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First published online 13 March 2008
doi: 10.1242/dev.012922
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1 Unité de Génétique des Déficits Sensoriels,
UMRS587 INSERM-Université Paris VI, Institut Pasteur, 25 rue du Dr
Roux, 75724 Paris cedex 15, France.
2 Johannes Gutenberg University of Mainz, Institute of Zoology, Cell and Matrix
Biology, Muellerweg 6, D-55099 Mainz, Germany.
3 Collège de France, 11 place Marcellin Berthelot, 75231 Paris cedex 05,
France.
* Author for correspondence (email: cpetit{at}pasteur.fr)
Accepted 7 February 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Usher syndrome, Hair bundle links, Planar polarity, Stereocilia growth, Harmonin (USH1C), Cadherin 23 (USH1D), Protocadherin 15 (USH1F), Sans (USH1G), Myosin VII (USH1B)
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Alagramam et al., 2001b;
Bitner-Glindzicz et al., 2000;
Bolz et al., 2001;
Bork et al., 2001;
Verpy et al., 2000;
Weil et al., 1995;
Weil et al., 2003).
The cochlear and vestibular sensory cells (hair cells) respond to sound and
head movements, respectively. The hair bundle located at their apex is the
mechanosensitive organelle. The cochlear hair bundle consists of three to four
graded rows of F-actin-filled, large and stiff microvilli, known as
stereocilia, which are interconnected by fibrous links and together form a
`V'-shaped staircase (Fig. 1B).
During its development, the cochlear hair bundle also includes a single
transient genuine cilium, the kinocilium, which is located at the vertex of
the `V' and is also connected to the tallest stereocilia row by fibrous links
(Fig. 1B). All vertices of
cochlear hair bundles point to the lateral wall of the cochlear duct, thereby
defining the planar cell polarity (PCP) axis of the sensory epithelium (organ
of Corti) (Fig. 1B). Such an
organization allows the uniform deflection of hair bundles along the PCP axis
in response to a sound stimulus. Indeed, deflection along this axis, which is
also the hair bundle symmetry axis, is the only effective direction to gate
the mechanoelectrical transducer ion channels. According to the `gating
spring' model, hair bundle deflection towards the tallest row of stereocilia
stretches the tip link (a single apical link that connects the tip of each
stereocilium to the side of its adjacent taller neighbor). This in turn opens
the transduction channel that is tethered to this link and causes hair cell
depolarization (Hudspeth,
1985). Increasing evidence indicates that cadherin 23 and
protocadherin 15 make up the upper and lower parts of the tip link,
respectively (Ahmed et al.,
2006; Kazmierczak et al.,
2007; Siemens et al.,
2004).
Disorganization of the inner ear hair bundles has been observed in
spontaneous mouse mutants carrying allelic variants of each USH1 gene
ortholog, namely shaker 1 (Myo7ash1), deaf circler
(Ush1cdfcr), waltzer (Cdh23v), Ames
waltzer (Pcdh15av) and Jackson shaker
(Ush1gjs) (Alagramam et
al., 2001a; Di Palma et al.,
2001; Gibson et al.,
1995; Johnson et al.,
2003; Kikkawa et al.,
2003; Kitamura et al.,
1992; Pawlowski et al.,
2006; Wilson et al.,
2001). The abnormal hair bundle architecture of these mutants
accounts for their hearing and balance impairments. In addition, it has led to
discovery of the existence of transient lateral links that connect the
stereocilia together and with the kinocilium during early hair bundle
development (Goodyear et al.,
2005). Some of these links are indeed composed of cadherin 23
(Michel et al., 2005) and
protocadherin 15 (Ahmed et al.,
2006). The role played by these early connectors in the
differentiation of the hair bundle is, however, poorly understood. A
scaffolding role has been ascribed to harmonin, as its PDZ domains are
expected to allow the formation of large molecular complexes. Myosin VIIa is
an actin filament plus-end-directed myosin, which is therefore expected to
move from the base to the apex of stereocilia. Moreover, genetic evidence
supports its involvement in the targeting of various hair bundle proteins,
including harmonin, during postnatal stages
(Boeda et al., 2002;
Michalski et al., 2007;
Senften et al., 2006).
Several in vitro interactions between USH1 proteins have been reported
(Fig. 1C). Harmonin can bind to
the four other USH1 proteins (Adato et al.,
2005; Boeda et al.,
2002; Reiners et al.,
2005; Siemens et al.,
2002; Weil et al.,
2003), and myosin VIIa can interact with sans and protocadherin 15
(Adato et al., 2005;
Senften et al., 2006). By
contrast, only sparse evidence has been collected to support direct
interactions between these proteins within the hair bundle. In addition, the
precise developmental processes in which these proteins are involved are still
unknown. We addressed these issues by searching for common hair bundle
anomalies in mouse models for each of the five USH1 genetic forms, and by
investigating the possible co-distributions and interdependent localizations
of the Ush1 proteins.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Rabbit polyclonal antibodies used to detect myosin VIIa (here named
Myo7a-F1; 1:1000), harmonin-b (harmonin-H1b; 1:100), cadherin 23a (Cdh23-N1;
1:100) and protocadherin 15a/b (here named Pcdh15-cter; 1:500) have been
described (Boeda et al., 2002;
el-Amraoui et al., 1996;
Reiners et al., 2005). The
rabbit anti-Vangl2 antibody (1:200) was kindly provided by Drs Yao and Noda
(JFCR Cancer Institute, Tokyo, Japan). Additional antibodies are commercially
available: goat anti-Scrib1 (1:100; Santa Cruz Biotechnology, Santa Cruz, CA),
rabbit anti-frizzled 3 (1:200; Sigma, Evry, France), mouse anti-β-catenin
(1:200; Transduction Laboratories, BD Biosciences, Le Pont de Claix, France),
mouse anti-acetylated-tubulin (1:200; Sigma), Alexa Fluor 488 or 546
F(ab')2 fragment of goat anti-rabbit IgG, Alexa Fluor 488
F(ab')2 fragment of goat anti-mouse IgG, Alexa Fluor 488
donkey anti-goat IgG (1:500; Molecular Probes-Invitrogen, Cergy Pontoise,
France) and TRITC-conjugated phalloidin (1:1000; Sigma).
