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Regulation of PCDH15 Function in Mechanosensory Hair Cells by Alternative Splicing of the Cytoplasmic Domain Stuart W

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Regulation of PCDH15 Function in Mechanosensory Hair Cells by Alternative Splicing of the Cytoplasmic Domain Stuart W 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 ear and in photoreceptors of the retina. Mutations in PCDH15 cause Usher Syndrome (deaf-blindness) and recessive deafness. In developing hair cells, PCDH15 localizes to extracellular linkages that connect the stereocilia and kinocilium into a bundle and regulate its morphogenesis. In mature hair cells, PCDH15 is a component of tip links, which gate mechanotransduction 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 hearing 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) proteins 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 gene encoding the intraflagellar and intraciliary Hair cells of the inner ear are mechanosensors for the perception transport protein Ift88 (Jones et al., 2008). Hair bundle of sound, head movement and gravity. The mechanically polarization is also affected in mice with mutations in genes that sensitive organelle of a hair cell 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 cochlea, 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 cadherin 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 ears 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
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