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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.
INTRODUCTION
Hair cells of the inner ear are mechanosensors for the perception
of sound, head movement and gravity. The mechanically
sensitive organelle of a hair cell is the hair bundle, which
consists of rows of stereocilia of graded heights polarized across
the apical cell surface. Bundle polarity is essential for normal
hair cell function, as only deflections of the bundle in the
direction of the tallest stereocilia increase the open probability
of mechanotransduction channels (Shotwell et al., 1981). Hair
bundle polarity is established through a complex series of
morphogenetic events. At the onset of hair bundle development,
microvilli cover the apical hair cell surface and surround a
centrally positioned kinocilium. The kinocilium moves to the
edge of the apical hair cell surface. Subsequently, the microvilli
next to the kinocilium elongate. This is followed by elongation
of the more distant rows, generating a hair bundle with a
staircase pattern that subsequently matures to attain its final
shape (Tilney et al., 1992; Schwander et al., 2010).
The kinocilium is important for establishing hair bundle
polarity as bundles are misoriented in mice carrying mutation in
orthologs of genes linked to the ciliopathy Bardet-Biedl
syndrome (Ross et al., 2005), and in mice with a conditional
1
Dorris Neuroscience Center and Department of Cell Biology, The Scripps Research
Institute, La Jolla, CA 92037, USA. 2Laboratory of Cell Structure and Dynamics,
National Institute of Deafness and other Communication Disorders, National
Institute of Health, Bethesda, MD 20892, USA. 3Department of
Otolaryngology–Head and Neck Surgery and Biomedical Engineering, Johns Hopkins
School of Medicine, Baltimore, MD 21287, USA.
*Authors for correspondence ([email protected]; [email protected];
[email protected])
Accepted 24 January 2011
mutation in the gene encoding the intraflagellar and intraciliary
transport protein Ift88 (Jones et al., 2008). Hair bundle
polarization is also affected in mice with mutations in genes that
regulate 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 is
known about the molecules that connect PCP signaling to the
kinocilium and bundle polarization. Candidate molecules are
proteins that form the extracellular filaments connecting the
stereocilia of a hair cell to each other and to the kinocilium
because they are appropriately positioned to coordinate bundle
polarization across different rows of stereocilia and in relation to
the kinocilium (Fig. 1A). In the mouse cochlea, developing hair
bundles contain ankle links, transient lateral links, top connectors
and tip links; mature cochlear hair bundles maintain top
connectors and tip links. Developing cochlear hair bundles also
contain one kinocilium that is connected to the longest
stereocilia by kinociliary links (Goodyear et al., 2005). Genes
linked to hearing loss encode several of the proteins forming
these linkages (Müller, 2008; Gillespie and Müller, 2009; Petit
and Richardson, 2009). Relevant to the current study are
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.,
2006; Kazmierczak et al., 2007). Tip links are heterophilic
adhesion complexes consisting of PCDH15 homodimers
interacting with CDH23 homodimers (Kazmierczak et al., 2007)
(Fig. 1A). Kinociliary links show a similar asymmetry
(Goodyear et al., 2010) (Fig. 1A). Functional null mutations in
PCDH15 and CDH23 lead to defects in hair bundle structure and
polarity, indicating the importance of linkages containing these
proteins for bundle morphogenesis (Alagramam et al., 2001a; Di
DEVELOPMENT
KEY WORDS: Hair cells, PCDH15, Deafness, Mouse
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 PCDH15DCD1, 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
C57BL6⫻129SvEv 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
Palma et al., 2001; Wilson et al., 2001; Pawlowski et al., 2006;
Senften et al., 2006; Lefevre et al., 2008). Tip links are thought
to gate mechanotransduction channels in hair cells (Gillespie and
Müller, 2009). However, the specific aspects of bundle
development regulated by kinociliary links, transient lateral links
and tip links are not known.
One way a single gene could contribute to the formation of
functionally distinct linkages in hair bundles is by alternative
splicing of the primary transcript. Hair cells express three
prominent PCDH15 splice variants (PCDH15-CD1, PCDH15CD2 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 to
published data, PCDH15-CD1 and PCDH15-CD3 are abundantly
detectable in hair bundles as they mature, whereas PCDH15CD2 is abundant only in developing bundles (Ahmed et al.,
2006). It has been hypothesized that PCDH15-CD1 and
PCDH15-CD2 regulate hair bundle development, while
PCDH15-CD3 has been proposed to be a tip-link component
RNA was isolated using Trizol (Invitrogen, Carlsbad, CA). cDNA was
synthesized from 500 ng of RNA with Superscript III reverse transcriptase
(Invitrogen) and oligo(dT) primers. RT-PCR analysis and qPCR was
performed as described (Belvindrah et al., 2007) (primers are listed in
Table S1 in the supplementary material).