Whole-mount immunofluorescence
Mouse inner ears were dissected from temporal bones at different
developmental stages. The cochlear shell was pierced at its apex, and the
round and oval windows opened. Generally, inner ears were then immersed for
fixation in 4% paraformaldehyde in PBS for 1 hour. After several washes in
PBS, the cochlear shell was finely dissected and the organ of Corti processed
as described (Michel et al.,
2005). For Vangl2, the cochlear shell was dissected prior to
fixation and the organ of Corti was only quickly fixed for 5 minutes in cold
(-20°C) methanol. In this staining procedure, an anti-β-catenin
antibody was used to label the apical region of hair cells because methanol
has deleterious effects on the actin cytoskeleton. Fluorescence images were
obtained with a confocal microscope (Zeiss LSM 510 META) equipped with a Plan
Apochromat 63x/1.4 oil immersion objective. z-stack images were
deconvoluted and reconstructed with Huygens (Scientific Volume Imaging,
Hilversum, The Netherlands) and Image J (Rasband WS, US NIH, Bethesda, MD;
http://rsb.info.nih.gov/ij/)
software, respectively.
Scanning electron microscopy
Inner ears of Ush1 mutant mice were processed as for
immunofluorescence, except that they were fixed by immersion in 2.5%
glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) at room temperature for 2
hours. After several washes with buffer alone, they were finely dissected to
provide direct access to the cochlear and vestibular sensory areas. The
samples were then dehydrated by successive washes in ethanol (50, 70, 80, 90
and 100%), critical-point dried, mounted on a stub, sputter-coated with
gold-palladium and examined under a Jeol JSM6700F scanning electron
microscope.
Angular deviations were measured between the expected and effective positions of the kinocilia at the apical surfaces of hair cells from the cochlear apical turn, using the graphic tools of the Jeol JSM6700F software. Briefly, for each hair cell, the kinociliary deviation was determined by the angle formed by two crossing lines. The first line was drawn mediolaterally along the symmetry/PCP axis of the cell, thereby running through the expected position of the kinocilium. The second line was traced between the center of the hair cell surface and the effective position of the kinocilium. Data were analyzed using Excel (Microsoft).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), whereas virtually all USH1C mutations reported so
far in patients are expected to be `functional null alleles'
(Bitner-Glindzicz et al., 2000;
Verpy et al., 2000;
Zwaenepoel et al., 2001).
Therefore, we engineered a harmonin-null (Ush1c-/-) mouse
lacking Ush1c exon 1, which contains the translation initiation site
common to all reported harmonin isoforms (see Fig. S1 in the supplementary
material).
Ush1 mutant mice exhibit fragmented and misoriented hair bundles from E17.5 onwards
In the mouse cochlea, postmitotic cells of the primordial sensory
epithelium differentiate in concurrent medial-to-lateral (neural-to-abneural)
and basal-to-apical gradients along the cochlear duct between E14.5 and 18.5.
Sensory cells concomitantly become organized into one row of inner hair cells
(IHCs, the genuine sensory cells) and three rows of outer hair cells (OHCs,
the cochlear amplifiers) (Fig.
1B), by cell intercalations. This convergent extension process
results in the shortening in width and extension in length of the sensory
epithelium. Actin-rich protrusions, which will ultimately form the
stereocilia, soon emerge at the hair cell apical surface
(Chen and Segil, 1999;
Lim and Anniko, 1985;
Sher, 1971). In the mouse,
temporal information regarding the differentiation of these protrusions into
fully mature stereocilia is limited. In the rat and hamster, the protrusions
rapidly assemble into a bundle and grow uniformly until late embryonic stages.
Then, the growing stereocilia elongate differentially according to their
location within the bundle, so that the staircase-like pattern of the
stereocilia rows is settled a few days after birth. While supernumerary
stereocilia are reabsorbed, the remaining stereocilia continue to grow
simultaneously in length and width, until the hair bundle reaches its mature
shape (Kaltenbach et al.,
1994; Zine and Romand,
1996). Therefore, initial, intermediate and final periods of hair
bundle development can be distinguished, in which the growth of the
stereocilia row is uniform, differential and simultaneous, respectively. From
our observations (see below), we established that the first two periods extend
from E15.5 to P0, and from P1 to around P5, respectively, whereas the third
lasts until P15, in the mouse. To determine and characterize hair bundle
morphogenetic defects in mice defective in myosin VIIa
(Myo7a4626SB/4626SB), harmonin
(Ush1c-/-), cadherin 23
(Cdh23v2J/v2J), protocadherin 15
(Pcdh15av3J/av3J) and sans
(Ush1gjs/js), we undertook a systematic and comparative
analysis of the different Ush1 mutant hair bundles during these three
developmental periods.
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Examination by confocal microscopy of E18.5 cochleas stained with actin and
microtubule markers revealed that a large proportion of the hair cells
displayed a mispositioned kinocilium in Ush1 mouse mutants
(Fig. 3A and data not shown).
