1607RESEARCH ARTICLE

INTRODUCTIONHair cells of the inner ear are mechanosensors for the perceptionof sound, head movement and gravity. The mechanicallysensitive organelle of a hair cell is the hair bundle, whichconsists of rows of stereocilia of graded heights polarized acrossthe apical cell surface. Bundle polarity is essential for normalhair cell function, as only deflections of the bundle in thedirection of the tallest stereocilia increase the open probabilityof mechanotransduction channels (Shotwell et al., 1981). Hairbundle polarity is established through a complex series ofmorphogenetic events. At the onset of hair bundle development,microvilli cover the apical hair cell surface and surround acentrally positioned kinocilium. The kinocilium moves to theedge of the apical hair cell surface. Subsequently, the microvillinext to the kinocilium elongate. This is followed by elongationof the more distant rows, generating a hair bundle with astaircase pattern that subsequently matures to attain its finalshape (Tilney et al., 1992; Schwander et al., 2010).

The kinocilium is important for establishing hair bundlepolarity as bundles are misoriented in mice carrying mutation inorthologs of genes linked to the ciliopathy Bardet-Biedlsyndrome (Ross et al., 2005), and in mice with a conditional

mutation in the gene encoding the intraflagellar and intraciliarytransport protein Ift88 (Jones et al., 2008). Hair bundlepolarization is also affected in mice with mutations in genes thatregulate planar cell polarity (PCP) (Curtin et al., 2003;Montcouquiol et al., 2003; Lu et al., 2004; Wang et al., 2005;Montcouquiol et al., 2006; Wang et al., 2006). However, little isknown about the molecules that connect PCP signaling to thekinocilium and bundle polarization. Candidate molecules areproteins that form the extracellular filaments connecting thestereocilia of a hair cell to each other and to the kinociliumbecause they are appropriately positioned to coordinate bundlepolarization across different rows of stereocilia and in relation tothe kinocilium (Fig. 1A). In the mouse cochlea, developing hairbundles contain ankle links, transient lateral links, top connectorsand tip links; mature cochlear hair bundles maintain topconnectors and tip links. Developing cochlear hair bundles alsocontain one kinocilium that is connected to the longeststereocilia by kinociliary links (Goodyear et al., 2005). Geneslinked to hearing loss encode several of the proteins formingthese linkages (Müller, 2008; Gillespie and Müller, 2009; Petitand Richardson, 2009). Relevant to the current study areprotocadherin 15 (PCDH15) and cadherin 23 (CDH23), whichare components of kinociliary links, transient lateral links and tiplinks (Siemens et al., 2004; Lagziel et al., 2005; Michel et al.,2005; Rzadzinska et al., 2005; Ahmed et al., 2006; Senften et al.,2006; Kazmierczak et al., 2007). Tip links are heterophilicadhesion complexes consisting of PCDH15 homodimersinteracting with CDH23 homodimers (Kazmierczak et al., 2007)(Fig. 1A). Kinociliary links show a similar asymmetry(Goodyear et al., 2010) (Fig. 1A). Functional null mutations inPCDH15 and CDH23 lead to defects in hair bundle structure andpolarity, indicating the importance of linkages containing theseproteins for bundle morphogenesis (Alagramam et al., 2001a; Di

Development 138, 1607-1617 (2011) doi:10.1242/dev.060061© 2011. Published by The Company of Biologists Ltd

1Dorris Neuroscience Center and Department of Cell Biology, The Scripps ResearchInstitute, La Jolla, CA 92037, USA. 2Laboratory of Cell Structure and Dynamics,National Institute of Deafness and other Communication Disorders, NationalInstitute of Health, Bethesda, MD 20892, USA. 3Department ofOtolaryngology–Head and Neck Surgery and Biomedical Engineering, Johns HopkinsSchool of Medicine, Baltimore, MD 21287, USA.

*Authors for correspondence (swwebb@scripps.edu; kacharb@nidce.nih.gov;umueller@scripps.edu)

Accepted 24 January 2011

SUMMARYProtocadherin 15 (PCDH15) is expressed in hair cells of the inner ear and in photoreceptors of the retina. Mutations in PCDH15cause Usher Syndrome (deaf-blindness) and recessive deafness. In developing hair cells, PCDH15 localizes to extracellular linkagesthat connect the stereocilia and kinocilium into a bundle and regulate its morphogenesis. In mature hair cells, PCDH15 is acomponent of tip links, which gate mechanotransduction channels. PCDH15 is expressed in several isoforms differing in theircytoplasmic domains, suggesting that alternative splicing regulates PCDH15 function in hair cells. To test this model, wegenerated three mouse lines, each of which lacks one out of three prominent PCDH15 isoforms (CD1, CD2 and CD3). Surprisingly,mice lacking PCDH15-CD1 and PCDH15-CD3 form normal hair bundles and tip links and maintain hearing function. Tip links arealso present in mice lacking PCDH15-CD2. However, PCDH15-CD2-deficient mice are deaf, lack kinociliary links and haveabnormally polarized hair bundles. Planar cell polarity (PCP) proteins are distributed normally in the sensory epithelia of themutants, suggesting that PCDH15-CD2 acts downstream of PCP components to control polarity. Despite the absence of kinociliarylinks, vestibular function is surprisingly intact in the PCDH15-CD2 mutants. Our findings reveal an essential role for PCDH15-CD2in the formation of kinociliary links and hair bundle polarization, and show that several PCDH15 isoforms can functionredundantly at tip links.

KEY WORDS: Hair cells, PCDH15, Deafness, Mouse

Regulation of PCDH15 function in mechanosensory hair cellsby alternative splicing of the cytoplasmic domainStuart W. Webb1,*, Nicolas Grillet1, Leonardo R. Andrade2, Wei Xiong1, Lani Swarthout3,Charley C. Della Santina3, Bechara Kachar2,* and Ulrich Müller1,*

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Palma et al., 2001; Wilson et al., 2001; Pawlowski et al., 2006;Senften et al., 2006; Lefevre et al., 2008). Tip links are thoughtto gate mechanotransduction channels in hair cells (Gillespie andMüller, 2009). However, the specific aspects of bundledevelopment regulated by kinociliary links, transient lateral linksand tip links are not known.

