A mouse model for nonsyndromic deafness (DFNB12)links hearing loss to defects in tip links ofmechanosensory hair cellsMartin Schwandera,b, Wei Xionga,b, Joshua Tokitac, Andrea Lellid, Heather M. Elledgea,b, Piotr Kazmierczaka,b,Anna Sczanieckaa,b, Anand Kolatkara, Tim Wiltshiree,1, Peter Kuhna, Jeffrey R. Holtd, Bechara Kacharc, Lisa Tarantinoe,1,and Ulrich Mullera,b,2

aDepartment of Cell Biology and bInstitute of Childhood and Neglected Disease, The Scripps Research Institute, La Jolla, CA 92037; cLaboratory of CellularBiology, National Institute of Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, MD 20892; dDepartment ofNeuroscience, University of Virginia School of Medicine, Charlottesville, VA 22908; and eGenomics Institute of the Novartis Research Foundation,San Diego, CA 92121

Communicated by Bruce A. Beutler, The Scripps Research Institute, La Jolla, CA, January 26, 2009 (received for review January 9, 2009)

Deafness is the most common form of sensory impairment inhumans and is frequently caused by single gene mutations. Inter-estingly, different mutations in a gene can cause syndromic andnonsyndromic forms of deafness, as well as progressive andage-related hearing loss. We provide here an explanation for thephenotypic variability associated with mutations in the cadherin 23gene (CDH23). CDH23 null alleles cause deaf-blindness (Ushersyndrome type 1D; USH1D), whereas missense mutations causenonsyndromic deafness (DFNB12). In a forward genetic screen, wehave identified salsa mice, which suffer from hearing loss due to aCdh23 missense mutation modeling DFNB12. In contrast to waltzermice, which carry a CDH23 null allele mimicking USH1D, hair celldevelopment is unaffected in salsa mice. Instead, tip links, whichare thought to gate mechanotransduction channels in hair cells, areprogressively lost. Our findings suggest that DFNB12 belongs to anew class of disorder that is caused by defects in tip links. Wepropose that mutations in other genes that cause USH1 andnonsyndromic deafness may also have distinct effects on hair celldevelopment and function.

cadherin 23 � Cdh23 � Usher syndrome � progressive hearing loss

Dramatic progress has been made in the identification of genemutations that cause hearing loss, but we know compara-

tively little about the mechanisms by which the mutations lead todisease. Interestingly, different mutations in a gene can causedistinct disease outcomes. The cadherin 23 gene (CDH23)provides a striking example. Predicted CDH23 null mutationslead to deaf-blindness (USH1D), whereas missense mutationscause nonsyndromic deafness (DFNB12) (1–13). A polymor-phism in Cdh23 is linked to age-related hearing loss (14).Similarly, mutations in the genes for myosin VIIa (MYO7A) andprotocadherin 15 (PCDH15) cause USH1 and nonsyndromicdeafness (http://webh01.ua.ac.be/hhh/).

Recent studies in mice suggest that USH1 is caused by defectsin hair cell development. Each developing hair cell contains atthe apical surface a single kinocilium and rows of stereocilia,which form the mechanically sensitive organelle of a hair cell(Fig. 1A). Extracellular filaments connect the stereocilia andkinocilium of a developing hair cell (15). CDH23 and PCDH15are components of transient lateral links, kinociliary links, andtip links (Fig. 1 A) (16–20), and their cytoplasmic domains bindto protein complexes containing harmonin, MYO7A, and sans(21–25). Predicted null mutations in murine USH1 genes causedefects in hair bundle development (24, 26–34), suggesting thatUSH1 proteins form transmembrane complexes that regulatehair bundle morphogenesis. Failure of these complexes likelycauses USH1.

So far, there are no animal models for nonsyndromicdeafness caused by mutations in any USH1 gene, and the

mechanism by which such mutations cause disease is unclear.In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen (35),we have now identified salsa mice, which suffer from progres-sive hearing loss and carry a Cdh23 missense mutation that ispredicted to affect Ca2� binding by the extracellular CDH23domain. Similar mutations in the human CDH23 gene causeDFNB12 (1, 5, 6, 10, 13, 36). Unlike in mice with predictedCdh23 null alleles, hair bundle development appears unaf-fected in salsa mice. Instead, tip links are progressively lost,suggesting that similar mutations in DFNB12 patients lead todeafness by affecting tip links.