Moreover, observation of Ush1 mutant hair bundles by SEM at P0 showed
that the stereociliary clumps were also often misoriented (Figs
2,
3). According to the current
view, the kinocilium leads the establishment of the stereociliary bundle
polarity (Jones and Chen,
2007). Therefore, we quantified the hair bundle polarity defect of
the different Ush1 mutants by measuring, on each hair cell surface,
the kinocilium deviation (in degrees) from its expected position along the PCP
axis (arbitrarily fixed as 0°) (Fig.
3B; see Materials and methods for details). Consistent with the
previous study by Dabdoub et al. (Dabdoub
et al., 2003), kinocilia had mean absolute deviations ranging from
6.5° in IHCs to 12.5° in OHCs of the third row in P0 wild-type mice.
They were equally distributed on both sides of the PCP axis, and more than 80%
of them were present within ±15° of the PCP axis
(Fig. 3C and data not shown).
By contrast, most hair cells of the Ush1 mutants displayed
mispositioned kinocilia. Both the IHCs and the three rows of OHCs showed
larger mean kinociliary deviations than in wild-type mice
(Fig. 3C). Only 37% of
kinocilia at best (Ush1gjs/js), and 14% at worst
(Cdh23v2J/v2J), were present within ±15° of the
PCP axis in the Ush1 mutant hair cells
(Fig. 3C). The mean absolute
kinociliary deviations of IHCs and OHCs were 25°
(Ush1gjs/js), 26°
(Myo7a4626SB/4626SB), 26°
(Ush1c-/-), 38° (Pcdh15av3J/av3J)
and 52° (Cdh23v2J/v2J)
(Fig. 3C). Nevertheless,
virtually all kinocilia, apart from a small proportion in
Cdh23v2J/v2J and Pcdh15av3J/av3J hair
cells, were located within the lateral half and near the edge of the apical
cell surface. Notably, in Cdh23v2J/v2J and
Pcdh15av3J/av3J mice, the kinocilia were also often
dissociated from the stereociliary clumps
(Fig. 3C).
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). We therefore analyzed the distribution of two of these
proteins, vang-like 2 (Vangl2) and frizzled 3, which are required for the
establishment of inner ear PCP
(Montcouquiol et al., 2003;
Wang et al., 2006). In E18.5
Ush1c-/-, Cdh23v2J/v2J and
Pcdh15av3J/av3J mice, Vangl2 and frizzled 3 were
asymmetrically distributed on the apical, medial side of hair cells, as in
wild-type littermates (see Fig. S2 in the supplementary material). In
addition, scribble 1 (Scrib1; scribbled), which has also been found to be
involved in inner ear PCP (Montcouquiol et
al., 2003), was normally distributed along the hair cell
basolateral membrane in all Ush1 mutant mice (see Fig. S2 in the
supplementary material). Of note, PCP proteins are also required for cochlear
convergent extension (Jones and Chen,
2007). In agreement with the normal distributions of Vangl2,
frizzled 3 and Scrib1, organs of Corti in all Ush1 mouse mutants had
normal length and width at P0, and they did not show extra hair cell rows
(data not shown).
Ush1 proteins localize at stereocilia tips during initial hair bundle development
The early core phenotype that we identified in Ush1 mutant mice as
early as E17.5 suggests that the Ush1 proteins cooperate during the initial
period of hair bundle development. To determine whether Ush1 proteins
colocalize during this period, we studied their distribution by
immunofluorescence analysis of whole-mount cochleas. For each Ush1 protein, we
focused on specific isoform classes that have been shown to be either
prominent in, or largely restricted to, the inner ear and retina
(Ahmed et al., 2006;
Ahmed et al., 2003;
Hasson et al., 1995;
Lagziel et al., 2005;
Michel et al., 2005;
Reiners et al., 2003;
Rzadzinska et al., 2005;
Verpy et al., 2000). Thus,
submembrane actin-binding class b harmonin (harmonin-b), transmembrane class a
cadherin 23 (cadherin 23a), transmembrane class a and class b protocadherin 15
(protocadherin 15a/b), and myosin VIIa isoforms were specifically stained by
the harmonin-H1b, Cdh23-N1, Pcdh15-cter and Myo7a-F1 antibodies, respectively
(Fig. 4). The specificity of
these antibodies was established by the loss of hair bundle immunoreactivity
in each mouse mutant deficient for the corresponding protein (see Fig. S3 in
the supplementary material). In the absence of a similar specificity
indication for our anti-sans antibody, we did not study the distribution of
this protein.
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Harmonin-b localization to the tips of emerging stereocilia is dependent on the presence of myosin VIIa and sans
The simultaneous presence of four Ush1 proteins at stereocilia tips during
the initial phase of hair bundle morphogenesis, in conjunction with their
well-documented in vitro interactions, suggested that they can interact during
this period of development. Because of the central role played by harmonin in
these in vitro interactions (Fig.
1C) (Adato et al.,
2005; Boeda et al.,
2002; Reiners et al.,
2005; Siemens et al.,
2002; Weil et al.,
2003), we further investigated the relationship between harmonin
and the other Ush1 proteins. To this purpose, we analyzed the effect of the
absence of harmonin on the distribution of myosin VIIa, cadherin 23a and
protocadherin 15a/b, and analyzed the distribution of harmonin-b in the
different Ush1 mutants, during the initial period of hair bundle
development, i.e. until P0.
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The intermediate phase of stereociliary growth is defective in Ush1 mutant mice
Between P0 and P5, the three outward-most stereocilia rows of wild-type
hair bundles showed differential row-specific elongation, ultimately leading
to the characteristic staircase-like pattern of mature hair bundles
(Fig. 6A). Strikingly, SEM
analysis of Ush1 mutant hair bundles during this period of
development showed that many stereocilia were of abnormal length
(Fig. 6A,B). Many stereocilia
of Myo7a4626SB/4626SB mice were taller than those in
wild-type mice (Fig. 6B). By
contrast, in all the other Ush1 mouse mutants, many stereocilia of
the small and, to a lesser extent, medium rows were shorter than expected,
whereas stereocilia of the tall row were of normal height
(Fig. 6A,B). In particular, in
OHCs defective in harmonin, the majority of stereocilia from the small and
medium rows did not elongate further from P0. At P5 they were in the process
of regressing, and at P15 they had disappeared
(Fig. 6A).