Histology, immunohistochemistry and electron microscopy
Whole-mount staining was carried out as described previously (Grillet et
al., 2009b). For scanning electron microscopy (SEM), inner ears were
dissected and fixed by local perfusion with 2.5% glutaraldehyde, 4%
formaldehyde, 50 mM HEPES buffer (pH 7.2), 2 mM CaCl2, 1 mM MgCl2
and 140 mM NaCl for 2 hours at room temperature. Inner ear sensory
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.
Samples were dehydrated, critical-point dried (Bal-Tec CPD 030), coated
with a 4 nm platinum layer (Balzers BAF 300) and observed in a SEM
Hitachi S-4800, operated at 5 kV. Transmission electron microscopy was
carried out as described previously (Grillet et al., 2009a).
In situ hybridization
PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3 were amplified from
murine cochlear mRNA and cloned into pcDNA-SK+ (Invitrogen). In situ
probes were generated towards a common sequence in the 5⬘ region and
toward unique regions in each of the isoforms and used for in situ
hybridization as described (Schwander et al., 2007; Grillet et al., 2009a)
(primers used to generate probes listed in Table S1 in the supplementary
material). The 5⬘ probe was generating by cloning a PstI fragment of
PCDH15 (bp 1456-2196 of mRNA, Accession Number NM_023115) into
pBS-SK+.
ABR and DPOAE measurement and mechanotransduction currents
ABR and DPOAE measurements were carried out as described previously
(Schwander et al., 2007; Grillet et al., 2009b). Mechanotransduction
currents from cochlear outer hair cells (OHCs) were recorded as described
previously (Grillet et al., 2009b).
DEVELOPMENT
Fig. 1. Generation of PCDH15-DCD1, PCDH15-DCD2 and PCDH15DCD3 mice. (A)Extracellular linkages in developing and mature murine
hair cells. The asymmetric distribution of PCDH15 and CDH23 at tip
links and kinociliary links is indicated. (B)Arrangement of known
PCDH15 protein domains: 11 extracellular cadherin repeats (EC),
transmembrane spanning region (TM), poly-proline repeats (PP) and
unique C-terminal PDZ-binding interfaces. (C)Arrangement of Pcdh15
exons encoding the cytoplasmic domain in wild-type (wt), PCDH15DCD1, PCDH15-DCD2 and PCDH15-DCD3 alleles. Exons are numbered.
Deleted exons are shown as triangles. (D)Gel images show examples of
genotyping by PCR from genomic DNA (sizes are indicated in Table S1
in the supplementary material).
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 5⬘ 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. PCDH15CD2 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.
We measured eye movements in response to whole-body head rotations in
alert mice. A head bolt was placed for animal restraint. The post was
oriented so that when the animal was placed into a restraining device atop
a servo-controlled rotating table, the plane tangent to the flat part of the
dorsal skull was pitched 30° ‘nose-down’ from Earth horizontal. The axes
of the animal’s horizontal semicircular canals aligned to within 10° of the
Earth-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 saturated
with fluorescent yellow ink were opaque except for three fluorescent
200⫻200 m windows separated by 200 m and arranged in a 45° right
triangle. Images were acquired at a rate of 180 frames/second using IEEE1394
(‘Firewire’) cameras [500⫻400 pixel frame size; Dragonfly Express, Point
Grey Research, Canada; resolution <0.3°; camera/lens resolution (263
pixels/mm) at the eye surface aligned with the center of corneal curvature to
within 72 m (Migliaccio et al., 2005)]. Data were interpolated on a 1 kHz
time base using a nonlinear filter based on a running spline (LabVIEW). We
adjusted the spline smoothness parameter so that the correlation coefficient
R2 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 the
motor’s Earth-vertical axis. Transient yaw whole body rotation stimuli were
delivered at 3000°/s2 constant acceleration for 100 ms to a peak/plateau
velocity of 300°/s lasting 0.8-1.0 s, followed by a 3000°/s2 deceleration for
100 ms to rest at 90° from the starting position. Eye rotation data were
converted to rotation vectors in head coordinates and analyzed as described
previously (Migliaccio and Todd, 1999; Migliaccio et al., 2004; Migliaccio
et al., 2005). Yaw (horizontal) component eye velocity data were inverted
prior to gain calculation. Quick phases and saccades were removed. The start
and end of quick phases/saccades were defined as the points at which eye
acceleration rose above or fell below manually estimated maximum slow
phase eye acceleration. Data from at least 10 stimulus trials were averaged
to obtain data for determining response parameters. For each trial, response
latency was computed as the time difference between the zero-velocityintercept times for lines fit in a least-mean-square sense to eye and head
velocity during the constant-acceleration stimulus portion.
