RESEARCH ARTICLE 1607

Development 138, 1607-1617 (2011) doi:10.1242/dev.060061 © 2011. Published by The Company of Biologists Ltd Regulation of PCDH15 function in mechanosensory hair cells by alternative splicing of the cytoplasmic domain Stuart W. Webb1,*, Nicolas Grillet1, Leonardo R. Andrade2, Wei Xiong1, Lani Swarthout3, Charley C. Della Santina3, Bechara Kachar2,* and Ulrich Müller1,*

SUMMARY Protocadherin 15 (PCDH15) is expressed in hair cells of the inner and in photoreceptors of the retina. Mutations in PCDH15 cause (deaf-blindness) and recessive deafness. In developing hair cells, PCDH15 localizes to extracellular linkages that connect the and into a bundle and regulate its morphogenesis. In mature hair cells, PCDH15 is a component of tip links, which gate channels. PCDH15 is expressed in several isoforms differing in their cytoplasmic domains, suggesting that alternative splicing regulates PCDH15 function in hair cells. To test this model, we generated 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 function. Tip links are also present in mice lacking PCDH15-CD2. However, PCDH15-CD2-deficient mice are deaf, lack kinociliary links and have abnormally polarized hair bundles. Planar cell polarity (PCP) are distributed normally in the sensory epithelia of the mutants, suggesting that PCDH15-CD2 acts downstream of PCP components to control polarity. Despite the absence of kinociliary links, vestibular function is surprisingly intact in the PCDH15-CD2 mutants. Our findings reveal an essential role for PCDH15-CD2 in the formation of kinociliary links and hair bundle polarization, and show that several PCDH15 isoforms can function redundantly at tip links.

KEY WORDS: Hair cells, PCDH15, Deafness, Mouse

INTRODUCTION mutation in the encoding the intraflagellar and intraciliary Hair cells of the are mechanosensors for the perception transport Ift88 (Jones et al., 2008). Hair bundle of sound, head movement and gravity. The mechanically polarization is also affected in mice with mutations in that sensitive organelle of a is the hair bundle, which regulate planar cell polarity (PCP) (Curtin et al., 2003; consists of rows of stereocilia of graded heights polarized across Montcouquiol et al., 2003; Lu et al., 2004; Wang et al., 2005; the apical cell surface. Bundle polarity is essential for normal Montcouquiol et al., 2006; Wang et al., 2006). However, little is hair cell function, as only deflections of the bundle in the known about the molecules that connect PCP signaling to the direction of the tallest stereocilia increase the open probability kinocilium and bundle polarization. Candidate molecules are of mechanotransduction channels (Shotwell et al., 1981). Hair proteins that form the extracellular filaments connecting the bundle polarity is established through a complex series of stereocilia of a hair cell to each other and to the kinocilium morphogenetic events. At the onset of hair bundle development, because they are appropriately positioned to coordinate bundle microvilli cover the apical hair cell surface and surround a polarization across different rows of stereocilia and in relation to centrally positioned kinocilium. The kinocilium moves to the the kinocilium (Fig. 1A). In the mouse , developing hair edge of the apical hair cell surface. Subsequently, the microvilli bundles contain ankle links, transient lateral links, top connectors next to the kinocilium elongate. This is followed by elongation and tip links; mature cochlear hair bundles maintain top of the more distant rows, generating a hair bundle with a connectors and tip links. Developing cochlear hair bundles also staircase pattern that subsequently matures to attain its final contain one kinocilium that is connected to the longest shape (Tilney et al., 1992; Schwander et al., 2010). stereocilia by kinociliary links (Goodyear et al., 2005). Genes The kinocilium is important for establishing hair bundle linked to hearing loss encode several of the proteins forming polarity as bundles are misoriented in mice carrying mutation in these linkages (Müller, 2008; Gillespie and Müller, 2009; Petit orthologs of genes linked to the ciliopathy Bardet-Biedl and Richardson, 2009). Relevant to the current study are syndrome (Ross et al., 2005), and in mice with a conditional protocadherin 15 (PCDH15) and 23 (CDH23), which are components of kinociliary links, transient lateral links and tip links (Siemens et al., 2004; Lagziel et al., 2005; Michel et al., 2005; Rzadzinska et al., 2005; Ahmed et al., 2006; Senften et al., 1Dorris Neuroscience Center and Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. 2Laboratory of Cell Structure and Dynamics, 2006; Kazmierczak et al., 2007). Tip links are heterophilic National Institute of Deafness and other Communication Disorders, National adhesion complexes consisting of PCDH15 homodimers 3 Institute of Health, Bethesda, MD 20892, USA. Department of interacting with CDH23 homodimers (Kazmierczak et al., 2007) Otolaryngology–Head and Neck Surgery and Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, MD 21287, USA. (Fig. 1A). Kinociliary links show a similar asymmetry (Goodyear et al., 2010) (Fig. 1A). Functional null mutations in *Authors for correspondence ([email protected]; [email protected]; PCDH15 and CDH23 lead to defects in hair bundle structure and [email protected]) polarity, indicating the importance of linkages containing these

Accepted 24 January 2011 proteins for bundle morphogenesis (Alagramam et al., 2001a; Di DEVELOPMENT 1608 RESEARCH ARTICLE Development 138 (8)

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

MATERIALS AND METHODS Genetically modified mice The strategy for generating knockout mice (see Fig. S1 in the supplementary 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). Exons 35, 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 into 129P2/OlaHsd embryonic stem cells. Targeted clones were used to generate germline-transmitting chimera and crossed to FLP deleter mice to remove the selection cassette. Mice were maintained on a mixed C57BL6ϫ129SvEv background (for genotyping primers, see Table S1 in the supplementary material).

Antibodies The 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 agarose beads. 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 Alexa Fluor 488- and 568-phalloidin, Alexa Fluor 488 and 594 goat anti-rabbit, and Alexa Fluor 647 goat anti-mouse.

