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Development of Outer Hair Cells in Ames Waltzer MiceMutation in Protocadherin 15 Affects Development of Cuticular Plate and Associated Structures.

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THE ANATOMICAL RECORD 291:224–232 (2008)
Development of Outer Hair Cells in Ames
Waltzer Mice: Mutation in Protocadherin
15 Affects Development of Cuticular
Plate and Associated Structures
Department of Otolaryngology-Head and Neck Surgery, University of Texas Southwestern
Medical Center, Dallas, Texas
Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine,
Kyoto University, Kyoto, Japan
Graduate School of Biomedical Sciences, University of Texas Southwestern
Medical Center, Dallas, Texas
Callier Center, Behavior and Brain Sciences, University of Texas at Dallas, Dallas, Texas
Department of Otolaryngology-Head and Neck Surgery, University Hospitals of
Cleveland, Case Western Reserve University, Cleveland, Ohio
The Ames waltzer (av) mouse mutant harbors a mutation in the protocadherin 15 gene (Pcdh15) and is a model for deafness in Usher syndrome
1F and nonsyndromic deafness DFNB23. Mutation in Pcdh15 affects stereocilia morphogenesis and polarity. Disruptions of apical cellular components in outer hair cells have also been described in av mutants. Organization of stereocilia and cell polarization may be dependent on proper orientation of structural components residing in the apical portion of the cell
during development. We used electron and immunofluorescent microscopy
to examine structural maturation of outer hair cells in av3J mice with emphasis on the fonticulus, basal body/centriole complex, actin mesh, and the
microtubule network during initiation of bundle organization, between embryonic day (E) 16.5 and postnatal day 5 (P5). We found major ultrastructural rearrangements near the hair cell surface in av3J mice. Earliest
changes were in kinocilia, basal body, and stereocilia positioning and
microtubule arrangement once the kinocilia had lateralized to the side of
the cell (between E16.5 and postnatal day [P] 0, before cuticular plate formation and stereocilia elongation). By P0, the developing fonticulus in av
mice appeared enlarged, with a normal vesicle density. Stereocilia bundle
disorganization increased after P0, with disruptions of the actin mesh
within the cuticular plate. These observations support the hypothesis that
mutations in Pcdh15 in av3J mice adversely affect coordinated maturation
of apical cell components, resulting in disturbed stereocilia bundle polarity
in av mice. Anat Rec, 291:224–232, 2008. Ó 2007 Wiley-Liss, Inc.
Key words: kinocilia; stereocilia; polarity; basal body/centriole;
fonticulus; Ames waltzer; protocadherin 15 (Pcdh15)
Grant sponsor: The NIDCD; Grant number: DC05385; Grant
sponsor: Deafness Research Foundation.
Drs. Kikkawa and Pawlowski contributed equally to this work.
*Correspondence to: Karen S. Pawlowski, Department of Otolaryngology – Head and Neck Surgery, University of Texas
Southwestern Medical Center, Dallas, TX, 75390-9035. Fax:
1-214-648-9122. E-mail:
Received 19 July 2007; Accepted 5 November 2007
DOI 10.1002/ar.20632
Published online 18 December 2007 in Wiley InterScience (www.
Fig. 1. Schematic diagram of a normal mature outer hair cell
detailing the architecture of the apical portion of the cell. There are
three rows of stereocilia (Sc), with a distinct gradation of height from
the tallest row to the shortest. The rows are arranged in a ‘‘W’’ formation or a ‘‘V’’ formation with a notch in the V at the fonticulus, the area
void of cuticular-plate actin, which is occupied by a kinocilium earlier
in cell development. The fonticulus (F) is located at the lateral-most
side of the cell. The primary and daughter basal bodies (BB) maintain
an almost perpendicular orientation to each other. They are located
near the surface, but deeper than the position they occupy earlier in
cell development. Complex tight junctions (JC) can be seen at the
level of the cuticular plate (CP) on either side of the cell. Vesicles (V)
are seen within the fonticulus at the surface to just beneath the level
of the bottom of the CP. Microtubules (MT) extend from this area toward the base of the cell. The CP, composed of actin fibers, is well
formed with an increased density of actin at the surface of the cell
surrounding the stereocilia rootlets, called the actin mesh (A). The
rootlets (R) of the short stereocilia extend from a short distance above,
to some distance into the cuticular plate. Rootlets from the taller rows
of stereocilia appear hollow and often extend through the curricular
plate, into the cell cytoplasm.