As row-specific elongation proceeded, the tips of small and medium
stereocilia evolved from a round, oblate shape into an asymmetric, prolate
shape in wild-type hair bundles (Fig.
6C). Such a morphological change is thought to result from tension
forces applied, via the tip link, to the apical membrane of these stereocilia
(Rzadzinska et al., 2004). In
Ush1 mutant hair bundles, especially in IHCs, stereocilia of the
small and medium rows virtually never had prolate tips, but rather had round,
oblate tips (Fig. 6C),
indicating that these putative tension forces might not develop properly in
the absence of Ush1 proteins.
We therefore examined the distribution of interstereociliary links in the
different Ush1 mutant hair bundles. During late embryonic and early
postnatal stages, the lateral fibrous links that are initially present all
along the shafts of the growing stereocilia progressively become restricted
towards the distal end of the stereocilia (forming apical lateral links),
while two subsets of interstereociliary links can progressively be
distinguished at their tips (tip links) and bases (ankle links)
(Goodyear et al., 2005). Ankle
links could be seen in most hair bundles of all Ush1 mutants (data
not shown), except Myo7a4626SB/4626SB. This is consistent
with the requirement of myosin VIIa for the stereociliary targeting of
molecular components of the ankle links
(Michalski et al., 2007). By
contrast, in P5 Cdh23v2J/v2J and
Pcdh15av3J/av3J mice, we failed to detect the presence of
any apical (lateral or tip) links. In myosin VIIa-, harmonin- and
sans-deficient mice, however, some of these links were still visible, but they
appeared sparser than in wild-type mice
(Fig. 6C).
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Remarkably, we observed that the harmonin-H1b immunoreactivity remained at stereocilia tips and was not detected in the region of the tip link upper end in P5 Cdh23v2J/v2J and Pcdh15av3J/av3J hair bundles (Fig. 7A), suggesting that the two cadherins directly or indirectly control the harmonin-b switch. By contrast, Cdh23-N1 and Pcdh15-cter labelings were detected at stereocilia tips in harmonin-null mice (Fig. 7B), as in wild-type mice. This suggests that cadherin 23a and protocadherin 15a/b do not rely on the presence of harmonin isoforms for their apical distribution. Whether they require harmonin to form functional apical links, however, remains to be examined. In Myo7a4626SB/4626SB and sans-deficient Ush1gjs/js mice, harmonin-b was again not detected in the stereocilia (Fig. 7A).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Hair bundle polarization has been described as a two-step process. From
E15.5 in the mouse, the kinocilium migrates towards a lateral position, from
the center to the periphery of the cell apical surface. Then, once stereocilia
have differentiated and assembled into a bundle, a reorientation step occurs
in which the hair bundle progressively reaches its final location on the cell
apical surface, pointing towards the distal pole
(Cotanche and Corwin, 1991;
Denman-Johnson and Forge,
1999; Dabdoub et al.,
2003). The role of the kinocilium in the differentiation, growth
and assembly of the closest microvilli into a polarized, V-shaped
stereociliary bundle is still obscure. However, its directional migration
towards the cell periphery before any hair bundle is recognizable strongly
suggests that it has a leader role in the establishment of hair bundle
polarity. Moreover, a lack of polarization or a mispolarization of the
stereocilia bundles has recently been reported in mutant hair cells, in which
the basal body remained in a central position or was mispositioned,
respectively (Jones et al.,
2008). In all Ush1 mouse mutants analyzed here, kinocilia
were most frequently mispositioned. Nevertheless, virtually no kinocilia were
found at the center or within the medial half of the hair cell apical surface,
indicating that the first step of hair bundle polarization is not affected in
Ush1 mutant mice. Moreover, the polarization of Vangl2 and frizzled
3, two essential components of the core PCP pathway (for a review, see
Wang and Nathans, 2007) that
precedes and participates in the polarized positioning of the kinocilium
(Montcouquiol et al., 2003;
Wang et al., 2006;
Deans et al., 2007), occurred
normally at cell-cell junctions in Ush1 mutant hair cells.
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).
Moreover, in Cdh23v2J/v2J and
Pcdh15av3J/av3J mutant mice that lack these isoforms,
kinocilia were often dissociated from the stereociliary bundles and showed, on
average, greater deviations than those of other Ush1 mutant hair
cells. From our results, we can draw the general conclusion that the part of
the hair bundle emerging from the apical cell surface, together with the
expected cytoskeletal connection between the basal body and cell-cell
junctions (Jones et al.,
2008), contributes to the determination of the hair bundle final
position. Incidentally, the observation that hair bundles in
Cdh23v2J/v2J and Pcdh15av3J/av3J
mutant mice often have abnormal `S' or line shapes, together with disconnected
kinocilia, also points to an important role of the connection between the
kinocilium and stereocilia in the establishment of the normal hair bundle `V'
shape. Finally, because harmonin-b can bind to actin filaments and is
colocalized with cadherin 23a and protocadherin 15a/b at stereocilia tips
during the initial period of hair bundle development, it is likely to have an
essential role in maintaining hair bundle cohesiveness, by anchoring the
apical-most cadherin 23- and protocadherin 15-composed links to the
stereocilia cytoskeleton.