One way a single gene could contribute to the formation offunctionally distinct linkages in hair bundles is by alternativesplicing of the primary transcript. Hair cells express threeprominent PCDH15 splice variants (PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3) differing in their cytoplasmic domains(Ahmed et al., 2001; Alagramam et al., 2001a; Alagramam et al.,2001b; Ahmed et al., 2006; Senften et al., 2006). According topublished data, PCDH15-CD1 and PCDH15-CD3 are abundantlydetectable in hair bundles as they mature, whereas PCDH15-CD2 is abundant only in developing bundles (Ahmed et al.,2006). It has been hypothesized that PCDH15-CD1 andPCDH15-CD2 regulate hair bundle development, whilePCDH15-CD3 has been proposed to be a tip-link component

(Ahmed et al., 2006). To test this model and to define thefunction of PCDH15 isoforms in hair cells, we generated andcharacterized isoform-specific knockout mice.

MATERIALS AND METHODSGenetically modified miceThe strategy for generating knockout mice (see Fig. S1 in thesupplementary material) followed published procedures (Grillet et al.,2009b). Homology arms were amplified from genomic DNA by PCR(Phusion High-Fidelity DNA Polymerase, New England Biolabs). Exons35, 38 and 39 were replaced with a pGK-neomycin cassette in PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 targeting constructs,respectively. The linearized targeting vectors were electroporated into129P2/OlaHsd embryonic stem cells. Targeted clones were used togenerate germline-transmitting chimera and crossed to FLP deleter mice toremove the selection cassette. Mice were maintained on a mixedC57BL6�129SvEv background (for genotyping primers, see Table S1 inthe supplementary material).

AntibodiesThe PCDH15-CD2 rabbit antiserum was raised by Covance (Denver, PA)against a peptide specific for exon 38 (CSEGEKARKNIVLARRRP).Antibodies were affinity purified against the peptide coupled to agarosebeads. Other antibodies used were anti-acetylated-a-tubulin (mouse,Sigma, 6-11B-1), pericentrin (rabbit, Covance), Vangl2 (rabbit, Santa Cruz,H-55) and Fzd6 (mouse, R&D Systems). Additional reagents were AlexaFluor 488- and 568-phalloidin, Alexa Fluor 488 and 594 goat anti-rabbit,and Alexa Fluor 647 goat anti-mouse.

RT-PCR and qPCRRNA was isolated using Trizol (Invitrogen, Carlsbad, CA). cDNA wassynthesized from 500 ng of RNA with Superscript III reverse transcriptase(Invitrogen) and oligo(dT) primers. RT-PCR analysis and qPCR wasperformed as described (Belvindrah et al., 2007) (primers are listed inTable S1 in the supplementary material).

Histology, immunohistochemistry and electron microscopyWhole-mount staining was carried out as described previously (Grillet etal., 2009b). For scanning electron microscopy (SEM), inner ears weredissected and fixed by local perfusion with 2.5% glutaraldehyde, 4%formaldehyde, 50 mM HEPES buffer (pH 7.2), 2 mM CaCl2, 1 mM MgCl2and 140 mM NaCl for 2 hours at room temperature. Inner ear sensoryorgans were fine dissected and processed by a modified OTOTO method(Waguespack et al., 2007). To increase structural stability and imagecontrast, we substituted thiocarbohydrazide by 1% tannic acid, resulting inalternate baths of 1% osmium tetroxide and 1% tannic acid for 1 hour each.Samples were dehydrated, critical-point dried (Bal-Tec CPD 030), coatedwith a 4 nm platinum layer (Balzers BAF 300) and observed in a SEMHitachi S-4800, operated at 5 kV. Transmission electron microscopy wascarried out as described previously (Grillet et al., 2009a).

In situ hybridizationPCDH15-CD1, PCDH15-CD2 and PCDH15-CD3 were amplified frommurine cochlear mRNA and cloned into pcDNA-SK+ (Invitrogen). In situprobes were generated towards a common sequence in the 5� region andtoward unique regions in each of the isoforms and used for in situhybridization as described (Schwander et al., 2007; Grillet et al., 2009a)(primers used to generate probes listed in Table S1 in the supplementarymaterial). The 5� probe was generating by cloning a PstI fragment ofPCDH15 (bp 1456-2196 of mRNA, Accession Number NM_023115) intopBS-SK+.

ABR and DPOAE measurement and mechanotransduction currentsABR and DPOAE measurements were carried out as described previously(Schwander et al., 2007; Grillet et al., 2009b). Mechanotransductioncurrents from cochlear outer hair cells (OHCs) were recorded as describedpreviously (Grillet et al., 2009b).

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Fig. 1. Generation of PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice. (A)Extracellular linkages in developing and mature murinehair cells. The asymmetric distribution of PCDH15 and CDH23 at tiplinks and kinociliary links is indicated. (B)Arrangement of knownPCDH15 protein domains: 11 extracellular cadherin repeats (EC),transmembrane spanning region (TM), poly-proline repeats (PP) andunique C-terminal PDZ-binding interfaces. (C)Arrangement of Pcdh15exons encoding the cytoplasmic domain in wild-type (wt), PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 alleles. Exons are numbered.Deleted exons are shown as triangles. (D)Gel images show examples ofgenotyping by PCR from genomic DNA (sizes are indicated in Table S1in the supplementary material).

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Vestibular functionWe measured eye movements in response to whole-body head rotations inalert mice. A head bolt was placed for animal restraint. The post wasoriented so that when the animal was placed into a restraining device atopa servo-controlled rotating table, the plane tangent to the flat part of thedorsal skull was pitched 30° ‘nose-down’ from Earth horizontal. The axesof the animal’s horizontal semicircular canals aligned to within 10° of theEarth-vertical axis (Calabrese and Hullar, 2006).