RT-PCR and qPCR RNA was isolated using Trizol (Invitrogen, Carlsbad, CA). cDNA was Fig. 1. Generation of PCDH15-DCD1, PCDH15-DCD2 and PCDH15- synthesized from 500 ng of RNA with Superscript III reverse transcriptase DCD3 mice. (A)Extracellular linkages in developing and mature murine (Invitrogen) and oligo(dT) primers. RT-PCR analysis and qPCR was hair cells. The asymmetric distribution of PCDH15 and CDH23 at tip performed as described (Belvindrah et al., 2007) (primers are listed in links and kinociliary links is indicated. (B)Arrangement of known Table S1 in the supplementary material). PCDH15 protein domains: 11 extracellular cadherin repeats (EC), transmembrane spanning region (TM), poly-proline repeats (PP) and Histology, immunohistochemistry and electron microscopy unique C-terminal PDZ-binding interfaces. (C)Arrangement of Pcdh15 Whole-mount staining was carried out as described previously (Grillet et exons encoding the cytoplasmic domain in wild-type (wt), PCDH15- al., 2009b). For scanning electron microscopy (SEM), inner were DCD1, PCDH15-DCD2 and PCDH15-DCD3 alleles. Exons are numbered. dissected and fixed by local perfusion with 2.5% glutaraldehyde, 4% Deleted exons are shown as triangles. (D)Gel images show examples of formaldehyde, 50 mM HEPES buffer (pH 7.2), 2 mM CaCl2, 1 mM MgCl2 genotyping by PCR from genomic DNA (sizes are indicated in Table S1 and 140 mM NaCl for 2 hours at room temperature. Inner ear sensory in the supplementary material). organs were fine dissected and processed by a modified OTOTO method (Waguespack et al., 2007). To increase structural stability and image contrast, we substituted thiocarbohydrazide by 1% tannic acid, resulting in alternate baths of 1% osmium tetroxide and 1% tannic acid for 1 hour each. Palma et al., 2001; Wilson et al., 2001; Pawlowski et al., 2006; Samples were dehydrated, critical-point dried (Bal-Tec CPD 030), coated Senften et al., 2006; Lefevre et al., 2008). Tip links are thought with a 4 nm platinum layer (Balzers BAF 300) and observed in a SEM to gate mechanotransduction channels in hair cells (Gillespie and Hitachi S-4800, operated at 5 kV. Transmission electron microscopy was Müller, 2009). However, the specific aspects of bundle carried out as described previously (Grillet et al., 2009a). development regulated by kinociliary links, transient lateral links In situ hybridization and tip links are not known. PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3 were amplified from One way a single gene could contribute to the formation of murine cochlear mRNA and cloned into pcDNA-SK+ (Invitrogen). In situ functionally distinct linkages in hair bundles is by alternative probes were generated towards a common sequence in the 5Ј region and splicing of the primary transcript. Hair cells express three toward unique regions in each of the isoforms and used for in situ prominent PCDH15 splice variants (PCDH15-CD1, PCDH15- hybridization as described (Schwander et al., 2007; Grillet et al., 2009a) CD2 and PCDH15-CD3) differing in their cytoplasmic domains (primers used to generate probes listed in Table S1 in the supplementary (Ahmed et al., 2001; Alagramam et al., 2001a; Alagramam et al., material). The 5Ј probe was generating by cloning a PstI fragment of 2001b; Ahmed et al., 2006; Senften et al., 2006). According to PCDH15 (bp 1456-2196 of mRNA, Accession Number NM_023115) into published data, PCDH15-CD1 and PCDH15-CD3 are abundantly pBS-SK+. detectable in hair bundles as they mature, whereas PCDH15- ABR and DPOAE measurement and mechanotransduction currents CD2 is abundant only in developing bundles (Ahmed et al., ABR and DPOAE measurements were carried out as described previously 2006). It has been hypothesized that PCDH15-CD1 and (Schwander et al., 2007; Grillet et al., 2009b). Mechanotransduction PCDH15-CD2 regulate hair bundle development, while currents from cochlear outer hair cells (OHCs) were recorded as described

PCDH15-CD3 has been proposed to be a tip-link component previously (Grillet et al., 2009b). DEVELOPMENT PCDH15 function and hair cells RESEARCH ARTICLE 1609

Fig. 2. Analysis of Pcdh15 isoform expression by in situ hybridization. (A)Examination of PCDH15 isoform expression in wild-type mice at P1. The diagrams summarize the data. A 5Ј probe (5P) detected PCDH15 expression in IHCs and OHCs (arrows), and in Kölliker’s organ; lower expression levels were evident in support cells. Probes specific for PCDH15-CD1 and PCDH15-CD3 revealed expression in hair cells, support cells and Kölliker’s organ. PCDH15- CD2 appeared to be restricted to IHCs and OHCs. (B)Examination of PCDH15 isoform expression in mutants at P1. As expected, only expression of the PCDH15 isoform targeted by the mutation was abolished in each genetically modified mouse line; expression of the remaining splice variants was maintained. In the PCDH15-DCD3 mouse line, ectopic PCDH15-CD2 expression was detected in support cells and Kölliker’s organ. Arrows point to hair cells. Scale bars: 50m.