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) would be
transmitted to the growing hair bundles through the apical cell junctions and
cytoskeleton. Hair bundle fragmentation would occur as a consequence of the
loss of some interstereociliary links, as in the case of
Cdh23v2J/v2J and Pcdh15av3J/av3J
mutants, or as a consequence of the weakening of the apical-most links, as in
the case of harmonin-deficient, sans-deficient and
Myo7a4626SB/4626SB mutants. Supporting this proposal, in
the vestibule, in which extensive epithelium remodeling by convergent
extension does not occur, hair bundles of Ush1 mutants are rarely
fragmented in clumps and display only minor orientation abnormalities, whereas
they show significant differential growth defects (G.L., unpublished).
During the intermediate phase of hair bundle morphogenesis, the phenotype
of all the Ush1 mutant mice examined includes abnormal stereociliary
elongation. At first glance, the absence of myosin VIIa has an opposite effect
on stereocilia growth (increased growth) compared with the absence of any
other Ush1 protein (decreased growth). The increased elongation of stereocilia
in the absence of myosin VIIa is reminiscent of that induced by mutant forms
of myosin IIIa (Schneider et al.,
2006). However, the distribution of myosin IIIa, which is
restricted to stereocilia tips, indicates a local role for this protein in
acting directly on the machinery controlling actin polymerization, whereas the
presence of myosin VIIa along the stereocilia shafts rather suggests that it
acts as a conveyor of key regulators of actin polymerization towards
stereocilia tips. The stereocilia growth defect observed in the other
Ush1 mutants is unprecedented. Indeed, it differs from that observed
in mice deficient for myosin XVa, whirlin or espin
(Mburu et al., 2003;
Probst et al., 1998;
Sjostrom and Anniko, 1992;
Zheng et al., 2000) in that it
spares stereocilia of the tallest row. The concomitant appearance of this
defect and switch of the harmonin-b staining from the stereocilia tip to the
upper attachment point of the tip link, in conjunction with the involvement of
cadherin 23 and protocadherin 15 as tip link components
(Ahmed et al., 2006;
Kazmierczak et al., 2007;
Siemens et al., 2004),
strongly suggest that the stereocilia elongation defect of Ush1
mutants results from insufficient tension forces applied by the tip links on
the tips of small and medium stereocilia. Along this line, pulling forces
applied to actin filaments have been predicted to control actin polymerization
(Hill and Kirschner, 1982),
and a mechanism involving formins in this process has been proposed
(Kozlov and Bershadsky, 2004).
Finally, regarding the differential growth of the tallest stereocilia row, the
observation that the length of these stereocilia is not affected when the
kinocilium either lacks its axonemal part
(Jones et al., 2008) or is
entirely disconnected from the stereocilia (Cdh23v2J/v2J
and Pcdh15av3J/av3J mutants, this study), suggests that
the kinocilium does not play a crucial role in this process.
Additional lines of evidence suggest that harmonin-b anchors the tip link
upper end (likely to be made of cadherin 23) to the stereocilia actin core,
hence participating in the transmission of the above-mentioned tension forces.
Firstly, direct interactions of harmonin-b with cadherin 23 and F-actin have
been shown in vitro (Adato et al.,
2005; Boeda et al.,
2002; Siemens et al.,
2002). Secondly, the harmonin-b immunoreactivity switch does not
occur in Cdh23v2J/v2J and
Pcdh15av3J/av3J mice that do not have any detectable tip
links. Thirdly, small and medium stereocilia have oblate-shaped tips in
Cdh23v2J/v2J, Pcdh15av3J/av3J and
Ush1c-/- mutants, instead of the normal prolate-shaped
tips that are believed to result from the traction force exerted by the tip
link on the apical membrane (Rzadzinska et
al., 2004; Prost et al.,
2007). Notably, the elongation defect in sans-deficient
Ush1gjs/js mice might result from the absence of
harmonin-b, which was never detected in the stereocilia of these mice.
In conclusion, our results on Ush1 mutant mice shed new light on the cellular mechanisms involved in hair bundle morphogenesis. In particular, they unravel the role of interstereociliary and stereokinociliary links in hair bundle cohesion and orientation at early developmental stages. Moreover, they point to a previously unrecognized role of the tip link in stereocilia differential growth, in addition to its well-established role in mechanoelectrical transduction.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/8/1427/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Adato, A., Michel, V., Kikkawa, Y., Reiners, J., Alagramam, K.
N., Weil, D., Yonekawa, H., Wolfrum, U., El-Amraoui, A. and Petit, C.
(2005). Interactions in the network of Usher syndrome type 1
proteins. Hum. Mol. Genet.
14,347
-356.
Ahmed, Z. M., Riazuddin, S., Bernstein, S. L., Ahmed, Z., Khan,
S., Griffith, A. J., Morell, R. J., Friedman, T. B., Riazuddin, S. and Wilcox,
E. R. (2001). Mutations of the protocadherin gene PCDH15
cause Usher syndrome type 1F. Am. J. Hum. Genet.
69, 25-34.[CrossRef][Medline]
Ahmed, Z. M., Riazuddin, S., Ahmad, J., Bernstein, S. L., Guo,
Y., Sabar, M. F., Sieving, P., Riazuddin, S., Griffith, A. J., Friedman, T. B.
et al. (2003). PCDH15 is expressed in the neurosensory
epithelium of the eye and ear and mutant alleles are responsible for both
USH1F and DFNB23. Hum. Mol. Genet.
12,3215
-3223.
Ahmed, Z. M., Goodyear, R., Riazuddin, S., Lagziel, A., Legan,
P. K., Behra, M., Burgess, S. M., Lilley, K. S., Wilcox, E. R., Riazuddin, S.
et al. (2006). The tip-link antigen, a protein associated
with the transduction complex of sensory hair cells, is protocadherin-15.