The video-oculography technique was used for eye movement recording(Migliaccio et al., 2005). Marker arrays fashioned from photo paper saturatedwith fluorescent yellow ink were opaque except for three fluorescent200�200 m windows separated by 200 m and arranged in a 45° righttriangle. Images were acquired at a rate of 180 frames/second using IEEE1394(‘Firewire’) cameras [500�400 pixel frame size; Dragonfly Express, PointGrey Research, Canada; resolution <0.3°; camera/lens resolution (263pixels/mm) at the eye surface aligned with the center of corneal curvature towithin 72 m (Migliaccio et al., 2005)]. Data were interpolated on a 1 kHztime base using a nonlinear filter based on a running spline (LabVIEW). Weadjusted the spline smoothness parameter so that the correlation coefficientR2 between the raw and spline-filtered data was greater than 0.80.

For rotational testing, the center of the animal’s skull was aligned with themotor’s Earth-vertical axis. Transient yaw whole body rotation stimuli weredelivered at 3000°/s2 constant acceleration for 100 ms to a peak/plateauvelocity of 300°/s lasting 0.8-1.0 s, followed by a 3000°/s2 deceleration for100 ms to rest at 90° from the starting position. Eye rotation data wereconverted to rotation vectors in head coordinates and analyzed as describedpreviously (Migliaccio and Todd, 1999; Migliaccio et al., 2004; Migliaccioet al., 2005). Yaw (horizontal) component eye velocity data were invertedprior to gain calculation. Quick phases and saccades were removed. The startand end of quick phases/saccades were defined as the points at which eyeacceleration rose above or fell below manually estimated maximum slowphase eye acceleration. Data from at least 10 stimulus trials were averagedto obtain data for determining response parameters. For each trial, responselatency was computed as the time difference between the zero-velocity-intercept times for lines fit in a least-mean-square sense to eye and headvelocity during the constant-acceleration stimulus portion.

For comparison to other species (Migliaccio et al., 2004), weparameterized the responses by computing the ratio of eye and headacceleration during the constant-head-acceleration segment (Ga) and the

ratio of eye and head velocity during the velocity plateau (Gv) when aplateau was evident. During calculation of Ga and Gv, the horizontalcomponent of eye and head velocity was used. Results are mean±s.d.; eachdata point represents the mean for one animal over at least 10 stimulustrials. Ga, Gv and latency data were analyzed using a one-way analysis ofvariance (ANOVA) with significance set at P0.05 (Diggle et al., 1994).

RESULTSGeneration of PCDH15-DCD1, PCDH15-DCD2 andPCDH15-DCD3 miceTo define the function of PCDH15 isoforms in hair cells, wegenerated mice lacking specific isoforms (Fig. 1C; see Fig. S1 inthe supplementary material). The three best-characterized PCDH15isoforms in hair cells are PCDH15-CD1, PCDH15-CD2 andPCDH15-CD3, which differ in their cytoplasmic domains (Ahmedet al., 2006). Exon 35 is specific for CD1, exon 38 for CD2 andexon 39 for CD3. We replaced exons 35, 38 and 39 in ES cells bygene targeting with a neomycin cassette flanked by Frt sites (Fig.1C; see Fig. S1 in the supplementary material). Geneticallymodified mice were generated and the neomycin cassette wasremoved by crossing the mice to a mouse line expressing FLP(Rodriguez et al., 2000). Heterozygous mice were intercrossed togenerate mice homozygous for the deletion of exon 35, 38 and 39.Mice were genotyped by PCR (Fig. 1D), and will be referred to asPCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3.

To confirm that the genetic modifications selectively affect theexpression of specific PCDH15 isoforms, we analyzed RNA of P1mice by PCR (see Fig. S1D,E in the supplementary material). Theidentity of the amplified DNA fragments was confirmed by DNAsequencing (data not shown). PCDH15-CD1, PCDH15-CD2 andPCDH15-CD3 were detectable in wild-type mice. PCDH15-DCD1mice lacked PCDH15-CD1 expression, whereas PCDH15-CD2and PCDH15-CD3 were maintained. PCDH15-DCD2 lackedPCDH15-CD2 expression but not PCDH15-CD1 or PCDH15-CD3; PCDH15-DCD3 mice lacked PCDH15-CD3 expression butnot PCDH15-CD1 or PCDH15-CD2.

1609RESEARCH ARTICLEPCDH15 function and hair cells

Fig. 2. Analysis of Pcdh15 isoformexpression by in situ hybridization.(A)Examination of PCDH15 isoformexpression in wild-type mice at P1. Thediagrams summarize the data. A 5� probe (5P)detected PCDH15 expression in IHCs andOHCs (arrows), and in Kölliker’s organ; lowerexpression levels were evident in support cells.Probes specific for PCDH15-CD1 andPCDH15-CD3 revealed expression in hair cells,support cells and Kölliker’s organ. PCDH15-CD2 appeared to be restricted to IHCs andOHCs. (B)Examination of PCDH15 isoformexpression in mutants at P1. As expected,only expression of the PCDH15 isoformtargeted by the mutation was abolished ineach genetically modified mouse line;expression of the remaining splice variantswas maintained. In the PCDH15-DCD3 mouseline, ectopic PCDH15-CD2 expression wasdetected in support cells and Kölliker’s organ.Arrows point to hair cells. Scale bars: 50m.

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Next, we carried out in situ hybridization on cochlear sectionsusing probes specific for PCDH15-CD1, PCDH15-CD2 andPCDH15-CD3. We also included a probe to the 5� end of PCDH15mRNA common to all three isoforms, and sense control probes(Fig. 2; see Fig. S2 in the supplementary material). Previous studieshave shown that PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3are expressed in hair cells (Ahmed et al., 2006). We confirmedthese findings and extended them. The probe to the 5� region ofPCDH15 revealed that in wild-type mice, PCDH15 was expressednot only in mechanosensory hair cells but also in Kölliker’s organ(Fig. 2A). PCDH15-CD1 and PCDH15-CD3 were expressed inhair cells and Kölliker’s organ, whereas PCDH15-CD2 appearedto be specific for hair cells (Fig. 2A). The analysis of PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice revealed that ineach mouse line, the two PCDH15 isoforms that were not mutatedwere still expressed (Fig. 2B). The expression pattern for theremaining isoforms appeared unaffected, with the exception ofPCDH15-CD2, which was ectopically expressed in Kölliker’sorgan of PCDH15-DCD3 mice (Fig. 2B). Quantitative RT-PCRshowed that expression of PCDH15-CD1 was not substantiallychanged in PCDH15-DCD2 and PCDH15-DCD3 mice. Likewise,PCDH15-CD3 expression was relatively normal in PCDH15-DCD1 and PCDH15-DCD3 mice. However, PCDH15-CD2 wasupregulated in PCDH15- DCD3 and downregulated in PCDH15-DCD1 mice (see Fig. S1E in the supplementary material).