For comparison to other species (Migliaccio et al., 2004), we
parameterized the responses by computing the ratio of eye and head
acceleration during the constant-head-acceleration segment (Ga) and the
ratio of eye and head velocity during the velocity plateau (Gv) when a
plateau was evident. During calculation of Ga and Gv, the horizontal
component of eye and head velocity was used. Results are mean±s.d.; each
data point represents the mean for one animal over at least 10 stimulus
trials. Ga, Gv and latency data were analyzed using a one-way analysis of
variance (ANOVA) with significance set at P0.05 (Diggle et al., 1994).
RESULTS
Generation of PCDH15-DCD1, PCDH15-DCD2 and
PCDH15-DCD3 mice
To define the function of PCDH15 isoforms in hair cells, we
generated mice lacking specific isoforms (Fig. 1C; see Fig. S1 in
the supplementary material). The three best-characterized PCDH15
isoforms in hair cells are PCDH15-CD1, PCDH15-CD2 and
PCDH15-CD3, which differ in their cytoplasmic domains (Ahmed
et al., 2006). Exon 35 is specific for CD1, exon 38 for CD2 and
exon 39 for CD3. We replaced exons 35, 38 and 39 in ES cells by
gene targeting with a neomycin cassette flanked by Frt sites (Fig.
1C; see Fig. S1 in the supplementary material). Genetically
modified mice were generated and the neomycin cassette was
removed by crossing the mice to a mouse line expressing FLP
(Rodriguez et al., 2000). Heterozygous mice were intercrossed to
generate mice homozygous for the deletion of exon 35, 38 and 39.
Mice were genotyped by PCR (Fig. 1D), and will be referred to as
PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3.
To confirm that the genetic modifications selectively affect the
expression of specific PCDH15 isoforms, we analyzed RNA of P1
mice by PCR (see Fig. S1D,E in the supplementary material). The
identity of the amplified DNA fragments was confirmed by DNA
sequencing (data not shown). PCDH15-CD1, PCDH15-CD2 and
PCDH15-CD3 were detectable in wild-type mice. PCDH15-DCD1
mice lacked PCDH15-CD1 expression, whereas PCDH15-CD2
and PCDH15-CD3 were maintained. PCDH15-DCD2 lacked
PCDH15-CD2 expression but not PCDH15-CD1 or PCDH15CD3; PCDH15-DCD3 mice lacked PCDH15-CD3 expression but
not PCDH15-CD1 or PCDH15-CD2.
DEVELOPMENT
Vestibular function
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 5⬘ 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 5⬘ 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 PCDH15DCD1, 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,
PCDH15-CD3 expression was relatively normal in PCDH15DCD1 and PCDH15-DCD3 mice. However, PCDH15-CD2 was
upregulated in PCDH15- DCD3 and downregulated in PCDH15DCD1 mice (see Fig. S1E in the supplementary material).
Analysis of auditory function
PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice
survived into adulthood. To measure auditory function, we
recorded in 1-month- and 6-month-old mice the auditory brain
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).
Analysis of hearing function in response to pure tones confirmed
that hearing function in PCDH15-DCD2 mice was affected across
the entire analyzed frequency spectrum (Fig. 3C).
To study OHC function, we measured the distortion product
otoacoustic emissions (DPOAEs). In wild-type, PCDH15-DCD1
and PCDH15-DCD3 mice, DPOAEs were dependent on the
stimulus intensity at a given frequency, with no obvious difference
between wild-type and mutants (Fig. 3D). By contrast, DPOAEs
were not detectable in PCDH15-DCD2 mice (Fig. 3D). Similar
observations were made at all frequencies analyzed (6-28 kHz)
(Fig. 3D), indicating that in PCDH15-DCD2 the function of OHCs
was impaired.
Defects in hair bundle polarity in PCDH15-DCD2
mice
Ames waltzerav3J mice, which carry a predicted functional null
allele of PCDH15 that affects all isoforms, are deaf, circle and
show defects in hair bundle morphogenesis (Alagramam et al.,
2001a; Pawlowski et al., 2006; Senften et al., 2006). We wondered
whether the auditory phenotype of PCDH15-DCD2 mice was
caused by a similar mechanism and whether hair bundle
morphology was normal in PCDH15-DCD1 and PCDH15-DCD3
mice. We stained P6 cochlear wholemounts with phalloidin to label
F-actin in stereocilia (Fig. 4A-D). The sensory epithelium of the
three mutant mouse lines was patterned into one row of inner hair
cells (IHCs) and three rows of OHCs. Although hair bundle
morphology appeared normal in PCDH15-DCD1 and PCDH15DCD3 mice, hair bundles of OHCs were affected in PCDH15DCD2 mice, showing defects in polarization (Fig. 4C,F,I,J). This
Development 138 (8)
Fig. 3. Analysis of auditory function. (A)Auditory thresholds of
PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice at 1 month of
age compared with wild-type littermates (wild type, n19; DCD1, n11;
DCD2, n10; DCD3, n7; mean±s.d.) (B) Auditory thresholds at 6
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 1month-old PCDH15-DCD2 mutants were elevated at all frequencies
analyzed (wild type, n13; DCD1, n7; DCD2, n4; DCD3, n6;
mean±s.d.). Student’s t-test was performed on ABR data in A and B
(***P<0.0001).
observation was confirmed by SEM (see Fig. S3A in the
supplementary material). Unlike in Ames waltzerav3J mice, hair
bundles were with few exceptions not fragmented (Fig. 4E-J; see
Fig. S3A,D and Fig. S4A-C in the supplementary material).