J. Neurosci. 26,7022
-7034.
Alagramam, K. N., Murcia, C. L., Kwon, H. Y., Pawlowski, K. S.,
Wright, C. G. and Woychik, R. P. (2001a). The mouse Ames
waltzer hearing-loss mutant is caused by mutation of Pcdh15, a novel
protocadherin gene. Nat. Genet.
27, 99-102.[Medline]
Alagramam, K. N., Yuan, H., Kuehn, M. H., Murcia, C. L., Wayne,
S., Srisailpathy, C. R., Lowry, R. B., Knaus, R., Van Laer, L., Bernier, F. P.
et al. (2001b). Mutations in the novel protocadherin PCDH15
cause Usher syndrome type 1F. Hum. Mol. Genet.
10,1709
-1718.
Bitner-Glindzicz, M., Lindley, K. J., Rutland, P., Blaydon, D.,
Smith, V. V., Milla, P. J., Hussain, K., Furth-Lavi, J., Cosgrove, K. E.,
Shepherd, R. M. et al. (2000). A recessive contiguous gene
deletion causing infantile hyperinsulinism, enteropathy and deafness
identifies the Usher type 1C gene. Nat. Genet.
26, 56-60.[CrossRef][Medline]
Boeda, B., El-Amraoui, A., Bahloul, A., Goodyear, R., Daviet,
L., Blanchard, S., Perfettini, I., Fath, K. R., Shorte, S., Reiners, J. et
al. (2002). Myosin VIIa, harmonin and cadherin 23, three
Usher I gene products that cooperate to shape the sensory hair cell bundle.
Embo J. 21,6689
-6699.[CrossRef][Medline]
Bolz, H., von Brederlow, B., Ramirez, A., Bryda, E. C., Kutsche,
K., Nothwang, H. G., Seeliger, M., del C-Salcedó Cabrera, M., Vila, M.
C., Molina, O. P. et al. (2001). Mutation of CDH23, encoding
a new member of the cadherin gene family, causes Usher syndrome type 1D.
Nat. Genet. 27,108
-112.[CrossRef][Medline]
Bork, J. M., Peters, L. M., Riazuddin, S., Bernstein, S. L.,
Ahmed, Z. M., Ness, S. L., Polomeno, R., Ramesh, A., Schloss, M.,
Srisailpathy, C. R. et al. (2001). Usher syndrome 1D and
nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic
mutations of the novel cadherin-like gene CDH23. Am. J. Hum.
Genet. 68,26
-37.[CrossRef][Medline]
Chen, P. and Segil, N. (1999). p27(Kip1) links
cell proliferation to morphogenesis in the developing organ of Corti.
Development 126,1581
-1590.[Abstract]
Chen, Z. Y., Hasson, T., Kelley, P. M., Schwender, B. J.,
Schwartz, M. F., Ramakrishnan, M., Kimberling, W. J., Mooseker, M. S. and
Corey, D. P. (1996). Molecular cloning and domain structure
of human myosin-VIIa, the gene product defective in Usher syndrome 1B.
Genomics 36,440
-448.[CrossRef][Medline]
Cotanche, D. A. and Corwin, J. T. (1991).
Stereociliary bundles reorient during hair cell development and regeneration
in the chick cochlea. Hear. Res.
52,379
-402.[CrossRef][Medline]
Dabdoub, A., Donohue, M. J., Brennan, A., Wolf, V.,
Montcouquiol, M., Sassoon, D. A., Hseih, J. C., Rubin, J. S., Salinas, P. C.
and Kelley, M. W. (2003). Wnt signaling mediates
reorientation of outer hair cell stereociliary bundles in the mammalian
cochlea. Development
130,2375
-2384.
Deans, M. R., Antic, D., Suyama, K., Scott, M. P., Axelrod, J.
D. and Goodrich, L. V. (2007). Asymmetric distribution of
prickle-like 2 reveals an early underlying polarization of vestibular sensory
epithelia in the inner ear. J. Neurosci.
27,3139
-3147.
Denman-Johnson, K. and Forge, A. (1999).
Establishment of hair bundle polarity and orientation in the developing
vestibular system of the mouse. J. Neurocytol.
28,821
-835.[CrossRef][Medline]
Di Palma, F., Holme, R. H., Bryda, E. C., Belyantseva, I. A.,
Pellegrino, R., Kachar, B., Steel, K. P. and Noben-Trauth, K.
(2001). Mutations in Cdh23, encoding a new type of cadherin,
cause stereocilia disorganization in waltzer, the mouse model for Usher
syndrome type 1D. Nat. Genet.
27,103
-107.[CrossRef][Medline]
el-Amraoui, A., Sahly, I., Picaud, S., Sahel, J., Abitbol, M.
and Petit, C. (1996). Human Usher 1B/mouse shaker-1: the
retinal phenotype discrepancy explained by the presence/absence of myosin VIIA
in the photoreceptor cells. Hum. Mol. Genet.
5,1171
-1178.
Gibson, F., Walsh, J., Mburu, P., Varela, A., Brown, K. A.,
Antonio, M., Beisel, K. W., Steel, K. P. and Brown, S. D.
(1995). A type VII myosin encoded by the mouse deafness gene
shaker-1. Nature 374,62
-64.[CrossRef][Medline]
Goodyear, R. J., Marcotti, W., Kros, C. J. and Richardson, G.
P. (2005). Development and properties of stereociliary link
types in hair cells of the mouse cochlea. J. Comp.
Neurol. 485,75
-85.[CrossRef][Medline]
Hasson, T., Heintzelman, M. B., Santos-Sacchi, J., Corey, D. P.
and Mooseker, M. S. (1995). Expression in cochlea and retina
of myosin VIIa, the gene product defective in Usher syndrome type 1B.