Analysis of auditory functionPCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 micesurvived into adulthood. To measure auditory function, werecorded in 1-month- and 6-month-old mice the auditory brainstem (ABR) response to click-stimuli. Auditory thresholds werenormal in PCDH15-DCD1 and PCDH15-DCD3 mice, but theywere elevated to above 90 dB in PCDH15-DCD2 mice (Fig. 3A,B).Analysis of hearing function in response to pure tones confirmedthat hearing function in PCDH15-DCD2 mice was affected acrossthe entire analyzed frequency spectrum (Fig. 3C).

To study OHC function, we measured the distortion productotoacoustic emissions (DPOAEs). In wild-type, PCDH15-DCD1and PCDH15-DCD3 mice, DPOAEs were dependent on thestimulus intensity at a given frequency, with no obvious differencebetween wild-type and mutants (Fig. 3D). By contrast, DPOAEswere not detectable in PCDH15-DCD2 mice (Fig. 3D). Similarobservations were made at all frequencies analyzed (6-28 kHz)(Fig. 3D), indicating that in PCDH15-DCD2 the function of OHCswas impaired.

Defects in hair bundle polarity in PCDH15-DCD2miceAmes waltzerav3J mice, which carry a predicted functional nullallele of PCDH15 that affects all isoforms, are deaf, circle andshow defects in hair bundle morphogenesis (Alagramam et al.,2001a; Pawlowski et al., 2006; Senften et al., 2006). We wonderedwhether the auditory phenotype of PCDH15-DCD2 mice wascaused by a similar mechanism and whether hair bundlemorphology was normal in PCDH15-DCD1 and PCDH15-DCD3mice. We stained P6 cochlear wholemounts with phalloidin to labelF-actin in stereocilia (Fig. 4A-D). The sensory epithelium of thethree mutant mouse lines was patterned into one row of inner haircells (IHCs) and three rows of OHCs. Although hair bundlemorphology appeared normal in PCDH15-DCD1 and PCDH15-DCD3 mice, hair bundles of OHCs were affected in PCDH15-DCD2 mice, showing defects in polarization (Fig. 4C,F,I,J). This

observation was confirmed by SEM (see Fig. S3A in thesupplementary material). Unlike in Ames waltzerav3J mice, hairbundles were with few exceptions not fragmented (Fig. 4E-J; seeFig. S3A,D and Fig. S4A-C in the supplementary material).

To quantify polarity defects, we determined the deviation of theposition of the bundle center, which contains the longest stereocilia,from their normal position at the tip of the V-shaped bundle (Fig.4K-M). Four percent of the mutant bundles formed circles andwere excluded from the quantification. IHCs and OHCs in wild-type mice at P1 and P6 showed little variation in orientation (Fig.4L,M), but bundle orientation of OHCs was highly variable inPCDH15-DCD2 mice, deviating up to 90° from the normalposition, sometimes more (Fig. 4L,M). No such defect was seen inIHCs (Fig. 4L,M).

Expression of PCDH15-CD2 in hair cellsTo define the mechanism that caused the hair bundle defect inPCDH15-DCD2 mice, we raised antibodies against PCDH15-CD2and analyzed its expression in hair cells at P1. As reported (Ahmedet al., 2006), PCDH15-CD2 was expressed throughout hair bundlesof OHCs; we also observed expression in the short microvillipresent at the hair cell surface at P1 (Fig. 5A,B,E-H). AlthoughOHCs were strongly positive for PCDH15-CD2, it was expressedat low levels in IHCs (Fig. 5A,B). All staining was abolished inPCDH15-DCD2 mice (Fig. 5C,D), attesting to the specificity of theantibody. Co-staining with antibodies to acetylated-a-tubulin, toreveal the kinocilium, confirmed expression of PCDH15-CD2 inproximity to the kinocilium of OHCs (Fig. 5E-H, arrows). In somehair bundles, staining was concentrated at kinociliary tips (Fig.5G,H, arrows). In vestibular hair cells, PCDH15-CD2 staining was

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Fig. 3. Analysis of auditory function. (A)Auditory thresholds ofPCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice at 1 month ofage compared with wild-type littermates (wild type, n19; DCD1, n11;DCD2, n10; DCD3, n7; mean±s.d.) (B) Auditory thresholds at 6months of age (wild type, n10; DCD1, n3; DCD2, n3; DCD3, n3;mean±s.d.). (C)Auditory thresholds (mean±s.d.) in 1-month-old mice asdetermined by pure tone ABR recordings. (D)DPOAE thresholds in 1-month-old PCDH15-DCD2 mutants were elevated at all frequenciesanalyzed (wild type, n13; DCD1, n7; DCD2, n4; DCD3, n6;mean±s.d.). Student’s t-test was performed on ABR data in A and B(***P<0.0001).

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strong throughout the bundles of immature hair cells and near theinterface between stereocilia and the kinocilium (Fig. 6). Weconclude that in OHCs, PCDH15-CD2 is targeted to the stereociliaand kinocilium, and is appropriately positioned to contribute to theformation of linkages between steoreocilia, and between thestereocilia and the kinocilium. A similar expression pattern isobserved in vestibular hair cells, but there is little expression inIHCs.