To quantify polarity defects, we determined the deviation of the
position 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 and
were excluded from the quantification. IHCs and OHCs in wildtype mice at P1 and P6 showed little variation in orientation (Fig.
4L,M), but bundle orientation of OHCs was highly variable in
PCDH15-DCD2 mice, deviating up to 90° from the normal
position, sometimes more (Fig. 4L,M). No such defect was seen in
IHCs (Fig. 4L,M).
Expression of PCDH15-CD2 in hair cells
To define the mechanism that caused the hair bundle defect in
PCDH15-DCD2 mice, we raised antibodies against PCDH15-CD2
and analyzed its expression in hair cells at P1. As reported (Ahmed
et al., 2006), PCDH15-CD2 was expressed throughout hair bundles
of OHCs; we also observed expression in the short microvilli
present at the hair cell surface at P1 (Fig. 5A,B,E-H). Although
OHCs were strongly positive for PCDH15-CD2, it was expressed
at low levels in IHCs (Fig. 5A,B). All staining was abolished in
PCDH15-DCD2 mice (Fig. 5C,D), attesting to the specificity of the
antibody. Co-staining with antibodies to acetylated-a-tubulin, to
reveal the kinocilium, confirmed expression of PCDH15-CD2 in
proximity to the kinocilium of OHCs (Fig. 5E-H, arrows). In some
hair bundles, staining was concentrated at kinociliary tips (Fig.
5G,H, arrows). In vestibular hair cells, PCDH15-CD2 staining was
DEVELOPMENT
1610 RESEARCH ARTICLE
Fig. 4. Analysis of hair bundle morphology in the cochlea.
(A-D)Cochlear wholemounts of the indicated genotypes at P6 were
stained with phalloidin (green). Hair bundle morphology was affected
in PCDH15-DCD2 mice. (E,G,H) Hair bundles in Ames waltzerav3j mice
at P6 were fragmented and misoriented. (F,I,J) PCDH15-DCD2 hair
bundles maintained cohesion and staircase arrangement but polarity
was compromised. (K-M)Analysis of hair bundle polarity in OHCs and
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-DCD2 OHCs showed polarity defects
(wild type, n599; DCD2, n849). Fifty percent of the OHCs examined
deviated by 15° or more. Scale bars: 10m in A-D; 5m in E,F; 2m
in G-J.
strong throughout the bundles of immature hair cells and near the
interface between stereocilia and the kinocilium (Fig. 6). We
conclude that in OHCs, PCDH15-CD2 is targeted to the stereocilia
and kinocilium, and is appropriately positioned to contribute to the
formation of linkages between steoreocilia, and between the
stereocilia and the kinocilium. A similar expression pattern is
observed in vestibular hair cells, but there is little expression in
IHCs.
Defects in kinociliary links in PCDH15-DCD2 mice
We next analyzed hair bundles of cochlear hair cells at P1 and P6
by staining with fluorescence labeled phalloidin to reveal actin, and
antibodies to acetylated-a-tubulin to reveal the kinocilium. In wildtype mice, the kinocilium was tightly associated to the longest
stereocilia at the bundle apex (Fig. 7A,A⬘,C,C⬘). By contrast, the
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
wild-type OHCs and IHCs (A,B), and wild-type OHCs (E-H), PCDH15CD2 was present throughout the hair bundle. No staining was
observed in PCDH15-DCD2 mice (C,D). (E-H)In wild-type mice,
prominent staining was evident in the region proximal to the kinociliastereocilia interface (arrows in E,F). Staining in OHCs was often
observed as a puncta at the distal kinociliary tips (arrows in G,H). Scale
bars: 4m in A-D; 2m in E-H.
position of the kinocilium was more variable in PCDH15-DCD2
mice (Fig. 7B,B⬘,D,D⬘), suggesting that kinociliary links might be
affected. This was confirmed by SEM analysis. Although kinocilia
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
supplementary material). A similar phenotype was observed at E15
(Fig. 10).
Previous studies have suggested that several PCDH15 isoforms
might be present at transient lateral links (Ahmed et al., 2006).
Unlike in Ames waltzerav3J mice, hair bundles of IHCs and OHCs
were not fragmented in PCDH15-DCD1, PCDH15-DCD2 and
PCDH15-DCD3 mice (Fig. 4A-D,F,I,J; Fig. 7B,B⬘,D,D⬘ and see
Fig. S4 in the supplementary material), suggesting that some of
these isoforms have redundant functions at transient lateral links.