Vestibular function ratio of eye and head velocity during the velocity plateau (Gv) when a We measured eye movements in response to whole-body head rotations in plateau was evident. During calculation of Ga and Gv, the horizontal alert mice. A head bolt was placed for animal restraint. The post was component of eye and head velocity was used. Results are mean±s.d.; each oriented so that when the animal was placed into a restraining device atop data point represents the mean for one animal over at least 10 stimulus a servo-controlled rotating table, the plane tangent to the flat part of the trials. Ga,Gv and latency data were analyzed using a one-way analysis of dorsal skull was pitched 30° ‘nose-down’ from Earth horizontal. The axes variance (ANOVA) with significance set at P0.05 (Diggle et al., 1994). of the animal’s horizontal aligned to within 10° of the Earth-vertical axis (Calabrese and Hullar, 2006). RESULTS The video-oculography technique was used for eye movement recording PCDH15- CD1 PCDH15- CD2 (Migliaccio et al., 2005). Marker arrays fashioned from photo paper saturated Generation of D , D and PCDH15- CD3 with fluorescent yellow ink were opaque except for three fluorescent D mice 200ϫ200 m windows separated by 200 m and arranged in a 45° right To define the function of PCDH15 isoforms in hair cells, we triangle. Images were acquired at a rate of 180 frames/second using IEEE1394 generated mice lacking specific isoforms (Fig. 1C; see Fig. S1 in (‘Firewire’) cameras [500ϫ400 pixel frame size; Dragonfly Express, Point the supplementary material). The three best-characterized PCDH15 Grey Research, Canada; resolution <0.3°; camera/lens resolution (263 isoforms in hair cells are PCDH15-CD1, PCDH15-CD2 and pixels/mm) at the eye surface aligned with the center of corneal curvature to PCDH15-CD3, which differ in their cytoplasmic domains (Ahmed within 72 m (Migliaccio et al., 2005)]. Data were interpolated on a 1 kHz et al., 2006). Exon 35 is specific for CD1, exon 38 for CD2 and time base using a nonlinear filter based on a running spline (LabVIEW). We exon 39 for CD3. We replaced exons 35, 38 and 39 in ES cells by adjusted the spline smoothness parameter so that the correlation coefficient 2 gene targeting with a neomycin cassette flanked by Frt sites (Fig. R between the raw and spline-filtered data was greater than 0.80. 1C; see Fig. S1 in the supplementary material). Genetically For rotational testing, the center of the animal’s skull was aligned with the motor’s Earth-vertical axis. Transient yaw whole body rotation stimuli were modified mice were generated and the neomycin cassette was delivered at 3000°/s2 constant acceleration for 100 ms to a peak/plateau removed by crossing the mice to a mouse line expressing FLP velocity of 300°/s lasting 0.8-1.0 s, followed by a 3000°/s2 deceleration for (Rodriguez et al., 2000). Heterozygous mice were intercrossed to 100 ms to rest at 90° from the starting position. Eye rotation data were generate mice homozygous for the deletion of exon 35, 38 and 39. converted to rotation vectors in head coordinates and analyzed as described Mice were genotyped by PCR (Fig. 1D), and will be referred to as previously (Migliaccio and Todd, 1999; Migliaccio et al., 2004; Migliaccio PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3. et al., 2005). Yaw (horizontal) component eye velocity data were inverted To confirm that the genetic modifications selectively affect the prior to gain calculation. Quick phases and saccades were removed. The start expression of specific PCDH15 isoforms, we analyzed RNA of P1 and end of quick phases/saccades were defined as the points at which eye mice by PCR (see Fig. S1D,E in the supplementary material). The acceleration rose above or fell below manually estimated maximum slow identity of the amplified DNA fragments was confirmed by DNA phase eye acceleration. Data from at least 10 stimulus trials were averaged sequencing (data not shown). PCDH15-CD1, PCDH15-CD2 and to obtain data for determining response parameters. For each trial, response PCDH15-CD3 were detectable in wild-type mice. PCDH15- CD1 latency was computed as the time difference between the zero-velocity- D intercept times for lines fit in a least-mean-square sense to eye and head mice lacked PCDH15-CD1 expression, whereas PCDH15-CD2 velocity during the constant-acceleration stimulus portion. and PCDH15-CD3 were maintained. PCDH15-DCD2 lacked For comparison to other species (Migliaccio et al., 2004), we PCDH15-CD2 expression but not PCDH15-CD1 or PCDH15- parameterized the responses by computing the ratio of eye and head CD3; PCDH15-DCD3 mice lacked PCDH15-CD3 expression but

acceleration during the constant-head-acceleration segment (Ga) and the not PCDH15-CD1 or PCDH15-CD2. DEVELOPMENT 1610 RESEARCH ARTICLE Development 138 (8)