Defects in kinociliary links in PCDH15-DCD2 miceWe next analyzed hair bundles of cochlear hair cells at P1 and P6by staining with fluorescence labeled phalloidin to reveal actin, andantibodies to acetylated-a-tubulin to reveal the kinocilium. In wild-type mice, the kinocilium was tightly associated to the longeststereocilia at the bundle apex (Fig. 7A,A�,C,C�). By contrast, the

position of the kinocilium was more variable in PCDH15-DCD2mice (Fig. 7B,B�,D,D�), suggesting that kinociliary links might beaffected. This was confirmed by SEM analysis. Although kinociliain wild-type mice at P1 were connected to the stereociliary bundle(Fig. 7E-G), they were separated from the bundle of OHCs andIHCs of PCDH15-DCD2 mice (Fig. 7H-J; see Fig. S4 in thesupplementary material). A similar phenotype was observed at E15(Fig. 10).

Previous studies have suggested that several PCDH15 isoformsmight be present at transient lateral links (Ahmed et al., 2006).Unlike in Ames waltzerav3J mice, hair bundles of IHCs and OHCswere not fragmented in PCDH15-DCD1, PCDH15-DCD2 andPCDH15-DCD3 mice (Fig. 4A-D,F,I,J; Fig. 7B,B�,D,D� and seeFig. S4 in the supplementary material), suggesting that some ofthese isoforms have redundant functions at transient lateral links.This was confirmed by SEM analysis. In PCDH15-DCD2 mice, thekinocilium was separated from the stereocilia, but linkages alongthe length of stereocilia were visible (Fig. 7H-J; Fig. 8A-D). Suchlinkages were also observed in hair bundles from PCDH15-DCD1and PCDH15-DCD3 mice (see Fig. S5 in the supplementarymaterial).

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Fig. 4. Analysis of hair bundle morphology in the cochlea.(A-D)Cochlear wholemounts of the indicated genotypes at P6 werestained with phalloidin (green). Hair bundle morphology was affectedin PCDH15-DCD2 mice. (E,G,H) Hair bundles in Ames waltzerav3j miceat P6 were fragmented and misoriented. (F,I,J) PCDH15-DCD2 hairbundles maintained cohesion and staircase arrangement but polaritywas compromised. (K-M)Analysis of hair bundle polarity in OHCs andIHCs from wild-type and PCDH15-DCD2 mice at P1 and P6. Orientationwas determined by drawing a line through the axis of the bundle with0° indicating the normal mediolateral axis (K). Angular deviation fromthis axis was determined. PCDH15-DCD2 OHCs showed polarity defects(wild type, n599; DCD2, n849). Fifty percent of the OHCs examineddeviated by 15° or more. Scale bars: 10m in A-D; 5m in E,F; 2min G-J.

Fig. 5. PCDH15-CD2 expression in OHCs. (A-H)Cochlearwholemounts at P1 were stained using a PCDH15-CD2 antibody (red).(A,C,E,G) Stereocilia were labeled with phalloidin (green). (A-H)Kinociliawere stained using an acetylated-a-tubulin antibody (blue). (A-D)Inwild-type OHCs and IHCs (A,B), and wild-type OHCs (E-H), PCDH15-CD2 was present throughout the hair bundle. No staining wasobserved in PCDH15-DCD2 mice (C,D). (E-H)In wild-type mice,prominent staining was evident in the region proximal to the kinocilia-stereocilia interface (arrows in E,F). Staining in OHCs was oftenobserved as a puncta at the distal kinociliary tips (arrows in G,H). Scalebars: 4m in A-D; 2m in E-H.

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At P96, there was widespread degeneration of hair bundles inthe cochlea of PCDH15-DCD2 mice (see Fig. S3C in thesupplementary material), although some hair bundles weremaintained (see Fig. S3D in the supplementary material). Thedegenerative changes probably contribute to deafness of PCDH15-DCD2 mice. Stereocilia of OHCs that were maintained in themutants formed connections to the tectorial membrane, as evidentfrom their imprint in the tectorial membrane (see Fig. S3B in thesupplementary material).

Tip links and mechanotransductionPCDH15-DCD1 and PCDH15-DCD3 mice showed normalhearing, suggesting that tip links were unaffected. Deafness inPCDH15-DCD2 mice is probably caused by defects in hair bundlemorphology and maintenance, but tip-link function may also beimpaired. We therefore analyzed tip links by SEM in P7 mice.Linkages running from the top of stereocilia to the side of the nexttaller stereocilia were observed in PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice (Fig. 8C-G), indicating that tiplinks were preserved.

To determine whether hair cells from PCDH15-DCD2 mice hadfunctional transduction, we measured transducer currents in P7-P8OHCs from the apical/middle part of the cochlea using whole cell

recordings. We focused our analysis on hair bundles with minimalpolarity defects. In agreement with previous findings (Kennedy etal., 2003; Kros et al., 2002; Stauffer and Holt, 2007; Waguespacket al., 2007), control OHCs had rapidly activating transducercurrents, which subsequently adapted (Fig. 8H, blue traces); similarcurrent traces were obtained with OHCs from PCDH15-DCD2mice (Fig. 8H, red traces). The amplitudes of saturatedmechanotransduction currents at maximal deflection were similarin controls and mutants (Fig. 8H,I), suggesting that the totalnumber of transducer channels was not altered in mutants. We alsoplotted the current/displacement relationships and channel openprobability/displacement relationship and observed no significantdifference between wild-type and mutants (Fig. 8I,J), indicatingthat the transduction machinery of hair cells in PCDH15-DCD2mice functioned normally. Finally, as PCDH15-CD3 has previously

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Fig. 6. PCDH15-CD2 expression in vestibular hair cells.(A-L)Vestibular wholemounts at P4 stained using a PCDH15-CD2antibody (red). In immature wild-type vestibular hair cells (A-C�,G-L),PCDH15-CD2 was present throughout the hair bundle. No staining wasobserved in PCDH15-DCD2 mice (D-F). In wild-type mice, prominentstaining was evident in the region proximal to the kinocilia-stereociliainterface (arrows in G-I). Staining in immature hair cells was oftenobserved as a puncta at the distal end of the tallest stereocilia (arrowsin A-C,J-L). Scale bars: 10m in A-F; 5m in G-L.