Proc. Natl. Acad. Sci. USA
92,9815
-9819.
Hill, T. L. and Kirschner, M. W. (1982).
Subunit treadmilling of microtubules or actin in the presence of cellular
barriers: possible conversion of chemical free energy into mechanical work.
Proc. Natl. Acad. Sci. USA
79,490
-494.
Hudspeth, A. J. (1985). Models for
mechanoelectrical transduction by hair cells. Prog. Clin. Biol.
Res. 176,193
-205.[Medline]
Johnson, K. R., Gagnon, L. H., Webb, L. S., Peters, L. L.,
Hawes, N. L., Chang, B. and Zheng, Q. Y. (2003). Mouse models
of USH1C and DFNB18: phenotypic and molecular analyses of two new spontaneous
mutations of the Ush1c gene. Hum. Mol. Genet.
12,3075
-3086.
Jones, C. and Chen, P. (2007). Planar cell
polarity signaling in vertebrates. BioEssays
29,120
-132.[CrossRef][Medline]
Jones, C., Roper, V. C., Foucher, I., Qian, D., Banizs, B.,
Petit, C., Yoder, B. and Chen, P. (2008). Ciliary proteins
link basal body polarization to planar cell polarity regulation.
Nat. Genet. 40,69
-77.[CrossRef][Medline]
Kaltenbach, J. A., Falzarano, P. R. and Simpson, T. H.
(1994). Postnatal development of the hamster cochlea. II. Growth
and differentiation of stereocilia bundles. J. Comp.
Neurol. 350,187
-198.[CrossRef][Medline]
Kazmierczak, P., Sakaguchi, H., Tokita, J., Wilson-Kubalek, E.
M., Milligan, R. A., Müller, U. and Kachar, B. (2007).
Cadherin 23 and protocadherin 15 interact to form tip-link filaments in
sensory hair cells. Nature
449, 87-91.[CrossRef][Medline]
Keller, R. (2006). Mechanisms of elongation in
embryogenesis. Development
133,2291
-2302.
Kikkawa, Y., Shitara, H., Wakana, S., Kohara, Y., Takada, T.,
Okamoto, M., Taya, C., Kamiya, K., Yoshikawa, Y., Tokano, H. et al.
(2003). Mutations in a new scaffold protein Sans cause deafness
in Jackson shaker mice. Hum. Mol. Genet.
12,453
-461.
Kitamura, K., Kakoi, H., Yoshikawa, Y. and Ochikubo, F.
(1992). Ultrastructural findings in the inner ear of Jackson
shaker mice. Acta Otolaryngol.
112,622
-627.[Medline]
Kozlov, M. M. and Bershadsky, A. D. (2004).
Processive capping by formin suggests a force-driven mechanism of actin
polymerization. J. Cell Biol.
167,1011
-1017.
Lagziel, A., Ahmed, Z. M., Schultz, J. M., Morell, R. J.,
Belyantseva, I. A. 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. Biol.
280,295
-306.[CrossRef][Medline]
Lim, D. J. and Anniko, M. (1985). Developmental
morphology of the mouse inner ear. A scanning electron microscopic
observation. Acta Otolaryngol. Suppl.
422, 1-69.
Mburu, P., Mustapha, M., Varela, A., Weil, D., El-Amraoui, A.,
Holme, R. H., Rump, A., Hardisty, R. E., Blanchard, S., Coimbra, R. S. et
al. (2003). Defects in whirlin, a PDZ domain molecule
involved in stereocilia elongation, cause deafness in the whirler mouse and
families with DFNB31. Nat. Genet.
34,421
-428.[CrossRef][Medline]
Michalski, N., Michel, V., Bahloul, A., Lefevre, G., Barral, J.,
Yagi, H., Chardenoux, S., Weil, D., Martin, P., Hardelin, J. P. et al.
(2007). Molecular characterization of the ankle-link complex in
cochlear hair cells and its role in the hair bundle functioning. J.
Neurosci. 27,6478
-6488.
Michel, V., Goodyear, R. J., Weil, D., Marcotti, W., Perfettini,
I., Wolfrum, U., Kros, C. J., Richardson, G. P. and Petit, C.
(2005). Cadherin 23 is a component of the transient lateral links
in the developing hair bundles of cochlear sensory cells. Dev.
Biol. 280,281
-294.[CrossRef][Medline]
Montcouquiol, M., Rachel, R. A., Lanford, P. J., Copeland, N.
G., Jenkins, N. A. and Kelley, M. W. (2003). Identification
of Vangl2 and Scrb1 as planar polarity genes in mammals.
Nature 423,173
-177.[CrossRef][Medline]
Pawlowski, K. S., Kikkawa, Y. S., Wright, C. G. and Alagramam,
K. N. (2006). Progression of inner ear pathology in Ames
waltzer mice and the role of protocadherin 15 in hair cell development.
J. Assoc. Res. Otolaryngol.
7, 83-94.[CrossRef][Medline]
Probst, F. J., Fridell, R. A., Raphael, Y., Saunders, T. L.,
Wang, A., Liang, Y., Morell, R. J., Touchman, J. W., Lyons, R. H.,
Noben-Trauth, K. et al. (1998). Correction of deafness in
shaker-2 mice by an unconventional myosin in a BAC transgene.
Science 280,1444
-1447.
Prost, J., Barbetta, C. and Joanny, J. F.
(2007). Dynamical control of the shape and size of stereocilia
and microvilli. Biophys. J.