ResultsProgressive Hearing Loss in salsa Mice. In an ENU mutagenesisscreen, we identified salsa mice, which show no auditory startleresponse (35). Recordings of the auditory brainstem response(ABR) revealed that salsa mice suffer from progressive hearingloss. Wild-type mice at 3 weeks and 2 months of age had ABRthresholds to click stimuli at 20 � 5 dB. Thresholds in salsa micewere by 3 weeks at 78 � 15 dB and by 2 months at 100 � 5 dB(Fig. 1 C and D). salsa mice were hearing-impaired across allfrequencies (Fig. 1E). No defect was observed in heterozygoussalsa mice, demonstrating recessive mode of inheritance (35).Distortion product otoacoustic emissions were not detected insalsa mice (Fig. S1), indicating that outer hair cell function wasperturbed. Movement and the ability to swim were unaffected(Fig. 1F) (35), indicating that the vestibular system of salsa micewas intact.

salsa Mice Carry a Cdh23 Point Mutation. salsa mice were derived ona C57BL/6J background (35). To identify the affected gene, wecrossed salsa mice to 129S1/SvImJ mice. Offspring were inter-crossed to obtain F2 mice for ABR measurements and DNApreparation. By using single-nucleotide polymorphisms (SNPs),the affected genomic locus was mapped to a 4-MB interval onchromosome 10 containing Cdh23 (Fig. S2 A). Sequencing re-vealed a single point mutation, A2210T, in exon 22 of Cdh23

Author contributions: M.S., W.X., A.L., H.M.E., P. Kazmierczak, A.S., T.W., J.R.H., B.K., L.T.,and U.M. designed research; M.S., W.X., J.T., A.L., H.M.E., P. Kazmierczak, A.S., B.K., and L.T.performed research; A.K. and P. Kuhn contributed new reagents/analytic tools; M.S., W.X.,J.T., A.L., H.M.E., P. Kazmierczak, A.S., A.K., T.W., P. Kuhn, J.R.H., B.K., L.T., and U.M.analyzed data; and M.S. and U.M. wrote the paper.

The authors declare no conflict of interest.

See Commentary on page 4959.

1Present address: Department of Psychiatry, University of North Carolina, Chapel Hill, NC27516.

2To whom correspondence should be addressed. E-mail: umueller@scripps.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0900691106/DCSupplemental.

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(Fig. 2A). Compound heterozygous mice carrying 1 salsa alleleand 1 previously reported mutant Cdh23 allele (waltzerv2J) (27)were deaf (Fig. S2B), confirming that the salsa mutation causesdeafness. The salsa mutation leads to a Glu737Val substitutionwithin an LDRE motif in the seventh cadherin repeat of CDH23(Fig. 2B), which is conserved in CDH23 across species and incadherin repeats of other cadherins (Fig. 2B), and is required forCa2� chelation (Fig. 2C) (37, 38). Several mutations that causeDFNB12 resemble the salsa mutation and affect Ca2�-bindingmotifs (LDRE, DXND, DXD; Fig. 2D and Table S1) (1, 5–7, 10,13, 36). salsa mice are therefore a model for some forms ofDFNB12. Mutations that lead to predicted CDH23 null allelescause USH1D (Fig. 2D) (1–5, 7–12), which is modeled bymutations in waltzer mice (Fig. 2D) (27, 33, 34).

Progressive Tip-Link Loss in salsa Mice. Although hair bundles aredisrupted in waltzer mice at early postnatal ages (Fig. 3F) (27,34), hair bundles were unaffected in salsa mice at postnatal day5 (P5) and P28 (Fig. 3). This suggests that CDH23 plays animportant role in steps beyond hair bundle development.

Staining with CDH23 antibodies revealed that CDH23 wastargeted to the stereocilia of hair cells in salsa mice by P5 andP10 (Fig. 4 A–F and K), but expression was only occasionallydetectable by P30 (Fig. 4 G–K). However, no defects in CDH23cell surface transport were observed (Fig. S3). BecauseCDH23 is a tip-link component (16, 20), tip-link maintenancemay be affected. Tips of stereocilia show characteristic tentingthat is thought to be the consequence of tip-link-mediatedtension (39–42). Tenting can therefore be used to quantify thepresence of tip links. Tenting was observed in wild-type haircells at P5, P21, and P60 (Fig. 5 A, C, and H and Figs. S4 andS5). In salsa mice, tenting was observed throughout thecochlear duct at P5 (Fig. 5 B and H and Fig. S4); by P21, tentingwas no longer observed in the basal turn of the cochlea, andwas absent in all hair cells by P60 (Fig. 5D and Fig. S5). salsamice showed no signs of vestibular dysfunction (Fig. 1F), andtenting of stereociliary tips and tip links were maintained investibular hair cells (Fig. 5 F and G). Finally, by P90, the organof Corti and spiral ganglion degenerated in salsa mice, whereasthe vestibule was unaffected (Fig. S6). Staining for activated