Fig. 7. Kinocilia position in OHCs from PCDH15-DCD2 mice.(A-D�) Staining of cochlear wholemounts from P6 wild-type andPCDH15-DCD2 mice with phalloidin (green) and antibodies to acetylateda-tubulin (red). Kinocilia (arrows) in wild-type hair cells (A,A�,C,C�) werelinked to the stereociliary bundles; those of PCDH15-DCD2 hair cells(B,B�,D,D�) were separated from the bundle and misplaced. Cilia onsupport cells are marked with an asterisk. (E-J)SEM examination of wild-type and PCDH15-DCD2 OHCs at P1. (E-G)Wild-type hair cells showingtight association between the stereocilia bundle and kinocilium (arrowsin E,F; asterisk in G). (H-J)Kinocila (arrows in H,I; asterisk in J) in PCDH15-DCD2 mice were disconnected from the stereocilia. Scale bars: 5m inA-D; 5m in E,F,H; 2m in I; 0.5m in G,J.

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been proposed as a component of tip links (Ahmed et al., 2006),we also measured transducer currents in PCDH15-DCD3 mice butobserved no defects (see Fig. S6 in the supplementary material).

Defects in kinociliary positioning in PCDH15-DCD2miceThe kinocilium of hair cells is thought to be important for thedevelopment of hair bundle polarity. To determine the extent towhich kinocilia position and bundle rotation in PCDH15-DCD2mice correlates, we determined kinociliary position relative tostereociliary bundles (Fig. 9A). In IHCs and OHCs of wild-typemice, the position of the kinocilium predicts the orientation of thebundle (Fig. 9B,C). By contrast, kinocilia position did not predictbundle orientation in PCDH15-DCD2 mice and frequently deviated90° or more from the normal position (Fig. 9B,C).

In wild-type hair cells, the centrioles of the basal body undergoa similar polarized movement to the kinocilium (Jones et al., 2008).Consistent with these data, staining for pericentrin revealed thepolar localization of the centrioles in OHCs from wild-type miceat P1 (Fig. 9D-F�). In PCDH15-DCD2 mice, the two centrioles andkinocilium were coordinately mislocalized (Fig. 9G-I�) and the twocentrioles were no longer aligned appropriately relative to eachother along the axis of bundle polarity (Fig. 9G-I�). We concludethat kinociliary links are required to coordinate the polarization ofthe kinocilium and their basal bodies with the polarization of thestereociliary bundle.

Normal localization of components of the corePCP pathwayRandom polarization of hair bundles, similar to that observed inPCDH15-DCD2 mice, is a hallmark of mice with mutations inorthologs of genes linked to the Bardet-Biedl syndrome (Ross etal., 2005), and in mice with mutations that affect the intraciliarytransport protein Ift88 (Jones et al., 2008). The ciliary genes arethought to act downstream of components of the core PCPpathway. We therefore asked whether core PCP proteins showednormal asymmetric localization in PCDH15-DCD2 mice. Westained cochlear wholemounts of P1 mice with phalloidin and

antibodies to Fzd6 and Vangl2, which are normally localized atthe medial edge of OHCs (Fig. 9J-M�; data not shown). Theasymmetric distribution of Fzd6 (Fig. 9L-M�) and Vangl2 (data notshown) was not altered in PCDH15-DCD2 mice, suggesting thatPCDH15-CD2 acts downstream of the PCP pathway.

Analysis of vestibular hair cell morphology andfunctionIt is thought that the kinocilium of vestibular hair cells is requiredfor mechanical coupling between the hair bundle and the overlyinggelatinous matrix (Roberts et al., 1988). We therefore wonderedwhether kinociliary links and vestibular function were affected inPCDH15-DCD2 mice. Analysis at E15.5, P1 and P6 revealed thatalthough the kinocilium was tightly linked to hair bundles in wild-type mice (Fig. 10A-B�; data not shown), the kinocilium wasseparated from the stereociliary bundle in PCDH15-DCD2 mice inhair cells in the utricle, saccule and semicircular canals (Fig. 10C-D�; see Fig. S7 in the supplementary material; data not shown).Stereocilia were also in contact with the overlying gelatinousmatrix (see Fig. S7C-E in the supplementary material).

We next analyzed vestibular function. Unlike in Ames waltzerav3J

mice, which lack all three PCDH15 isoforms, PCDH15-DCD2mice (and PCDH15-DCD1 and PCDH15-DCD3 mice) did notcircle (data not shown). To evaluate vestibular functionquantitatively, we measured eye movements in alert mice mediatedby the vestibulo-ocular reflex in response to whole-body headrotations (Fig. 10E-G). Mice were immobilized in a superstructuremounted atop a motor able to deliver 3000°/s constant accelerationsto a plateau velocity of 300°/s about the dorsoventral axis of theanimal through the stereotaxic origin of the skull. Eye movementswere measured using a three-dimensional video-oculographysystem. We quantified responses to whole-body, passive, transientrotations in darkness using three parameters: Ga (‘constant-acceleration gain’) was computed as the ratio of eye to headvelocity averaged over the constant acceleration segment of thestimulus; Gv (‘constant-velocity gain’) was computed as the ratioof eye to head velocity averaged over the constant velocity segmentof the stimulus; latency is the delay from the onset of head

1613RESEARCH ARTICLEPCDH15 function and hair cells

Fig. 8. Linkages in hair bundles andmechanotransduction currents. (A-D)Hairbundles from PCDH15-DCD2 mice at P1 (A,B) andP6 (C,D) were examined by SEM for linkages(arrows). (D)Tip links (arrows) in cochlear hair cells.Asterisk in A indicates the kinocilium.(E-G)Vestibular hair cells of PCDH15-DCD2,PCDH15-DCD2 and PCDH15-DCD3 mice at P6,revealing tip links (arrows).(H-J)Mechanotransduction currents in P7 OHCsfrom PCDH15-DCD2 mice. (H)Examples oftransduction currents in respond to mechanicalstimulation. (I)Current-displacement [I(X)]relationships were plotted and fitted with a second-order Boltzman function. (J)The relationshipbetween open probability (Popen) obtained withpeak currents following deflection revealed nosignificant difference between wild type andmutants. Data are mean±s.e.m. Scale bars: 1.5min A; 0.2m in B; 0.5m in C; 150 nm in D-G.