Cochlear hair cells transduce the mechanical energy
of sound into neurochemical signals that are carried to
the brain. Bundles of stereocilia that reside at the apex
of these cells are essential to the mechanotransduction
process. Outer hair cell anatomy in adult mice is fairly
well known (Liberman, 1987; Liberman and Dodds,
1987). The observed structures in the apical portion in
adult mouse outer hair cell (OHC) are reviewed in Figure 1. However, detailed reports of these structures during outer hair cell morphogenesis in mammals are few
and the mechanisms governing their organizational
development are still being elucidated. With the arrival
of a wealth of molecular information unraveling the
genetic causes of deafness, it has become clear that
many deafness mutations affect the organizational
development of the hair cell. Ames waltzer (av) is a recessive mutation in mice that causes deafness and vestibular dysfunction. The inner ear defect in av mice is
associated with disorganized cochlear stereocilia (Alagramam et al., 2000). We have previously shown that
the av mutant has a genetic defect in a cadherin family
protein, protocadherin 15 (Pcdh15; Ahmed et al., 2001;
Alagramam et al., 2001a,b). The av mouse is a model for
deafness and vestibular dysfunction in Usher syndrome
1F (USH1F) and nonsyndromic deafness (DFNB23)
(Ahmed et al., 2001; Alagramam et al., 2001b). Protocadherin proteins are predicted to function as Ca21-dependent adhesion proteins, and Pcdh15 is reported to be
present along the stereocilia bundle and in the cuticular
plate of adult mice (Ahmed et al., 2003; Senften et al.,
2006). Morphologic observations on av mice during development show disruption of the cuticular plate and
abnormally positioned stereocilia on the outer hair cells,
followed by degeneration of the organ of Corti (Pawlowski et al., 2006). The lack of stereocilia bundle polarity in av mice suggests lack of coordination of apical cell
components during hair cell maturation.
The present study focused on early OHC development
in the av allele carrying a presumptive null mutation of
Pcdh15. Here, we followed the development of cellular
components of the OHC apex during the establishment
of polarity of the stereocilia bundle. Three different
methods were used in this study; scanning electron microscopy (SEM), transmission electron microscopy
(TEM), and confocal microscopy. Most ultrastructural
studies of the hair cell have been performed with SEM,
which can clearly show disorganization of stereocilia
that occurs in many of the hearing-affected mouse
mutants (Kitamura et al., 1992; Takumida et al., 1995).
However, SEM observations are limited to changes that
can be seen at the cell surface. By combining these three
methods, we can associate cell surface structure with internal architecture and distribution in the developing
hair cell, to better interpret ultrastructural alterations
caused by the mutation.
Here, we present ultrastructural details of the cuticular plate and associated elements of outer hair cells in
av mutants during the time of stereocilia development
compared with hair cells from age-matched controls. By
studying the morphology of the defective sensory cells in
the mutant animals, we can begin to elucidate the functional significance of Pcdh15 on these cellular components early in hair cell development.
C57BL/6J mice and Ames waltzer (av) heterozygote
mice (1/3J), age E16.5 to P5, were used to describe normal development of the cochlear hair cell, as no physiological or morphological changes have been observed in
these mice at this stage (Pawlowski et al., 2006; Zheng
et al., 2006). We set up mating the night before and
checked for vaginal plugs the next day. If the female
was plugged, we considered that as E0.5 or the midpoint
of the light cycle the next day, assuming that ovulation
occurred sometime during the middle of the dark cycle
(Goodyear et al., 2005). Sixteen days from the time the
plug was first noted, females showing clear signs of
pregnancy were euthanized and embryos were harvested
in the morning. These embryos were labeled E16.5, for
example. The day of birth was noted as P0.
To study changes associated with mutation in Pcdh15,
two alleles of av were used: Pcdh15av-J, which carries an
in-frame deletion and Pcdh15av-3J, which carries a stop
mutation (Alagramam et al., 2001b). Mice homozygous
for Pcdh15av-J (J/J) or Pcdh15av-3J (3J/3J) along with
sibling control were used. Sample size was two to six
mice for each age group/genotype. The Animal Care and
Use Committee of the Case Western Reserve University
approved the protocol for animal use.
Electron Microscopy
All animals were anesthetized and euthanized by
decapitation. The middle and inner ears were opened,
phosphate buffered 2.5% glutaraldehyde fixative was
flushed through the perilymphatic space, and the tissue
was placed in fixative for 14–18 hr at 48C (Alagramam
et al., 2000). Ear specimens prepared for TEM work
were fixed with warm (35–378C) phosphate buffered
2.5% glutaraldehyde to preserve microtubules configuration (Mandelkow et al., 1985). All tissues were then
rinsed in 0.1 M sodium phosphate buffer, pH 7.4–7.5,
and stored at 48C before processing for light and electron microscopic examination.