Next, we carried out in situ hybridization on cochlear sections using probes specific for PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3. We also included a probe to the 5Ј end of PCDH15 mRNA common to all three isoforms, and sense control probes (Fig. 2; see Fig. S2 in the supplementary material). Previous studies have shown that PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3 are expressed in hair cells (Ahmed et al., 2006). We confirmed these findings and extended them. The probe to the 5Ј region of PCDH15 revealed that in wild-type mice, PCDH15 was expressed not only in mechanosensory hair cells but also in Kölliker’s organ (Fig. 2A). PCDH15-CD1 and PCDH15-CD3 were expressed in hair cells and Kölliker’s organ, whereas PCDH15-CD2 appeared to be specific for hair cells (Fig. 2A). The analysis of PCDH15- DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice revealed that in each mouse line, the two PCDH15 isoforms that were not mutated were still expressed (Fig. 2B). The expression pattern for the remaining isoforms appeared unaffected, with the exception of PCDH15-CD2, which was ectopically expressed in Kölliker’s organ of PCDH15-DCD3 mice (Fig. 2B). Quantitative RT-PCR showed that expression of PCDH15-CD1 was not substantially changed in PCDH15-DCD2 and PCDH15-DCD3 mice. Likewise, Fig. 3. Analysis of auditory function. (A)Auditory thresholds of PCDH15-CD3 expression was relatively normal in PCDH15- PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice at 1 month of DCD1 and PCDH15-DCD3 mice. However, PCDH15-CD2 was age compared with wild-type littermates (wild type, n19; DCD1, n11; upregulated in PCDH15- DCD3 and downregulated in PCDH15- DCD2, n10; DCD3, n7; mean±s.d.) (B) Auditory thresholds at 6 DCD1 mice (see Fig. S1E in the supplementary material). months of age (wild type, n10; DCD1, n3; DCD2, n3; DCD3, n3; mean±s.d.). (C)Auditory thresholds (mean±s.d.) in 1-month-old mice as determined by pure tone ABR recordings. (D)DPOAE thresholds in 1- Analysis of auditory function month-old PCDH15-DCD2 mutants were elevated at all frequencies PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice analyzed (wild type, n13; DCD1, n7; DCD2, n4; DCD3, n6; survived into adulthood. To measure auditory function, we mean±s.d.). Student’s t-test was performed on ABR data in A and B recorded in 1-month- and 6-month-old mice the auditory brain (***P<0.0001). stem (ABR) response to click-stimuli. Auditory thresholds were normal in PCDH15-DCD1 and PCDH15-DCD3 mice, but they were elevated to above 90 dB in PCDH15-DCD2 mice (Fig. 3A,B). observation was confirmed by SEM (see Fig. S3A in the Analysis of hearing function in response to pure tones confirmed supplementary material). Unlike in Ames waltzerav3J mice, hair that hearing function in PCDH15-DCD2 mice was affected across bundles were with few exceptions not fragmented (Fig. 4E-J; see the entire analyzed frequency spectrum (Fig. 3C). Fig. S3A,D and Fig. S4A-C in the supplementary material). To study OHC function, we measured the distortion product To quantify polarity defects, we determined the deviation of the otoacoustic emissions (DPOAEs). In wild-type, PCDH15-DCD1 position of the bundle center, which contains the longest stereocilia, and PCDH15-DCD3 mice, DPOAEs were dependent on the from their normal position at the tip of the V-shaped bundle (Fig. stimulus intensity at a given frequency, with no obvious difference 4K-M). Four percent of the mutant bundles formed circles and between wild-type and mutants (Fig. 3D). By contrast, DPOAEs were excluded from the quantification. IHCs and OHCs in wild- were not detectable in PCDH15-DCD2 mice (Fig. 3D). Similar type mice at P1 and P6 showed little variation in orientation (Fig. observations were made at all frequencies analyzed (6-28 kHz) 4L,M), but bundle orientation of OHCs was highly variable in (Fig. 3D), indicating that in PCDH15-DCD2 the function of OHCs PCDH15-DCD2 mice, deviating up to 90° from the normal was impaired. position, sometimes more (Fig. 4L,M). No such defect was seen in IHCs (Fig. 4L,M). Defects in hair bundle polarity in PCDH15-DCD2 mice Expression of PCDH15-CD2 in hair cells Ames waltzerav3J mice, which carry a predicted functional null To define the mechanism that caused the hair bundle defect in allele of PCDH15 that affects all isoforms, are deaf, circle and PCDH15-DCD2 mice, we raised antibodies against PCDH15-CD2 show defects in hair bundle morphogenesis (Alagramam et al., and analyzed its expression in hair cells at P1. As reported (Ahmed 2001a; Pawlowski et al., 2006; Senften et al., 2006). We wondered et al., 2006), PCDH15-CD2 was expressed throughout hair bundles whether the auditory phenotype of PCDH15-DCD2 mice was of OHCs; we also observed expression in the short microvilli caused by a similar mechanism and whether hair bundle present at the hair cell surface at P1 (Fig. 5A,B,E-H). Although morphology was normal in PCDH15-DCD1 and PCDH15-DCD3 OHCs were strongly positive for PCDH15-CD2, it was expressed mice. We stained P6 cochlear wholemounts with phalloidin to label at low levels in IHCs (Fig. 5A,B). All staining was abolished in F-actin in stereocilia (Fig. 4A-D). The sensory of the PCDH15-DCD2 mice (Fig. 5C,D), attesting to the specificity of the three mutant mouse lines was patterned into one row of inner hair antibody. Co-staining with antibodies to acetylated-a-tubulin, to cells (IHCs) and three rows of OHCs. Although hair bundle reveal the kinocilium, confirmed expression of PCDH15-CD2 in morphology appeared normal in PCDH15-DCD1 and PCDH15- proximity to the kinocilium of OHCs (Fig. 5E-H, arrows). In some DCD3 mice, hair bundles of OHCs were affected in PCDH15- hair bundles, staining was concentrated at kinociliary tips (Fig.

DCD2 mice, showing defects in polarization (Fig. 4C,F,I,J). This 5G,H, arrows). In vestibular hair cells, PCDH15-CD2 staining was DEVELOPMENT PCDH15 function and hair cells RESEARCH ARTICLE 1611

Fig. 5. PCDH15-CD2 expression in OHCs. (A-H)Cochlear wholemounts at P1 were stained using a PCDH15-CD2 antibody (red). (A,C,E,G) Stereocilia were labeled with phalloidin (green). (A-H)Kinocilia were stained using an acetylated-a-tubulin antibody (blue). (A-D) In Fig. 4. Analysis of hair bundle morphology in the cochlea.  wild-type OHCs and IHCs (A,B), and wild-type OHCs (E-H), PCDH15- (A-D)Cochlear wholemounts of the indicated genotypes at P6 were CD2 was present throughout the hair bundle. No staining was stained with phalloidin (green). Hair bundle morphology was affected observed in PCDH15-DCD2 mice (C,D). (E-H)In wild-type mice, in PCDH15-DCD2 mice. (E,G,H) Hair bundles in Ames waltzerav3j mice prominent staining was evident in the region proximal to the kinocilia- at P6 were fragmented and misoriented. (F,I,J) PCDH15-DCD2 hair stereocilia interface (arrows in E,F). Staining in OHCs was often bundles maintained cohesion and staircase arrangement but polarity observed as a puncta at the distal kinociliary tips (arrows in G,H). Scale was compromised. (K-M)Analysis of hair bundle polarity in OHCs and bars: 4m in A-D; 2m in E-H. IHCs from wild-type and PCDH15-DCD2 mice at P1 and P6. Orientation was determined by drawing a line through the axis of the bundle with 0° indicating the normal mediolateral axis (K). Angular deviation from this axis was determined. PCDH15- CD2 OHCs showed polarity defects position of the kinocilium was more variable in PCDH15-DCD2 D Ј Ј (wild type, n599; DCD2, n849). Fifty percent of the OHCs examined mice (Fig. 7B,B ,D,D ), suggesting that kinociliary links might be deviated by 15° or more. Scale bars: 10m in A-D; 5m in E,F; 2m affected. This was confirmed by SEM analysis. Although kinocilia in G-J. in wild-type mice at P1 were connected to the stereociliary bundle (Fig. 7E-G), they were separated from the bundle of OHCs and IHCs of PCDH15-DCD2 mice (Fig. 7H-J; see Fig. S4 in the strong throughout the bundles of immature hair cells and near the supplementary material). A similar phenotype was observed at E15 interface between stereocilia and the kinocilium (Fig. 6). We (Fig. 10). conclude that in OHCs, PCDH15-CD2 is targeted to the stereocilia Previous studies have suggested that several PCDH15 isoforms and kinocilium, and is appropriately positioned to contribute to the might be present at transient lateral links (Ahmed et al., 2006). formation of linkages between steoreocilia, and between the Unlike in Ames waltzerav3J mice, hair bundles of IHCs and OHCs stereocilia and the kinocilium. A similar expression pattern is were not fragmented in PCDH15-DCD1, PCDH15-DCD2 and observed in vestibular hair cells, but there is little expression in PCDH15-DCD3 mice (Fig. 4A-D,F,I,J; Fig. 7B,BЈ,D,DЈ and see IHCs. Fig. S4 in the supplementary material), suggesting that some of these isoforms have redundant functions at transient lateral links. Defects in kinociliary links in PCDH15-DCD2 mice This was confirmed by SEM analysis. In PCDH15-DCD2 mice, the We next analyzed hair bundles of cochlear hair cells at P1 and P6 kinocilium was separated from the stereocilia, but linkages along by staining with fluorescence labeled phalloidin to reveal actin, and the length of stereocilia were visible (Fig. 7H-J; Fig. 8A-D). Such antibodies to acetylated-a-tubulin to reveal the kinocilium. In wild- linkages were also observed in hair bundles from PCDH15-DCD1 type mice, the kinocilium was tightly associated to the longest and PCDH15-DCD3 mice (see Fig. S5 in the supplementary Ј Ј stereocilia at the bundle apex (Fig. 7A,A ,C,C ). By contrast, the material). DEVELOPMENT 1612 RESEARCH ARTICLE Development 138 (8)