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movement to the onset of eye movement, computed through linearextrapolation of eye and head responses during the constant-acceleration segment of the stimulus. Measurements were carriedout with PCDH15-DCD2 mice (n6), C57BL6�129 wild-typelittermate controls (n8), C57BL/6 wild-type controls (n7), Ameswaltzerav3J mice (n3) and one dead mouse (to rule out technicalartifacts). Ames waltzerav3J mice and dead mice did not show anyresponse. However, there was no statistically significant differencein any of the parameter between PCDH15-DCD2 mice and wild-type mice (Fig. 9E-G). In addition, we analyzed the response tohead pitch tilts about the intreraural axis and observed normalresponses in the mutants (data not shown). These data show thatvestibular function was surprisingly intact in PCDH15-DCD2 mice.

DISCUSSIONHere, we show that alternative splicing of PCDH15 regulates itsfunction in hair cells. Using isoform-specific knockout mice, weshow that PCDH15-CD1 and PCDH15-CD3 are not essential for

hair cell function, whereas PCDH15-CD2 is required for theformation of kinociliary links. In the absence of PCDH15-CD2, hairbundles consisting of rows of stereocilia of graded heights developbut bundle polarity is affected, demonstrating that kinociliary links

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Fig. 9. Position of kinocilia and basal bodies in PCDH15-DCD2mice. (A-C)Examination of the relationship between kinocilia positionand bundle rotation in OHCs. In wild-type mice, there was a correlationbetween the kinocilia position and the apex of a stereocilia chevron. InPCDH15-DCD2 bundles, the kinocilia were frequently mislocalized (wildtype, n203; DCD2, n106). Of the OHCs examined, 48.2% hadkinocilia mislocalized by 15° or more. (D-I�) Cochlear wholemountswere stained with phalloidin (green), antibodies to pericentrin (red) andantibodies to acetylated a-tubulin (blue). In wild type but not in themutants, the basal bodies (arrows) aligned with the kinocilium alongthe hair bundle polarity axis. (J-M�) Cochlear wholemounts werestained with phalloidin (green) and antibodies to frizzled 6 (red).Frizzled 6 localization (arrows) was not affected in the mutants. Thepresumed position of the kinocilium is indicated by an asterisk (J�,L�).Scale bars: 5m.

Fig. 10. Analysis of vestibular hair cells from PCDH15-DCD2 mice.(A-B�) SEM of vestibular hair cells in wild type at E15.5 and P1 revealtight coupling of the kinocilia (arrows) to the stereocilia. (C-D�) Kinocilia(arrows) of PCDH15-DCD2 vestibular hair cells were not connected tothe stereocilia. Images of hair cells are from E15 crista (A,A�,C), E15utricle (C�) and P1 ampulla (B,B�,D,D�). (E-G)Vestibulo-ocular reflexresponse for PCDH15-DCD2, wild-type littermates (C57BL/6�129SvEv),C57BL/6 mice, Ames waltzerav3J mice and a dead mouse. (E)Eye versushead horizontal velocity ratio, Ga, during the constant acceleration(3000° s–2) segment of a passive transient whole-body rotation about avertical axis. (F)Velocity ratio, Gv, during the constant velocity (300° s–1)plateau of the same stimulus. (G)Response latency from onset of headmotion to onset of eye motion (mseconds). Diamonds and whiskersindicate mean±s.d.; each data point is an the mean response of ananimal; n≥10 head rotations in each direction. Excluding the Ameswaltzerav3J mice and dead controls (for which Ga�Gv�0), one-wayANOVA revealed no significant difference among groups in Ga, Gv orlatency (P>0.05). Including the Ames waltzerav3J mice, controls in theanalyses revealed that all other live mouse groups differed significantlyin Ga and latency from Ames waltzerav3J mice controls (P<0.001); Gv

showed a similar trend but did not reach significance. There was atrend towards PCDH15-DCD2 mutants having longer response latencythan control mice, but this trend did not reach significance (P0.062).These data suggest that PCDH15-DCD2 mice have horizontalsemicircular canal function similar to both of the wild-type controlgroups but significantly better than Ames waltzerav3J mice. Scale bars:5m in A-D; 2.5m in A�-D�.

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are not essential for the development for the stereociliary staircasebut for the morphological transformation that lead to bundlepolarization. Components of the PCP pathway are normallydistributed in the sensory epithelia of PCDH15-DCD2 mice,suggesting that PCDH15-CD2 acts downstream of the PCPpathway. Hair bundles in PCDH15-DCD2 mice are gradually lostpostnatally, indicating that the morphological defects affect hair cellfunction causing deafness. In addition, we cannot exclude thatcochlear hair cells do not function normally because their stereociliaare not connected to the kinocilium. Unlike in Ames waltzerav3J

mice, which carry a predicted PCDH15 null allele, stereociliarybundles are not fragmented in mice individually lacking PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3, suggesting that severalPCDH15 isoforms contribute to the formation of transient laterallinks. We also observed that tip links are present in PCDH15-DCD1,PCDH15-DCD2 and PCDH15-DCD3 mice, providing evidence thatnone of these isoforms is uniquely required at tip links.

