL/6J mice. Hear. Res. 114, 83–92 33 Zheng, Q.Y. and Johnson, K.R. (2001) Hearing loss associated with the modifier of deaf waddler (mdfw) locus corresponds with age-related hearing loss in 12 inbred strains of mice. Hear. Res. 154, 45–53 34 Johnson, K.R. et al. (2001) A nuclear-mitochondrial DNA interaction affecting hearing impairment in mice. Nat. Genet. 27, 191–194 35 Di Palma, F. et al. (2001) Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D. Nat. Genet. 27, 103–107 36 McGuirt, W.T. et al. (1999) Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13). Nat. Genet. 23, 413–419

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37 Minowa, O. et al. (1999) Altered cochlear fibrocytes in a mouse model of DFN3 nonsyndromic deafness. Science 285, 1408–1411 38 Phippard, D. et al. (1999) Targeted mutagenesis of the POU-domain gene Brn4/Pou3f4 causes developmental defects in the inner ear. J. Neurosci. 19, 5980–5989 39 Phippard, D. et al. (2000) The sex-linked fidget mutation abolishes Brn4/Pou3f4 gene expression in the embryonic inner ear. Hum. Mol. Genet. 9, 79–85 40 Erkman, L. et al. (1996) Role of transcription factors Brn-3.1 and Brn-3.2 in auditory and visual system development. Nature 381, 603–606 41 Xiang, M. et al. (1997) Essential role of POU-domain factor Brn-3c in auditory and vestibular hair cell development. Proc. Natl. Acad. Sci. U. S. A. 93, 11950–11955 42 Wang, S.W. et al. (2002) Brn3b/Brn3c double knockout mice reveal an unsuspected role for Brn3c in retinal ganglion cell axon outgrowth. Development 129, 467–477 43 Everett, L.A. et al. (2001) Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome. Hum. Mol. Genet. 10, 153–161 44 Legan, P.K. et al. (2000) A targeted deletion in alpha-tectorin reveals that the tectorial membrane is required for the gain and timing of cochlear feedback. Neuron 28, 273–285

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