93,1124
-1133.[CrossRef][Medline]
Reiners, J., Reidel, B., El-Amraoui, A., Boeda, B., Huber, I.,
Petit, C. and Wolfrum, U. (2003). Differential distribution
of harmonin isoforms and their possible role in Usher-1 protein complexes in
mammalian photoreceptor cells. Invest. Ophthalmol. Vis.
Sci. 44,5006
-5015.
Reiners, J., Marker, T., Jurgens, K., Reidel, B. and Wolfrum,
U. (2005). Photoreceptor expression of the Usher syndrome
type 1 protein protocadherin 15 (USH1F) and its interaction with the scaffold
protein harmonin (USH1C). Mol. Vis.
11,347
-355.[Medline]
Rzadzinska, A. K., Schneider, M. E., Davies, C., Riordan, G. P.
and Kachar, B. (2004). An actin molecular treadmill and
myosins maintain stereocilia functional architecture and self-renewal.
J. Cell Biol. 164,887
-897.
Rzadzinska, A. K., Derr, A., Kachar, B. and Noben-Trauth, K.
(2005). Sustained cadherin 23 expression in young and adult
cochlea of normal and hearing-impaired mice. Hear.
Res. 208,114
-121.[CrossRef][Medline]
Schneider, M. E., Dose, A. C., Salles, F. T., Chang, W.,
Erickson, F. L., Burnside, B. and Kachar, B. (2006). A new
compartment at stereocilia tips defined by spatial and temporal patterns of
myosin IIIa expression. J. Neurosci.
26,10243
-10252.
Senften, M., Schwander, M., Kazmierczak, P., Lillo, C., Shin, J.
B., Hasson, T., Geleoc, G. S., Gillespie, P. G., Williams, D., Holt, J. R. et
al. (2006). Physical and functional interaction between
protocadherin 15 and myosin VIIa in mechanosensory hair cells. J.
Neurosci. 26,2060
-2071.
Sher, A. E. (1971). The embryonic and postnatal
development of the inner ear of the mouse. Acta
Otolaryngol. Suppl.
285, 1-77.
Siemens, J., Kazmierczak, P., Reynolds, A., Sticker, M.,
Littlewood-Evans, A. and Muller, U. (2002). The Usher
syndrome proteins cadherin 23 and harmonin form a complex by means of
PDZ-domain interactions. Proc. Natl. Acad. Sci. USA
99,14946
-14951.
Siemens, J., Lillo, C., Dumont, R. A., Reynolds, A., Williams,
D. S., Gillespie, P. G. and Muller, U. (2004). Cadherin 23 is
a component of the tip link in hair-cell stereocilia.
Nature 428,950
-955.[CrossRef][Medline]
Sjostrom, B. and Anniko, M. (1992). Genetically
induced inner ear degeneration. A structural and functional study.
Acta Otolaryngol. Suppl.
493,141
-146.
Verpy, E., Leibovici, M., Zwaenepoel, I., Liu, X. Z., Gal, A.,
Salem, N., Mansour, A., Blanchard, S., Kobayashi, I., Keats, B. J. et al.
(2000). A defect in harmonin, a PDZ domain-containing protein
expressed in the inner ear sensory hair cells, underlies Usher syndrome type
1C. Nat. Genet. 26,51
-55.[CrossRef][Medline]
Wang, Y. and Nathans, J. (2007). Tissue/planar
cell polarity in vertebrates: new insights and new questions.
Development 134,647
-658.
Wang, Y., Guo, N. and Nathans, J. (2006). The
role of Frizzled3 and Frizzled6 in neural tube closure and in the planar
polarity of inner-ear sensory hair cells. J. Neurosci.
26,2147
-2156.
Weil, D., Blanchard, S., Kaplan, J., Guilford, P., Gibson, F.,
Walsh, J., Mburu, P., Varela, A., Levilliers, J., Weston, M. D. et al.
(1995). Defective myosin VIIA gene responsible for Usher syndrome
type 1B. Nature 374,60
-61.[CrossRef][Medline]
Weil, D., El-Amraoui, A., Masmoudi, S., Mustapha, M., Kikkawa,
Y., Laine, S., Delmaghani, S., Adato, A., Nadifi, S., Zina, Z. B. et al.
(2003). Usher syndrome type I G (USH1G) is caused by mutations in
the gene encoding SANS, a protein that associates with the USH1C protein,
harmonin. Hum. Mol. Genet.
12,463
-471.
Wilson, S. M., Householder, D. B., Coppola, V., Tessarollo, L.,
Fritzsch, B., Lee, E. C., Goss, D., Carlson, G. A., Copeland, N. G. and
Jenkins, N. A. (2001). Mutations in Cdh23 cause nonsyndromic
hearing loss in waltzer mice. Genomics
74,228
-233.[CrossRef][Medline]
Zheng, L., Sekerkova, G., Vranich, K., Tilney, L. G., Mugnaini,
E. and Bartles, J. R. (2000). The deaf jerker mouse has a
mutation in the gene encoding the espin actin-bundling proteins of hair cell
stereocilia and lacks espins. Cell
102,377
-385.[CrossRef][Medline]
Zine, A. and Romand, R. (1996). Development of
the auditory receptors of the rat: a SEM study. Brain
Res. 721,49
-58.[CrossRef][Medline]
Zwaenepoel, I., Verpy, E., Blanchard, S., Meins, M.,
Apfelstedt-Sylla, E., Gal, A. and Petit, C. (2001).
Identification of three novel mutations in the USH1C gene and detection of
thirty-one polymorphisms used for haplotype analysis. Hum.
Mutat. 17,34
-41.[CrossRef][Medline]
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-helix domain and two repeats,
each composed of a myosin tail homology 4 (MyTH4) domain and a 4.1 ezrin
radixin moesin (FERM) domain, separated by a Src homology 3 (SH3) domain
(Chen et al., 1996
0.05,
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