Fig. 1. Analysis of auditory function. (A) Diagram of a developing hairbundle. Stereocilia are connected to each other and to the kinocilium byvarious linkages. Linkages that contain PCDH15 and CDH23 are indicated. (B)The diagram shows the localization of CDH23 and PCDH15 at tip links. (C)Click-evoked ABR for a 2-month-old wild-type (black lines) and a salsa mouse(red line) at different sound intensities (dB). ABR waves I–IV are indicated. (D)Average auditory thresholds for 3-week-old and 2-month-old mice [wild-typen � 4 for 3 weeks (white) and 2 months (gray); salsa n � 5 for 3 weeks (orange)and 2 months (red)]. The mean � SD is indicated; a Student’s t test wasperformed. (E) Auditory thresholds in 3-week-old (triangles) and 2-month-old(circles) mice as determined by pure tone ABR recordings. In contrast towild-type (gray and black lines) salsa mutants (orange and red lines) showedprogressive hearing loss. (F) Analysis of movement in the open field. salsa mice(red) showed normal numbers of small-diameter rotations (�2.75-cm radius)and were not hyperactive. As a positive control, the sirtaki mouse line (gray)that has vestibular defects (35) is shown. Values are mean � SD. A Student ttest was performed. **, P � 0.01; ***, P � 0.001.

Fig. 2. The mutation in salsa mice maps to a Ca2�-binding motif in CDH23.(A) Sequence chromatograph from wild-type and salsa mice reveals an A-to-Tmutation in exon 22 of Cdh23. (B) The C-terminal part of EC7 of CDH23 fromdifferent species is shown. The Glu737Val substitution in salsa affects a con-served Ca2�-binding motif (yellow boxes). CDH1 and CDH2 are shown forcomparison. (C) CDH23 EC7/8 wild-type (blue) and salsa mutant (red) se-quences threaded onto the E-cadherin EC1/2 by using the automodel class anda sequence alignment produced by T-Coffee. Energy-minimized model showsthat the Glu737Val mutation affects Ca2� coordination. (D) Domain structureof CDH23 indicating the 27 extracellular cadherin repeats (blue). Missensemutations in Ca2�-binding motifs in the CDH23 extracellular domain causeDFNB12 in humans (purple shaded box). Nonsense and splice site mutationshave been identified in waltzer mice and USH1D (gray shaded box). SSindicates signal sequence; TM, transmembrane domain.

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caspase 3 showed that cochlear hair cells and spiral ganglionneurons died by apoptosis (Fig. S6).

Defects in Hair Cell Function by P7/8. To test whether hair cellfunction was affected before detectable morphological changes,we recorded mechanotransduction currents from P7/8 cochlearhair cells in response to 5-ms bundle deflections ranging from�400 to 1000 nm. Current-displacement [I(X)] relationship plotsdid not reveal a difference between wild-type and salsa mice(Fig. 6 A and B). Peak currents at maximal deflection weresimilar in controls (419 � 40 pA) and mutants (427 � 35 pA).We next analyzed the adaptation kinetics of mechanotransduc-tion currents in outer hair cells stimulated with 100-ms deflec-tions of 100–800 nm. Normalization of each current trace to itspeak current showed faster transduction current decline in salsacompared with wild type, which was only observed at deflectionsabove 300 nm (Fig. 6 C and D). We conclude that hair cellfunction was mildly affected by P7/8.

The salsa Mutation Affects CDH23 Adhesive Function. CDH23 inter-acts with PCDH15 to form tip links (16). To test the effects ofthe salsa mutation on CDH23 properties, we incubated thepurified extracellular domains of CDH23 with or without thesalsa mutation fused to Fc tags (referred to as CDH23wt-Fc andCDH23salsa-Fc, respectively) with the purified His-tagged ex-tracellular domain of protocadherin 15 (PCDH15-His; Fig. 7A).Protein complexes were analyzed by Western blotting. As re-ported, PCDH15-His bound to CDH23wt-Fc in a Ca2�-dependent manner (Fig. 7B) (16). We also observed Ca2�-

dependent interactions between PCDH15-His and CDH23salsa-Fc, but interactions were diminished (Fig. 7B). To determinewhether mutations in CDH23 that cause DFNB12 reduce inter-actions with PCDH15, we engineered 2 human mutations intoCDH23-Fc (Fig. 7A) (1). Both mutations reduced interactions ofCDH23 with PCDH15 (Fig. 7B).