One of the central findings of our study is that PCDH15-CD2 isrequired for the development of kinociliary links and the normalpolarization of hair bundles. Initial studies by the Tilney laboratorysuggested that the kinocilium is important for bundle polarization(Tilney et al., 1992), but the mechanism by which the kinociliumdetermines polarity had remained unclear. Recent studies haveshown that mutations in components of the core PCP pathway andmutations affecting the kinocilium cause polarity defects. WhenPCP signaling is defective, hair bundles maintain intrinsic polaritybut are randomly polarized in the apical cell surface. When thekinocilium is affected, bundles show random polarization withinthe apical surface and loss of intrinsic polarity (Curtin et al., 2003;Montcouquiol et al., 2003; Lu et al., 2004; Ross et al., 2005; Wanget al., 2005; Montcouquiol et al., 2006; Wang et al., 2006; Jones etal., 2008). The asymmetric localization of PCP proteins at theborder between hair and support cells is not affected in ciliarymutants (Jones et al., 2008), suggesting that ciliary genes actdownstream of PCP components (Jones and Chen, 2008). InPCDH15-DCD2 mice, the PCP components Frz6 and Vangl2 arenormally distributed, and hair bundle morphology resembles thatof the ciliary mutants, suggesting that PCDH15-CD2 behaves likea ciliary mutant acting downstream of the PCP pathway. In thePCDH15-DCD2 mutants, the kinocilium and stereocilia bundle aremislocalized, but not always in same direction, suggesting thatkinociliary links are required to coordinate movement of thekinocilium and stereocilia.

We have previously shown that tip links in hair cells have anintrinsic asymmetry in the planar polarity axis of hair cells, wherePCDH15 forms the lower end of tip links and CDH23 the upperend (Kazmierczak et al., 2007). CDH23 and PCDH15 are alsoasymmetrically distributed at kinociliary links (Goodyear et al.,2010). However, polarity is reversed relative to tip links, withPCDH15 being present in the kinocilium and CDH23 in the longeststereocilia (Fig. 1A). Our findings indicate that PCDH15-CD2 isthe PCDH15 isoform in kinocilia and suggest a mechanism bywhich it might regulate polarity. As the kinocilium, unlike thestereocilia, contains microtubules, it seems likely that the CD2cytoplasmic domain contains specific sequence motifs for targetingto the kinocilia that are absent in CD1 and CD3. In this way, apolarity axis is established in kinociliary links. The fact that somePCDH15-CD2 is present in stereocilia could be explained becauseactin-based molecular motors might also transport PCDH15-CD2.Alternatively, as PCDH15 molecules form dimers (Kazmierczak etal., 2007), PCDH15-CD2 might enter stereocilia as a heterodimerwith PCDH15-CD1 and/or PCDH15-CD3.

Our data show that several PCDH15 isoforms have redundantfunction in hair cells. The hair bundles of Ames waltzerav3J mice,which lack all PCDH15 isoforms, show bundle fragmentation(Alagramam et al., 2001a; Pawlowski et al., 2006; Senften et al.,2006). As stereociliary bundles are not significantly fragmented inPCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice, itseems that several of these isoform contribute to transient laterallinks. Consistent with this finding, we observed by SEM abundantlinks between the stereocilia in all three mutants. Similarly, it seemsthat neither PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3are uniquely required for tip-link formation. Previousimmunolocalization studies have failed to clearly define thePCDH15 isoform at tip links. PCDH15-CD3 has been the bestcandidate, as one CD3-specific antibody bound to the region of thelower tip link end. However, the same antibody did not bind toadult murine hair cells and a second PCDH15-CD3 antibody boundto stereocilia more broadly (Ahmed et al., 2006). Using antibodiesagainst PCDH15 isoforms, we have also been unable to define thePCDH15 isoform at tip links, as different antibodies to the sameisoform revealed distinct expression patterns. This variation mightbe caused by differences in antibody affinity or epitope masking.Nevertheless, our genetic studies show that PCDH15-CD1,PCDH15-CD2 and PCDH15-CD3 individually are not essential fortip-link formation. Extracellular filaments with tip link features arepresent in PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3mice. In addition, auditory and vestibular function is preserved inPCDH15-DCD1 and PCDH15-DCD3 mice, and vestibular functionin PCDH15-DCD2 mice. OHCs in PCDH15-DCD2 and PCDH15-DCD3 mice also show normal responses to mechanical stimulationin vitro. An important implication of our findings is that themechanotransduction channel in hair cells, which is localized inproximity to the lower tip-link end (Beurg et al., 2009) might bindto the extracellular and/or transmembrane domain of PCDH15 thatis shared by several PCDH15 isoforms.

We were surprised to find that vestibular function was notnoticeably altered in PCDH15-DCD2 mice. The kinocilium ofvestibular hair cells is coupled to the kinocilium and the overlyinggelatinous extracellular matrices. Unlike in the cochlea, adultvestibular hair cells do not loose their kinocilium. It has thereforebeen assumed that the kinocilium mechanically couples the hairbundle to the overlying gelatinous matrix (Roberts et al., 1988).Our findings now suggest that murine vestibular hair cells functionnormally, even when linkages between the kinocilium andstereocilia are disrupted. Direct coupling of the stereocilia to thegelatinous matrices is probably sufficient to transmit motion tostereocilia and activate transduction channels.

Our functional data were collected with mice of a mixedC57BL6�129SvEv background. Although we did not observedifferences in PCDH15 isoform expression between inbred mousestrains, we cannot exclude the possibility that different phenotypesmight be observed on distinct genetic backgrounds.

Our findings could have important ramifications for the analysisof the genetic causes of sensory impairment. Several PCDH15mutations have been identified that cause Usher Syndrome andrecessive forms of deafness (Ahmed et al., 2001; Alagramam et al.,2001b; Ben-Yosef et al., 2003; Ouyang et al., 2005; Roux et al.,2006; Le Guedard et al., 2007). These mutations commonly map tothe extracellular PCDH15 domain. Our findings suggest that it wouldbe important to determine whether mutations that specifically affectthe PCDH15-CD2 isoform might lead to auditory impairment. Last,mutations specific for PCDH15-CD1 and PCDH15-CD3 might leadto retinal disease without auditory dysfunction.

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AcknowledgementsThis work was funded by NIDCD grants DC007704, DC005965, DC005965-S1(U.M.), DC9255, DC2390 (C.D.S.) and NIDCD-DIR-Z01DC000002 (B.K.); by theSkaggs Institute for Chemical Biology (U.M.); by the Dorris NeuroscienceCenter (U.M.); by a Ruth Kirschstein Predoctoral Fellowship award (S.W.W.);and by a CIRM training grant (S.W.W.). Deposited in PMC for release after 12months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.060061/-/DC1

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1617RESEARCH ARTICLEPCDH15 function and hair cells

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