Fig. 6. PCDH15-CD2 expression in vestibular hair cells. (A-L)Vestibular wholemounts at P4 stained using a PCDH15-CD2 antibody (red). In immature wild-type vestibular hair cells (A-CЈ,G-L), PCDH15-CD2 was present throughout the hair bundle. No staining was observed in PCDH15-DCD2 mice (D-F). In wild-type mice, prominent staining was evident in the region proximal to the kinocilia-stereocilia interface (arrows in G-I). Staining in immature hair cells was often Fig. 7. Kinocilia position in OHCs from PCDH15-DCD2 mice. observed as a puncta at the distal end of the tallest stereocilia (arrows (A-DЈ) Staining of cochlear wholemounts from P6 wild-type and in A-C,J-L). Scale bars: 10m in A-F; 5m in G-L. PCDH15-DCD2 mice with phalloidin (green) and antibodies to acetylated a-tubulin (red). Kinocilia (arrows) in wild-type hair cells (A,AЈ,C,CЈ) were linked to the stereociliary bundles; those of PCDH15-DCD2 hair cells At P96, there was widespread degeneration of hair bundles in (B,BЈ,D,DЈ) were separated from the bundle and misplaced. Cilia on the cochlea of PCDH15-DCD2 mice (see Fig. S3C in the support cells are marked with an asterisk. (E-J)SEM examination of wild- supplementary material), although some hair bundles were type and PCDH15-DCD2 OHCs at P1. (E-G)Wild-type hair cells showing maintained (see Fig. S3D in the supplementary material). The tight association between the stereocilia bundle and kinocilium (arrows degenerative changes probably contribute to deafness of PCDH15- in 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: 5m in CD2 mice. Stereocilia of OHCs that were maintained in the D A-D; 5m in E,F,H; 2m in I; 0.5m in G,J. mutants formed connections to the , as evident from their imprint in the tectorial membrane (see Fig. S3B in the supplementary material). recordings. We focused our analysis on hair bundles with minimal polarity defects. In agreement with previous findings (Kennedy et Tip links and mechanotransduction al., 2003; Kros et al., 2002; Stauffer and Holt, 2007; Waguespack PCDH15-DCD1 and PCDH15-DCD3 mice showed normal et al., 2007), control OHCs had rapidly activating transducer hearing, suggesting that tip links were unaffected. Deafness in currents, which subsequently adapted (Fig. 8H, blue traces); similar PCDH15-DCD2 mice is probably caused by defects in hair bundle current traces were obtained with OHCs from PCDH15-DCD2 morphology and maintenance, but tip-link function may also be mice (Fig. 8H, red traces). The amplitudes of saturated impaired. We therefore analyzed tip links by SEM in P7 mice. mechanotransduction currents at maximal deflection were similar Linkages running from the top of stereocilia to the side of the next in controls and mutants (Fig. 8H,I), suggesting that the total taller stereocilia were observed in PCDH15-DCD1, PCDH15- number of transducer channels was not altered in mutants. We also DCD2 and PCDH15-DCD3 mice (Fig. 8C-G), indicating that tip plotted the current/displacement relationships and channel open links were preserved. probability/displacement relationship and observed no significant To determine whether hair cells from PCDH15-DCD2 mice had difference between wild-type and mutants (Fig. 8I,J), indicating functional transduction, we measured transducer currents in P7-P8 that the transduction machinery of hair cells in PCDH15-DCD2

OHCs from the apical/middle part of the cochlea using whole cell mice functioned normally. Finally, as PCDH15-CD3 has previously DEVELOPMENT PCDH15 function and hair cells RESEARCH ARTICLE 1613

Fig. 8. Linkages in hair bundles and mechanotransduction currents. (A-D)Hair bundles from PCDH15-DCD2 mice at P1 (A,B) and P6 (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 OHCs from PCDH15-DCD2 mice. (H)Examples of transduction currents in respond to mechanical stimulation. (I)Current-displacement [I(X)] relationships were plotted and fitted with a second- order Boltzman function. (J)The relationship between open probability (Popen) obtained with peak currents following deflection revealed no significant difference between wild type and mutants. Data are mean±s.e.m. Scale bars: 1.5m in A; 0.2m in B; 0.5m in C; 150 nm in D-G.