DiscussionPrevious studies have shown that predicted Cdh23 null allelesperturb hair bundle development, likely as a consequence ofdefects in the filaments that connect the stereocilia and kinoci-lium of a developing hair cell. In contrast, we show here that aCdh23 missense mutation leads to progressive tip-link loss and,ultimately, to hair cell death. In salsa mice, a small defect inmechanotransduction by cochlear hair cells was apparent atP7/P8, indicating that hair cell function was already affected. Atsubsequent ages, tip links were lost, as revealed by diminishedlevels of CDH23 protein in stereocilia and loss of tenting ofstereociliary tips. Because predicted CDH23 null alleles areassociated with USH1D, whereas missense mutations similar tothe one observed in salsa mice cause DFNB12, we propose that

Fig. 3. Preserved hair bundle morphology in salsa mice. (A–F) Scanningelectron microscopy micrographs of cochlear whole mounts from P5 wild-typemice and salsa and waltzer mutants. (A and B) The organ of Corti in salsa micewas patterned normally in 3 rows of outer and 1 row of inner hair cells. (C–F)Hair bundles in salsa displayed the characteristic polarized morphology witha single kinocilium. Hair bundles in waltzer mice were fragmented. (G–L) AtP28, hair bundle morphology was indistinguishable in wild-type and salsamice. [Scale bars: A and B (5 �m), C–F (2 �m), G and H (10 �m), and I–L (2 �m).]

A B

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Fig. 4. Progressive loss of CDH23 expression in salsa mice. (A–J) Cochlearwhole mounts of wild-type and salsa mice were stained with an antibodyagainst the CDH23 cytoplasmic domain (green) and with phalloidin (red). (Aand B) Levels of CDH23 expression in homozygous salsa and wild-type mice atP5 were similar. (C and D) Higher-magnification view of CDH23 expression inhair bundles at P5. (E and F) At P10, CDH23 staining was maintained atstereociliary tips. (G–J) At P30, CDH23 staining was barely detectable in salsa.Arrowheads point to CDH23 in wild types and residual staining in salsa. (K)Quantification of CDH23 staining in hair cells at P10 and P30. salsa mice (black)showed reduced numbers of CDH23 puncta in hair bundles at P30. Values aremean � SD. A Student’s t test was performed. ***, P � 0.001. [Scale bar: A andB (8 �m), C–J (2 �m).]

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USH1D is caused by developmental defects in hair bundles andDFNB12 by tip-link defects.

Our biochemical studies show that the salsa mutation affectsinteractions of CDH23 and PCDH15, even though the muta-tion affects an amino acid outside the ligand-binding domainin EC1 (16). Hair cell function was also maintained for sometime in salsa mice, and tip links sustained substantial forces inelectrophysiological experiments. These findings seem contra-dictory at first glance, but the study of classical cadherinsprovides clues that likely explain the results. Ca2� bindingrigidifies the cadherin extracellular domain. Disruption ofCa2� binding inf luences protein structure, often perturbingadhesive function (37, 38). In analogy to classical cadherins, itis likely that defects in ligand binding caused by the salsamutation report changes in CDH23 structure that are propa-gated over a considerable distance within the extracellulardomain. Structural changes might predispose tip links tobreakage, necessitating frequent regeneration that cannot besustained. CDH23 and PCDH15 at tip links might also carryadditional posttranslational modifications, which stabilizetheir interaction but were not present in the recombinantproteins. An interesting implication of our findings is thatsome CDH23 mutations may predispose individuals to hearingloss caused by mechanical insults. In fact, polymorphisms inCDH23 are associated with noise-induced hearing loss (43),and mice heterozygous for the Cdh23 waltzer allele showincreased noise susceptibility (44). Finally, a polymorphism inCdh23 is associated with age-related hearing loss (14), whichmay be related to instability of tip links.

All USH1 proteins control hair bundle development (15).CDH23, PCDH15, and MYO7A are also expressed in maturehair cells (the mature expression pattern for harmonin andsans has yet to be determined). As tip-link proteins, CDH23and PCDH15 are thought to gate transduction channels,whereas MYO7A contributes to channel adaptation (45).Missense mutations in the genes for MYO7A and PCDH15,similar to the salsa mutation in CDH23, might also affect

tip-link function without effects on hair cell development. Theduality of USH1 protein function for hair cell development andmechanotransduction provides a plausible explanation for thedifferent disease phenotypes that are associated with distinctmutations in USH1 genes (http://webh01.ua.ac.be/hhh/).Based on our findings, we predict that some missense muta-tions in USH1 genes, which cause nonsyndromic deafness,affect the mechanotransduction machinery of hair cells with-out effects on hair bundle development. Null mutations in-stead affect hair cell morphogenesis. It has been proposed thatUSH1 genes also participate in photoreceptor morphogenesis(46), a process that is likely affected by USH1 null alleles butnot by missense mutations that affect mechanotransduction.Strikingly, tip links in vestibular hair cells and vestibularfunction are maintained in salsa mice and DFNB12 patients,which may be a consequence of the different mechanicalproperties of these low-frequency mechanoreceptors. Consis-tent with this model, loss of tip links in salsa mice was firstobserved in the basal part of the cochlea, which contains haircells that respond to the highest frequencies.