Organ of Corti samples from E16.5–P5 homozygous
and heterozygous mice were prepared for either SEM or
TEM to examine the ultrastructure of the cuticular plate
during its development. Glutaraldehyde-fixed and
buffer-rinsed tissue specimens were stained with 1–1.5%
osmium tetroxide, followed by a buffer rinse. Material to
be studied by TEM was then decalcified in 0.35 M ethylenediaminetetraacetic acid, rinsed in buffer, dehydrated
in a graded series of ethanols, and embedded in Spurrs’
resin. The mid-basal turn of the organ of Corti was thin
sectioned either in a plane parallel to the surface of the
reticular lamina or in a plane perpendicular to that surface and radial to the modiolus. Sections were then
stained with lead citrate and uranyl acetate and viewed
by using a JEOL 1200EX transmission electron microscope. For SEM, the organs of Corti were exposed by
cochlear microdissection and the tissue was dehydrated
in a series of ethanols, critical point dried, coated with
gold-palladium and studied using a JEOL 848 scanning
electron microscope.
Whole-Mount Confocal Microscopy
All animals studied were anesthetized and euthanized
as described above. The middle and inner ears were
opened, phosphate buffered 4% paraformaldehyde fixative was flushed through the perilymphatic space and
the tissue was placed in fixative for 14–18 hr at 48C. After fixation, organ of Corti was dissected out in phosphate-buffered saline (PBS) at room temperature using
a fine needle. Tissue samples were then permeabilized
and blocked in 0.2% Triton X-100 in 5% normal goat serum (Sigma, St. Louis, MO) and 2% bovine serum albumin (Sigma) for 60 min. Samples were incubated overnight in monoclonal anti-acetylated tubulin antisera
(Sigma) at a 1:100 dilution in 5% normal goat serum
and 2% bovine serum albumin solution. After three
rinses in PBS, samples were incubated in a 1:200 dilution of the Alexa-568 conjugated anti-mouse IgG second-
ary antibody (Molecular Probes, New Brunswick, NJ)
with 1:40 dilution of Alexa-488 conjugated phalloidin
(Molecular probes) for 45 min. After rinsing three times
with PBS, samples were mounted using PBS and Gold
Seal cover glass (Ted Pella, Redding, CA). Pictures
were taken with Leica TCS-SP2 or TCS-SP5 confocal
microscope. Three-dimensional reconstruction was performed.
In av mice, ultrastructural changes developed early
and progressively worsened during development of the
apical portion of the outer hair cell. Organization of
structural features, including the basal bodies, microtubules, and actin mesh, have not been described at
the ultrastructural level for all the time points in the
mouse. Therefore, to provide a better perspective of the
abnormalities observed in OHCs of av mice at the ultrastructural level, we have included a description of
the apical cell components in OHC in age-matched
normal controls, based on our data and known characteristics.
Outer Hair Cell Anatomy, Late Embryonic
Stage (E16.5–E18)
At the late embryonic stage (E16.5–E18), the apical
surface of the hair cell is very immature (Fig. 2A–C). No
alterations in cell morphology can be seen in av3J
mutants at this age. In controls, no cuticular plate is
recognizable by TEM (Fig. 2C). The kinocilium (black
arrowheads in Fig. 2B,C) can be seen at the cell surface,
in the middle of the cell, and is incorporated into the
cell membrane, continuous with the basal body (red
arrowheads in Fig. 2A,B). The other (daughter) basal
body lies slightly deeper and perpendicular to the first
(Fig. 2D arrows). Initially, stereocilia and microvilli of
similar height, surround the kinocilium. Next, the kinocilium relocates to the side of the cell, before stereocilia
elongation. Vesicles can be seen beneath the basal
bodies, in a region where microtubules branch out radially toward the edges and longitudinally toward the basal portion of the cell (Fig. 2D, arrowheads). The junctional complex between cells is not well developed at
this stage, often appearing as two junctional complexes,
one above the other (Fig. 2C, arrows).
At E16.5, confocal microscopic observation reveals a
kinocilium in the middle of the cell. No significant differences in the organization or the condition of the apical
hair cell organelles (kinocilia and stereocilia) were
observed between control (heterozygous, 1/3J) and
homozygous (3J/3J) animals (Fig. 2E,F). By E18, however, the hair cell kinocilium starts to move toward the
lateral side of the cell (Fig. 2G,H, arrows). At this stage,
slight differences of stereocilia organization and kinocilium position are visible in mutants compared with
controls. Stereocilia elongation has not yet occurred;
the staircase pattern of the stereocilia characteristic
of mature hair cells is not obvious at this stage
(Fig. 2B,G,H).
Outer Hair Cell Anatomy, at Birth (P0)
At P0, polarization of cilia becomes visible (Fig. 3A,B).