been proposed as a component of tip links (Ahmed et al., 2006), antibodies to Fzd6 and Vangl2, which are normally localized at we also measured transducer currents in PCDH15-DCD3 mice but the medial edge of OHCs (Fig. 9J-MЈ; data not shown). The observed no defects (see Fig. S6 in the supplementary material). asymmetric distribution of Fzd6 (Fig. 9L-MЈ) and Vangl2 (data not shown) was not altered in PCDH15-DCD2 mice, suggesting that Defects in kinociliary positioning in PCDH15-DCD2 PCDH15-CD2 acts downstream of the PCP pathway. mice The kinocilium of hair cells is thought to be important for the Analysis of vestibular hair cell morphology and development of hair bundle polarity. To determine the extent to function which kinocilia position and bundle rotation in PCDH15-DCD2 It is thought that the kinocilium of vestibular hair cells is required mice correlates, we determined kinociliary position relative to for mechanical coupling between the hair bundle and the overlying stereociliary bundles (Fig. 9A). In IHCs and OHCs of wild-type gelatinous matrix (Roberts et al., 1988). We therefore wondered mice, the position of the kinocilium predicts the orientation of the whether kinociliary links and vestibular function were affected in bundle (Fig. 9B,C). By contrast, kinocilia position did not predict PCDH15-DCD2 mice. Analysis at E15.5, P1 and P6 revealed that bundle orientation in PCDH15-DCD2 mice and frequently deviated although the kinocilium was tightly linked to hair bundles in wild- 90° or more from the normal position (Fig. 9B,C). type mice (Fig. 10A-BЈ; data not shown), the kinocilium was In wild-type hair cells, the centrioles of the basal body undergo separated from the stereociliary bundle in PCDH15-DCD2 mice in a similar polarized movement to the kinocilium (Jones et al., 2008). hair cells in the , and semicircular canals (Fig. 10C- Consistent with these data, staining for pericentrin revealed the DЈ; see Fig. S7 in the supplementary material; data not shown). polar localization of the centrioles in OHCs from wild-type mice Stereocilia were also in contact with the overlying gelatinous at P1 (Fig. 9D-FЈ). In PCDH15-DCD2 mice, the two centrioles and matrix (see Fig. S7C-E in the supplementary material). kinocilium were coordinately mislocalized (Fig. 9G-IЈ) and the two We next analyzed vestibular function. Unlike in Ames waltzerav3J centrioles were no longer aligned appropriately relative to each mice, which lack all three PCDH15 isoforms, PCDH15-DCD2 other along the axis of bundle polarity (Fig. 9G-IЈ). We conclude mice (and PCDH15-DCD1 and PCDH15-DCD3 mice) did not that kinociliary links are required to coordinate the polarization of circle (data not shown). To evaluate vestibular function the kinocilium and their basal bodies with the polarization of the quantitatively, we measured eye movements in alert mice mediated stereociliary bundle. by the vestibulo-ocular reflex in response to whole-body head rotations (Fig. 10E-G). Mice were immobilized in a superstructure Normal localization of components of the core mounted atop a motor able to deliver 3000°/s constant accelerations PCP pathway to a plateau velocity of 300°/s about the dorsoventral axis of the Random polarization of hair bundles, similar to that observed in animal through the stereotaxic origin of the skull. Eye movements PCDH15-DCD2 mice, is a hallmark of mice with mutations in were measured using a three-dimensional video-oculography orthologs of genes linked to the Bardet-Biedl syndrome (Ross et system. We quantified responses to whole-body, passive, transient al., 2005), and in mice with mutations that affect the intraciliary rotations in darkness using three parameters: Ga (‘constant- transport protein Ift88 (Jones et al., 2008). The ciliary genes are acceleration gain’) was computed as the ratio of eye to head thought to act downstream of components of the core PCP velocity averaged over the constant acceleration segment of the pathway. We therefore asked whether core PCP proteins showed stimulus; Gv (‘constant-velocity gain’) was computed as the ratio normal asymmetric localization in PCDH15-DCD2 mice. We of eye to head velocity averaged over the constant velocity segment

stained cochlear wholemounts of P1 mice with phalloidin and of the stimulus; latency is the delay from the onset of head DEVELOPMENT 1614 RESEARCH ARTICLE Development 138 (8)