This was confirmed by SEM analysis. In PCDH15-DCD2 mice, the
kinocilium was separated from the stereocilia, but linkages along
the length of stereocilia were visible (Fig. 7H-J; Fig. 8A-D). Such
linkages were also observed in hair bundles from PCDH15-DCD1
and PCDH15-DCD3 mice (see Fig. S5 in the supplementary
material).
DEVELOPMENT
PCDH15 function and hair cells
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-C⬘,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
observed as a puncta at the distal end of the tallest stereocilia (arrows
in A-C,J-L). Scale bars: 10m in A-F; 5m in G-L.
At P96, there was widespread degeneration of hair bundles in
the cochlea of PCDH15-DCD2 mice (see Fig. S3C in the
supplementary material), although some hair bundles were
maintained (see Fig. S3D in the supplementary material). The
degenerative changes probably contribute to deafness of PCDH15DCD2 mice. Stereocilia of OHCs that were maintained in the
mutants formed connections to the tectorial membrane, as evident
from their imprint in the tectorial membrane (see Fig. S3B in the
supplementary material).
Tip links and mechanotransduction
PCDH15-DCD1 and PCDH15-DCD3 mice showed normal
hearing, suggesting that tip links were unaffected. Deafness in
PCDH15-DCD2 mice is probably caused by defects in hair bundle
morphology and maintenance, but tip-link function may also be
impaired. We therefore analyzed tip links by SEM in P7 mice.
Linkages running from the top of stereocilia to the side of the next
taller stereocilia were observed in PCDH15-DCD1, PCDH15DCD2 and PCDH15-DCD3 mice (Fig. 8C-G), indicating that tip
links were preserved.
To determine whether hair cells from PCDH15-DCD2 mice had
functional transduction, we measured transducer currents in P7-P8
OHCs from the apical/middle part of the cochlea using whole cell
Development 138 (8)
Fig. 7. Kinocilia position in OHCs from PCDH15-DCD2 mice.
(A-D⬘) Staining of cochlear wholemounts from P6 wild-type and
PCDH15-DCD2 mice with phalloidin (green) and antibodies to acetylated
a-tubulin (red). Kinocilia (arrows) in wild-type hair cells (A,A⬘,C,C⬘) were
linked to the stereociliary bundles; those of PCDH15-DCD2 hair cells
(B,B⬘,D,D⬘) were separated from the bundle and misplaced. Cilia on
support cells are marked with an asterisk. (E-J)SEM examination of wildtype and PCDH15-DCD2 OHCs at P1. (E-G)Wild-type hair cells showing
tight association between the stereocilia bundle and kinocilium (arrows
in E,F; asterisk in G). (H-J)Kinocila (arrows in H,I; asterisk in J) in PCDH15DCD2 mice were disconnected from the stereocilia. Scale bars: 5m in
A-D; 5m in E,F,H; 2m in I; 0.5m in G,J.
recordings. We focused our analysis on hair bundles with minimal
polarity defects. In agreement with previous findings (Kennedy et
al., 2003; Kros et al., 2002; Stauffer and Holt, 2007; Waguespack
et al., 2007), control OHCs had rapidly activating transducer
currents, which subsequently adapted (Fig. 8H, blue traces); similar
current traces were obtained with OHCs from PCDH15-DCD2
mice (Fig. 8H, red traces). The amplitudes of saturated
mechanotransduction currents at maximal deflection were similar
in controls and mutants (Fig. 8H,I), suggesting that the total
number of transducer channels was not altered in mutants. We also
plotted the current/displacement relationships and channel open
probability/displacement relationship and observed no significant
difference between wild-type and mutants (Fig. 8I,J), indicating
that the transduction machinery of hair cells in PCDH15-DCD2
mice functioned normally. Finally, as PCDH15-CD3 has previously
DEVELOPMENT
1612 RESEARCH ARTICLE
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 secondorder 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.
Defects in kinociliary positioning in PCDH15-DCD2
mice
The kinocilium of hair cells is thought to be important for the
development of hair bundle polarity. To determine the extent to
which kinocilia position and bundle rotation in PCDH15-DCD2
mice correlates, we determined kinociliary position relative to
stereociliary bundles (Fig. 9A). In IHCs and OHCs of wild-type
mice, the position of the kinocilium predicts the orientation of the
bundle (Fig. 9B,C). By contrast, kinocilia position did not predict
bundle orientation in PCDH15-DCD2 mice and frequently deviated
90° or more from the normal position (Fig. 9B,C).
In wild-type hair cells, the centrioles of the basal body undergo
a similar polarized movement to the kinocilium (Jones et al., 2008).