Materials and MethodsAn extended section is provided as SI Materials and Methods.

ENU Mutagenesis, Functional Studies, Positional Cloning, Histology, and Bio-chemistry. ENU mutagenesis, analysis of ABRs and distortion product oto-acoustic emissions, test of vestibular function, staining of sections and wholemounts, electron microscopy, positional cloning, the expression and purifica-tion of recombinant CDH23 and PCDH15, and protein interaction studies havebeen described previously (16, 35, 47, 48). Tip tenting was quantified bycounting tented stereocilia in the medium row in inner hair cell bundles (in2–6 hair cells per time point and genotype). For quantification of tip staining,the number of CDH23 fluorescent puncta in bundles was determined (�27hair cells per time point and genotype).

Molecular Modeling. Using Modeler version 9v4 (49), cadherin repeat 7 and 8of CDH23 were threaded onto cadherin repeat 1 and 2 of CDH1 using theautomodel class and the sequence alignment produced by T-Coffee (50). The

A C E F

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Fig. 5. Progressive loss of tenting at stereociliary tips. (A and B) Scanning electron microscopy analysis in P5 wild-type and salsa mice revealed tip tenting instereocilia of cochlear hair cells (arrowheads). (C and D) Defects in tenting in salsa mice at P60 (arrowheads in C indicate tenting in wild-type). (E–G) Tips investibular hair cells display normal tenting. Tip links (arrowhead in G) were preserved. (H) Quantification of tip tenting at P5 and P21. Tenting was reduced inhair cells from the medial and basal cochlear turns at P21. *, P � 0.05. [Scale bars: A–D (300 nm), E and F (500 nm), and G (150 nm).]

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model was energy-minimized to remove bad contacts. The minimized,threaded coordinates were used to mutate Glu-66 to Val to produce a mutantmodel. Minimization consisted of 20 cycles of conjugate gradient minimiza-tion followed by 50 cycles of molecular dynamics optimization using the Verletalgorithm and finished with 20 cycles of conjugate gradient minimization.

Mechanotransduction Currents. Outer hair cells in the apical/middle turn wererecorded. Cells were whole-cell-patched, and hair bundles were deflectedwith a stiff glass probe.

ACKNOWLEDGMENTS. We thank T. Ricci for advice with electrophysiologyand K. Spencer for help with microscopy. This work was funded by NationalInstitutes of Health Grants DC005969 and DC007704 (to U.M.), the SkaggsInstitute for Chemical Biology (U.M.), and the Bruce Ford and Anne SmithBundy Foundation (W.X.).

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Fig. 6. Mechanotransduction currents. Data from wild type are representedin blue and from salsa in red. (A) Examples of transduction currents in cochlearouter hair cells at P7 in response to 5-ms mechanical stimulation. (B) Current-displacement [I(X)] relationships revealed no obvious difference between wildtype and salsa. (C) Examples of transduction currents in cochlear outer haircells at P7 in response to 100-ms mechanical stimulation. (D) Averaged trans-duction currents for deflections between 100 and 800 nm expressed as apercentage of peak currents at 800 nm. Current amplitude was lower inhomozygous salsa mice between 300-nm and 800-nm deflection. Data areshown as mean � SEM. Student’s 2-tailed unpaired t test was performed (*,P � 0.05; **, P � 0.01).

Fig. 7. salsa and DFNB12 mutations affect interactions between CDH23 andPCDH15. (A) Diagram of CDH23 and PCDH15 constructs. The extracellulardomains were fused to a His or Fc tag. (B) CDH23Fc and the mutant derivativeswere incubated with PCDH15-His in the presence of 1 mM EDTA or in thepresence of 10 �M and 100 �M Ca2�. Protein complexes were purified andanalyzed by Western blotting. Complex formation was observed in the pres-ence but not absence of Ca2�. The salsa and DFNB12 mutations weakenedinteractions between CDH23 and PCDH15. (Right) Controls for input amountsof CDH23Fc and mutant derivatives.

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