Fig. 9. Position of kinocilia and basal bodies in PCDH15-DCD2 mice. (A-C)Examination of the relationship between kinocilia position and bundle rotation in OHCs. In wild-type mice, there was a correlation between the kinocilia position and the apex of a stereocilia chevron. In Fig. 10. Analysis of vestibular hair cells from PCDH15-DCD2 mice. PCDH15-DCD2 bundles, the kinocilia were frequently mislocalized (wild (A-BЈ) SEM of vestibular hair cells in wild type at E15.5 and P1 reveal type, n203; DCD2, n106). Of the OHCs examined, 48.2% had tight coupling of the kinocilia (arrows) to the stereocilia. (C-DЈ) Kinocilia Ј kinocilia mislocalized by 15° or more. (D-I ) Cochlear wholemounts (arrows) of PCDH15-DCD2 vestibular hair cells were not connected to were stained with phalloidin (green), antibodies to pericentrin (red) and the stereocilia. Images of hair cells are from E15 crista (A,AЈ,C), E15 antibodies to acetylated a-tubulin (blue). In wild type but not in the utricle (CЈ) and P1 ampulla (B,BЈ,D,DЈ). (E-G)Vestibulo-ocular reflex mutants, the basal bodies (arrows) aligned with the kinocilium along response for PCDH15-DCD2, wild-type littermates (C57BL/6ϫ129SvEv), Ј the hair bundle polarity axis. (J-M ) Cochlear wholemounts were C57BL/6 mice, Ames waltzerav3J mice and a dead mouse. (E)Eye versus stained with phalloidin (green) and antibodies to frizzled 6 (red). head horizontal velocity ratio, Ga, during the constant acceleration Frizzled 6 localization (arrows) was not affected in the mutants. The (3000° s–2) segment of a passive transient whole-body rotation about a presumed position of the kinocilium is indicated by an asterisk (JЈ,LЈ). –1 vertical axis. (F)Velocity ratio, Gv, during the constant velocity (300° s ) Scale bars: 5m. plateau of the same stimulus. (G)Response latency from onset of head motion to onset of eye motion (mseconds). Diamonds and whiskers movement to the onset of eye movement, computed through linear indicate mean±s.d.; each data point is an the mean response of an extrapolation of eye and head responses during the constant- animal; n≥10 head rotations in each direction. Excluding the Ames av3J Ϸ Ϸ acceleration segment of the stimulus. Measurements were carried waltzer mice and dead controls (for which Ga Gv 0), one-way ϫ ANOVA revealed no significant difference among groups in Ga, Gv or out with PCDH15-DCD2 mice (n6), C57BL6 129 wild-type latency (P>0.05). Including the Ames waltzerav3J mice, controls in the littermate controls (n8), C57BL/6 wild-type controls (n7), Ames analyses revealed that all other live mouse groups differed significantly av3J av3J waltzer mice (n3) and one dead mouse (to rule out technical in Ga and latency from Ames waltzer mice controls (P<0.001); Gv av3J artifacts). Ames waltzer mice and dead mice did not show any showed a similar trend but did not reach significance. There was a response. However, there was no statistically significant difference trend towards PCDH15-DCD2 mutants having longer response latency in any of the parameter between PCDH15-DCD2 mice and wild- than control mice, but this trend did not reach significance (P0.062). type mice (Fig. 9E-G). In addition, we analyzed the response to These data suggest that PCDH15-DCD2 mice have horizontal head pitch tilts about the intreraural axis and observed normal semicircular canal function similar to both of the wild-type control av3J responses in the mutants (data not shown). These data show that groups but significantly better than Ames waltzer mice. Scale bars: 5m in A-D; 2.5m in AЈ-DЈ. vestibular function was surprisingly intact in PCDH15-DCD2 mice.

DISCUSSION hair cell function, whereas PCDH15-CD2 is required for the Here, we show that alternative splicing of PCDH15 regulates its formation of kinociliary links. In the absence of PCDH15-CD2, hair function in hair cells. Using isoform-specific knockout mice, we bundles consisting of rows of stereocilia of graded heights develop