Consistent with these data, staining for pericentrin revealed the
polar localization of the centrioles in OHCs from wild-type mice
at P1 (Fig. 9D-F⬘). In PCDH15-DCD2 mice, the two centrioles and
kinocilium were coordinately mislocalized (Fig. 9G-I⬘) and the two
centrioles were no longer aligned appropriately relative to each
other along the axis of bundle polarity (Fig. 9G-I⬘). We conclude
that kinociliary links are required to coordinate the polarization of
the kinocilium and their basal bodies with the polarization of the
stereociliary bundle.
Normal localization of components of the core
PCP pathway
Random polarization of hair bundles, similar to that observed in
PCDH15-DCD2 mice, is a hallmark of mice with mutations in
orthologs of genes linked to the Bardet-Biedl syndrome (Ross et
al., 2005), and in mice with mutations that affect the intraciliary
transport protein Ift88 (Jones et al., 2008). The ciliary genes are
thought to act downstream of components of the core PCP
pathway. We therefore asked whether core PCP proteins showed
normal asymmetric localization in PCDH15-DCD2 mice. We
stained cochlear wholemounts of P1 mice with phalloidin and
antibodies to Fzd6 and Vangl2, which are normally localized at
the medial edge of OHCs (Fig. 9J-M⬘; data not shown). The
asymmetric distribution of Fzd6 (Fig. 9L-M⬘) and Vangl2 (data not
shown) was not altered in PCDH15-DCD2 mice, suggesting that
PCDH15-CD2 acts downstream of the PCP pathway.
Analysis of vestibular hair cell morphology and
function
It is thought that the kinocilium of vestibular hair cells is required
for mechanical coupling between the hair bundle and the overlying
gelatinous matrix (Roberts et al., 1988). We therefore wondered
whether kinociliary links and vestibular function were affected in
PCDH15-DCD2 mice. Analysis at E15.5, P1 and P6 revealed that
although the kinocilium was tightly linked to hair bundles in wildtype mice (Fig. 10A-B⬘; data not shown), the kinocilium was
separated from the stereociliary bundle in PCDH15-DCD2 mice in
hair cells in the utricle, saccule and semicircular canals (Fig. 10CD⬘; see Fig. S7 in the supplementary material; data not shown).
Stereocilia were also in contact with the overlying gelatinous
matrix (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-DCD2
mice (and PCDH15-DCD1 and PCDH15-DCD3 mice) did not
circle (data not shown). To evaluate vestibular function
quantitatively, we measured eye movements in alert mice mediated
by the vestibulo-ocular reflex in response to whole-body head
rotations (Fig. 10E-G). Mice were immobilized in a superstructure
mounted atop a motor able to deliver 3000°/s constant accelerations
to a plateau velocity of 300°/s about the dorsoventral axis of the
animal through the stereotaxic origin of the skull. Eye movements
were measured using a three-dimensional video-oculography
system. We quantified responses to whole-body, passive, transient
rotations in darkness using three parameters: Ga (‘constantacceleration gain’) was computed as the ratio of eye to head
velocity averaged over the constant acceleration segment of the
stimulus; Gv (‘constant-velocity gain’) was computed as the ratio
of eye to head velocity averaged over the constant velocity segment
of the stimulus; latency is the delay from the onset of head
DEVELOPMENT
been proposed as a component of tip links (Ahmed et al., 2006),
we also measured transducer currents in PCDH15-DCD3 mice but
observed no defects (see Fig. S6 in the supplementary material).
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
PCDH15-DCD2 bundles, the kinocilia were frequently mislocalized (wild
type, n203; DCD2, n106). Of the OHCs examined, 48.2% had
kinocilia mislocalized by 15° or more. (D-I⬘) Cochlear wholemounts
were stained with phalloidin (green), antibodies to pericentrin (red) and
antibodies to acetylated a-tubulin (blue). In wild type but not in the
mutants, the basal bodies (arrows) aligned with the kinocilium along
the hair bundle polarity axis. (J-M⬘) Cochlear wholemounts were
stained with phalloidin (green) and antibodies to frizzled 6 (red).
Frizzled 6 localization (arrows) was not affected in the mutants. The
presumed position of the kinocilium is indicated by an asterisk (J⬘,L⬘).
Scale bars: 5m.
movement to the onset of eye movement, computed through linear
extrapolation of eye and head responses during the constantacceleration segment of the stimulus. Measurements were carried
out with PCDH15-DCD2 mice (n6), C57BL6⫻129 wild-type
littermate controls (n8), C57BL/6 wild-type controls (n7), Ames
waltzerav3J mice (n3) and one dead mouse (to rule out technical
artifacts). Ames waltzerav3J mice and dead mice did not show any
response. However, there was no statistically significant difference
in any of the parameter between PCDH15-DCD2 mice and wildtype mice (Fig. 9E-G). In addition, we analyzed the response to
head pitch tilts about the intreraural axis and observed normal
responses in the mutants (data not shown). These data show that
vestibular function was surprisingly intact in PCDH15-DCD2 mice.