show that PCDH15-CD1 and PCDH15-CD3 are not essential for but bundle polarity is affected, demonstrating that kinociliary links DEVELOPMENT PCDH15 function and hair cells RESEARCH ARTICLE 1615 are not essential for the development for the stereociliary staircase Our data show that several PCDH15 isoforms have redundant but for the morphological transformation that lead to bundle function in hair cells. The hair bundles of Ames waltzerav3J mice, polarization. Components of the PCP pathway are normally which lack all PCDH15 isoforms, show bundle fragmentation distributed in the sensory epithelia of PCDH15-DCD2 mice, (Alagramam et al., 2001a; Pawlowski et al., 2006; Senften et al., suggesting that PCDH15-CD2 acts downstream of the PCP 2006). As stereociliary bundles are not significantly fragmented in pathway. Hair bundles in PCDH15-DCD2 mice are gradually lost PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice, it postnatally, indicating that the morphological defects affect hair cell seems that several of these isoform contribute to transient lateral function causing deafness. In addition, we cannot exclude that links. Consistent with this finding, we observed by SEM abundant cochlear hair cells do not function normally because their stereocilia links between the stereocilia in all three mutants. Similarly, it seems are not connected to the kinocilium. Unlike in Ames waltzerav3J that neither PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3 mice, which carry a predicted PCDH15 null allele, stereociliary are uniquely required for tip-link formation. Previous bundles are not fragmented in mice individually lacking PCDH15- immunolocalization studies have failed to clearly define the CD1, PCDH15-CD2 and PCDH15-CD3, suggesting that several PCDH15 isoform at tip links. PCDH15-CD3 has been the best PCDH15 isoforms contribute to the formation of transient lateral candidate, as one CD3-specific antibody bound to the region of the links. We also observed that tip links are present in PCDH15-DCD1, lower end. However, the same antibody did not bind to PCDH15-DCD2 and PCDH15-DCD3 mice, providing evidence that adult murine hair cells and a second PCDH15-CD3 antibody bound none of these isoforms is uniquely required at tip links. to stereocilia more broadly (Ahmed et al., 2006). Using antibodies One of the central findings of our study is that PCDH15-CD2 is against PCDH15 isoforms, we have also been unable to define the required for the development of kinociliary links and the normal PCDH15 isoform at tip links, as different antibodies to the same polarization of hair bundles. Initial studies by the Tilney laboratory isoform revealed distinct expression patterns. This variation might suggested that the kinocilium is important for bundle polarization be caused by differences in antibody affinity or epitope masking. (Tilney et al., 1992), but the mechanism by which the kinocilium Nevertheless, our genetic studies show that PCDH15-CD1, determines polarity had remained unclear. Recent studies have PCDH15-CD2 and PCDH15-CD3 individually are not essential for shown that mutations in components of the core PCP pathway and tip-link formation. Extracellular filaments with tip link features are mutations affecting the kinocilium cause polarity defects. When present in PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 PCP signaling is defective, hair bundles maintain intrinsic polarity mice. In addition, auditory and vestibular function is preserved in but are randomly polarized in the apical cell surface. When the PCDH15-DCD1 and PCDH15-DCD3 mice, and vestibular function kinocilium is affected, bundles show random polarization within in PCDH15-DCD2 mice. OHCs in PCDH15-DCD2 and PCDH15- the apical surface and loss of intrinsic polarity (Curtin et al., 2003; DCD3 mice also show normal responses to mechanical stimulation Montcouquiol et al., 2003; Lu et al., 2004; Ross et al., 2005; Wang in vitro. An important implication of our findings is that the et al., 2005; Montcouquiol et al., 2006; Wang et al., 2006; Jones et mechanotransduction channel in hair cells, which is localized in al., 2008). The asymmetric localization of PCP proteins at the proximity to the lower tip-link end (Beurg et al., 2009) might bind border between hair and support cells is not affected in ciliary to the extracellular and/or transmembrane domain of PCDH15 that mutants (Jones et al., 2008), suggesting that ciliary genes act is shared by several PCDH15 isoforms. downstream of PCP components (Jones and Chen, 2008). In We were surprised to find that vestibular function was not PCDH15-DCD2 mice, the PCP components Frz6 and Vangl2 are noticeably altered in PCDH15-DCD2 mice. The kinocilium of normally distributed, and hair bundle morphology resembles that vestibular hair cells is coupled to the kinocilium and the overlying of the ciliary mutants, suggesting that PCDH15-CD2 behaves like gelatinous extracellular matrices. Unlike in the cochlea, adult a ciliary mutant acting downstream of the PCP pathway. In the vestibular hair cells do not loose their kinocilium. It has therefore PCDH15-DCD2 mutants, the kinocilium and stereocilia bundle are been assumed that the kinocilium mechanically couples the hair mislocalized, but not always in same direction, suggesting that bundle to the overlying gelatinous matrix (Roberts et al., 1988). kinociliary links are required to coordinate movement of the Our findings now suggest that murine vestibular hair cells function kinocilium and stereocilia. normally, even when linkages between the kinocilium and We have previously shown that tip links in hair cells have an stereocilia are disrupted. Direct coupling of the stereocilia to the intrinsic asymmetry in the planar polarity axis of hair cells, where gelatinous matrices is probably sufficient to transmit motion to PCDH15 forms the lower end of tip links and CDH23 the upper stereocilia and activate transduction channels. end (Kazmierczak et al., 2007). CDH23 and PCDH15 are also Our functional data were collected with mice of a mixed asymmetrically distributed at kinociliary links (Goodyear et al., C57BL6ϫ129SvEv background. Although we did not observe 2010). However, polarity is reversed relative to tip links, with differences in PCDH15 isoform expression between inbred mouse PCDH15 being present in the kinocilium and CDH23 in the longest strains, we cannot exclude the possibility that different phenotypes stereocilia (Fig. 1A). Our findings indicate that PCDH15-CD2 is might be observed on distinct genetic backgrounds. the PCDH15 isoform in kinocilia and suggest a mechanism by Our findings could have important ramifications for the analysis which it might regulate polarity. As the kinocilium, unlike the of the genetic causes of sensory impairment. Several PCDH15 stereocilia, contains , it seems likely that the CD2 mutations have been identified that cause Usher Syndrome and cytoplasmic domain contains specific sequence motifs for targeting recessive forms of deafness (Ahmed et al., 2001; Alagramam et al., to the kinocilia that are absent in CD1 and CD3. In this way, a 2001b; Ben-Yosef et al., 2003; Ouyang et al., 2005; Roux et al., polarity axis is established in kinociliary links. The fact that some 2006; Le Guedard et al., 2007). These mutations commonly map to PCDH15-CD2 is present in stereocilia could be explained because the extracellular PCDH15 domain. Our findings suggest that it would actin-based molecular motors might also transport PCDH15-CD2. be important to determine whether mutations that specifically affect Alternatively, as PCDH15 molecules form dimers (Kazmierczak et the PCDH15-CD2 isoform might lead to auditory impairment. Last, al., 2007), PCDH15-CD2 might enter stereocilia as a heterodimer mutations specific for PCDH15-CD1 and PCDH15-CD3 might lead

with PCDH15-CD1 and/or PCDH15-CD3. to retinal disease without auditory dysfunction. DEVELOPMENT 1616 RESEARCH ARTICLE Development 138 (8)

Acknowledgements protocadherin 15 interact to form tip-link filaments in sensory hair cells. This work was funded by NIDCD grants DC007704, DC005965, DC005965-S1 Nature 449, 87-91. (U.M.), DC9255, DC2390 (C.D.S.) and NIDCD-DIR-Z01DC000002 (B.K.); by the Kennedy, H. J., Evans, M. G., Crawford, A. C. and Fettiplace, R. (2003). Fast Skaggs Institute for Chemical Biology (U.M.); by the Dorris Neuroscience adaptation of mechanoelectrical transducer channels in mammalian cochlear Center (U.M.); by a Ruth Kirschstein Predoctoral Fellowship award (S.W.W.); hair cells. Nat. Neurosci. 6, 832-836. and by a CIRM training grant (S.W.W.). Deposited in PMC for release after 12 Kros, C. J., Marcotti, W., van Netten, S. M., Self, T. J., Libby, R. T., Brown, S. months. D., Richardson, G. P. and Steel, K. P. (2002). Reduced climbing and increased slipping adaptation in cochlear hair cells of mice with Myo7a mutations. Nat. Neurosci. 5, 41-47. Competing interests statement Lagziel, A., Ahmed, Z. M., Schultz, J. M., Morell, R. J., Belyantseva, I. A. The authors declare no competing financial interests. and Friedman, T. B. (2005). Spatiotemporal pattern and isoforms of cadherin 23 in wild type and waltzer mice during inner ear hair cell development. Dev. Supplementary material Biol. 280, 295-306. Supplementary material for this article is available at Le Guedard, S., Faugere, V., Malcolm, S., Claustres, M. and Roux, A. F. http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.060061/-/DC1 (2007). Large genomic rearrangements within the PCDH15 gene are a significant cause of USH1F syndrome. Mol. Vis. 13, 102-107. References Lefevre, G., Michel, V., Weil, D., Lepelletier, L., Bizard, E., Wolfrum, U., Ahmed, Z. M., Riazuddin, S., Bernstein, S. L., Ahmed, Z., Khan, S., Griffith, Hardelin, J. P. and Petit, C. (2008). A core cochlear phenotype in USH1 A. J., Morell, R. J., Friedman, T. B. and Wilcox, E. R. (2001). 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