DISCUSSION
Here, we show that alternative splicing of PCDH15 regulates its
function in hair cells. Using isoform-specific knockout mice, we
show that PCDH15-CD1 and PCDH15-CD3 are not essential for
Development 138 (8)
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 reveal
tight coupling of the kinocilia (arrows) to the stereocilia. (C-D⬘) Kinocilia
(arrows) of PCDH15-DCD2 vestibular hair cells were not connected to
the stereocilia. Images of hair cells are from E15 crista (A,A⬘,C), E15
utricle (C⬘) and P1 ampulla (B,B⬘,D,D⬘). (E-G)Vestibulo-ocular reflex
response for PCDH15-DCD2, wild-type littermates (C57BL/6⫻129SvEv),
C57BL/6 mice, Ames waltzerav3J mice and a dead mouse. (E)Eye versus
head horizontal velocity ratio, Ga, during the constant acceleration
(3000° s–2) segment of a passive transient whole-body rotation about a
vertical axis. (F)Velocity ratio, Gv, during the constant velocity (300° s–1)
plateau of the same stimulus. (G)Response latency from onset of head
motion to onset of eye motion (mseconds). Diamonds and whiskers
indicate mean±s.d.; each data point is an the mean response of an
animal; n≥10 head rotations in each direction. Excluding the Ames
waltzerav3J mice and dead controls (for which Ga⬇Gv⬇0), one-way
ANOVA revealed no significant difference among groups in Ga, Gv or
latency (P>0.05). Including the Ames waltzerav3J mice, controls in the
analyses revealed that all other live mouse groups differed significantly
in Ga and latency from Ames waltzerav3J mice controls (P<0.001); Gv
showed a similar trend but did not reach significance. There was a
trend towards PCDH15-DCD2 mutants having longer response latency
than control mice, but this trend did not reach significance (P0.062).
These data suggest that PCDH15-DCD2 mice have horizontal
semicircular canal function similar to both of the wild-type control
groups but significantly better than Ames waltzerav3J mice. Scale bars:
5m in A-D; 2.5m in A⬘-D⬘.
hair cell function, whereas PCDH15-CD2 is required for the
formation of kinociliary links. In the absence of PCDH15-CD2, hair
bundles consisting of rows of stereocilia of graded heights develop
but bundle polarity is affected, demonstrating that kinociliary links
DEVELOPMENT
1614 RESEARCH ARTICLE
are not essential for the development for the stereociliary staircase
but for the morphological transformation that lead to bundle
polarization. Components of the PCP pathway are normally
distributed in the sensory epithelia of PCDH15-DCD2 mice,
suggesting that PCDH15-CD2 acts downstream of the PCP
pathway. Hair bundles in PCDH15-DCD2 mice are gradually lost
postnatally, indicating that the morphological defects affect hair cell
function causing deafness. In addition, we cannot exclude that
cochlear hair cells do not function normally because their stereocilia
are not connected to the kinocilium. Unlike in Ames waltzerav3J
mice, which carry a predicted PCDH15 null allele, stereociliary
bundles are not fragmented in mice individually lacking PCDH15CD1, PCDH15-CD2 and PCDH15-CD3, suggesting that several
PCDH15 isoforms contribute to the formation of transient lateral
links. We also observed that tip links are present in PCDH15-DCD1,
PCDH15-DCD2 and PCDH15-DCD3 mice, providing evidence that
none of these isoforms is uniquely required at tip links.
One of the central findings of our study is that PCDH15-CD2 is
required for the development of kinociliary links and the normal
polarization of hair bundles. Initial studies by the Tilney laboratory
suggested that the kinocilium is important for bundle polarization
(Tilney et al., 1992), but the mechanism by which the kinocilium
determines polarity had remained unclear. Recent studies have
shown that mutations in components of the core PCP pathway and
mutations affecting the kinocilium cause polarity defects. When
PCP signaling is defective, hair bundles maintain intrinsic polarity
but are randomly polarized in the apical cell surface. When the
kinocilium is affected, bundles show random polarization within
the apical surface and loss of intrinsic polarity (Curtin et al., 2003;
Montcouquiol et al., 2003; Lu et al., 2004; Ross et al., 2005; Wang
et al., 2005; Montcouquiol et al., 2006; Wang et al., 2006; Jones et
al., 2008). The asymmetric localization of PCP proteins at the
border between hair and support cells is not affected in ciliary
mutants (Jones et al., 2008), suggesting that ciliary genes act
downstream of PCP components (Jones and Chen, 2008). In
PCDH15-DCD2 mice, the PCP components Frz6 and Vangl2 are
normally distributed, and hair bundle morphology resembles that
of the ciliary mutants, suggesting that PCDH15-CD2 behaves like
a ciliary mutant acting downstream of the PCP pathway. In the
PCDH15-DCD2 mutants, the kinocilium and stereocilia bundle are
mislocalized, but not always in same direction, suggesting that
kinociliary links are required to coordinate movement of the
kinocilium and stereocilia.
We have previously shown that tip links in hair cells have an
intrinsic asymmetry in the planar polarity axis of hair cells, where
PCDH15 forms the lower end of tip links and CDH23 the upper
end (Kazmierczak et al., 2007). CDH23 and PCDH15 are also
asymmetrically distributed at kinociliary links (Goodyear et al.,
2010). However, polarity is reversed relative to tip links, with
PCDH15 being present in the kinocilium and CDH23 in the longest
stereocilia (Fig. 1A). Our findings indicate that PCDH15-CD2 is
the PCDH15 isoform in kinocilia and suggest a mechanism by
which it might regulate polarity. As the kinocilium, unlike the
stereocilia, contains microtubules, it seems likely that the CD2
cytoplasmic domain contains specific sequence motifs for targeting
to the kinocilia that are absent in CD1 and CD3. In this way, a
polarity axis is established in kinociliary links. The fact that some
PCDH15-CD2 is present in stereocilia could be explained because
actin-based molecular motors might also transport PCDH15-CD2.
Alternatively, as PCDH15 molecules form dimers (Kazmierczak et
al., 2007), PCDH15-CD2 might enter stereocilia as a heterodimer
with PCDH15-CD1 and/or PCDH15-CD3.
RESEARCH ARTICLE 1615
Our data show that several PCDH15 isoforms have redundant
function 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 in
PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3 mice, it
seems that several of these isoform contribute to transient lateral
links. Consistent with this finding, we observed by SEM abundant
links between the stereocilia in all three mutants. Similarly, it seems
that neither PCDH15-CD1, PCDH15-CD2 and PCDH15-CD3
are uniquely required for tip-link formation. Previous
immunolocalization studies have failed to clearly define the
PCDH15 isoform at tip links. PCDH15-CD3 has been the best
candidate, as one CD3-specific antibody bound to the region of the
lower tip link end. However, the same antibody did not bind to
adult murine hair cells and a second PCDH15-CD3 antibody bound
to stereocilia more broadly (Ahmed et al., 2006). Using antibodies
against PCDH15 isoforms, we have also been unable to define the
PCDH15 isoform at tip links, as different antibodies to the same
isoform revealed distinct expression patterns. This variation might
be 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 for
tip-link formation. Extracellular filaments with tip link features are
present in PCDH15-DCD1, PCDH15-DCD2 and PCDH15-DCD3
mice. In addition, auditory and vestibular function is preserved in
PCDH15-DCD1 and PCDH15-DCD3 mice, and vestibular function
in PCDH15-DCD2 mice. OHCs in PCDH15-DCD2 and PCDH15DCD3 mice also show normal responses to mechanical stimulation
in vitro. An important implication of our findings is that the
mechanotransduction channel in hair cells, which is localized in
proximity to the lower tip-link end (Beurg et al., 2009) might bind
to the extracellular and/or transmembrane domain of PCDH15 that
is shared by several PCDH15 isoforms.
We were surprised to find that vestibular function was not
noticeably altered in PCDH15-DCD2 mice. The kinocilium of
vestibular hair cells is coupled to the kinocilium and the overlying
gelatinous extracellular matrices. Unlike in the cochlea, adult
vestibular hair cells do not loose their kinocilium. It has therefore
been assumed that the kinocilium mechanically couples the hair
bundle to the overlying gelatinous matrix (Roberts et al., 1988).
Our findings now suggest that murine vestibular hair cells function
normally, even when linkages between the kinocilium and
stereocilia are disrupted. Direct coupling of the stereocilia to the
gelatinous matrices is probably sufficient to transmit motion to
stereocilia and activate transduction channels.
Our functional data were collected with mice of a mixed
C57BL6⫻129SvEv background. Although we did not observe
differences in PCDH15 isoform expression between inbred mouse
strains, we cannot exclude the possibility that different phenotypes
might be observed on distinct genetic backgrounds.
Our findings could have important ramifications for the analysis
of the genetic causes of sensory impairment. Several PCDH15
mutations have been identified that cause Usher Syndrome and
recessive 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 to
the extracellular PCDH15 domain. Our findings suggest that it would
be important to determine whether mutations that specifically affect
the PCDH15-CD2 isoform might lead to auditory impairment. Last,
mutations specific for PCDH15-CD1 and PCDH15-CD3 might lead
to retinal disease without auditory dysfunction.
DEVELOPMENT
PCDH15 function and hair cells
Acknowledgements
This work was funded by NIDCD grants DC007704, DC005965, DC005965-S1
(U.M.), DC9255, DC2390 (C.D.S.) and NIDCD-DIR-Z01DC000002 (B.K.); by the
Skaggs Institute for Chemical Biology (U.M.); by the Dorris Neuroscience
Center (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 12
months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.060061/-/DC1
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PCDH15 function and hair cells