The beginnings of the cuticular plate can be seen with a
Fig. 2. Image set demonstrating anatomical structures in the apical
portion of outer hair cells in control and mutant animals at late embryonic stage E16.5–E18. A: Schematic diagram detailing the architecture
of the apical portion of a normal outer hair cell at E18. This drawing
shows stereocilia clustered in the center of the cell around a single
kinocilium (black arrowheads in A–C,E, arrows in G,H). At this stage,
the basal body is continuous with the kinocilium (red arrowheads in
A,C). The basal body complex resides near the apical cell surface (red
arrows in D), surrounded by associated vesicles (white arrowheads in
D). Microtubules radiate from a cluster of vesicles associated with the
basal body (yellow arrows in A,C,E). The cuticular plate is not yet
present, and the tight junctions at the side of the cell are immature
and often appear as two separate junctions at this early point in development (double arrow in C). B: Scanning electron microscopy
(SEM) image of three rows of outer hair cells (1,2,3) in a control (1/3J)
at E18; arrowheads 5 kinocilia. C,D: Transmission electron micrographs of a longitudinal section (C) and higher power tangential section (D) through normal E18 outer hair cells. Arrowhead in C 5 kinoci-
lia, double arrow in C 5 tight junction, red arrowhead in D 5 basal
bodies (centrioles) at the cell surface, with associated vesicles marked
by white arrowheads. E,F: Laser confocal images of whole mount
preparations of organ of Corti at E16.5. No clear difference between
the actin labeling (phalloidin-green) or anti-acetylated tubulin labeling
(red) was detected between the tissue from control (E, 1/3J) and
homozygous (F, 3J/3J) animals. The red stain radiating from the kinocilia (red lines) in E and F is tubulin that is associated with microtubules.
G,H: High power SEM images of organ of Corti tissue from E18 control (G, 1/3J) and homozygous (H, 3J/3J) animals showing the beginning of the repositioning of the kinocilia (black arrows) from the center
to the side of the cell. The kinocilia remain in the center of several hair
cells (top arrow in G, bottom arrow in H), but have moved to the side
of the cell in others (bottom arrow in G, top arrow in H). The repositioned kinocilia in H has moved to the left instead of toward the top of
the image, and there is an area void of nascent stereocilia to the left
of this kinocilium. Scale bars 5 1 mm in B, 500 nm in C,D, 1 mm in
very small amount of actin mesh visible by TEM in the
superficial layers (asterisk in Fig. 3C). The kinocilium is
now located laterally on the surface of the cell, with the
stereocilia bundle organized in a loose ‘‘V’’ shape medial
to the kinocilium with some gradation in height between
the rows of stereocilia (Fig. 3B). Microvilli are still present medial to the stereocilia. The basal bodies are
located at the cell surface, within the fonticulus (Fig. 3D,
With confocal imaging, stereocilia and cuticular plate
organization differs greatly at P0 between control and
3J/3J animals (Fig. 3E,F). Actin labeling of the apical
hair cell surface shows a more disorganized, bulging
appearance in 3J/3J animals, and abnormal organization of stereocilia becomes more apparent at this age.
The fonticulus (arrowheads in Fig. 3E,F) is seen as an
actin-void area of the hair cell surface. It has been associated with vesicular trafficking from the area adjacent
to the basal bodies to the cell surface structures (Raphael et al., 1993). It contains basal bodies, scattered pericentriolar bodies (vesicles), and an abundance of micro-
tubule ends. The fonticulus is enlarged and out of position in 3J/3J animals.
At P0 in normal developing OHCs, microtubules
extend radially from the area of the basal body toward
the periphery and longitudinally toward the base of the
cell (Fig. 3G–I). Anti-acetylated tubulin staining (red) is
heavy near the fonticulus and extends radially to the
cell walls running beneath the cuticular plate (Fig. 3G).
Microtubule organization in some hair cells shows a
more disrupted pattern in 3J/3J, and the hair cell surface itself appears more distended in 3J/3J (Fig. 3E,F,
J,K; 3D reconstructions available in Supplementary
OHC Anatomy, 2 Days After Birth (P2)
The rows of stereocilia at P2 have a slight indentation
at the apex of the ‘‘V’’ pattern and appear to be taller
than those seen at P0 (Fig. 4A,B). Some microvilli
remain. The cuticular plate (Fig. 4C) is an actin-rich
area of the hair cell surface in which stereocilia rootlets
Fig. 3. At birth (P0): The kinocilia have all repositioned to the side
of the cell and the architecture of the apical region of the cell is starting to mature. A difference in outer hair cell cuticular plate organization can clearly be seen between control (E,J) and 3J/3J (F,K) tissues
at this stage of development. A: Schematic diagram illustrating the
now laterally positioned kinocilia, with the basal body still close to the
apical surface (arrowheads in C,D) and microtubules (yellow arrows in
A,D,J,K) radiating from the vesicles associated with the basal body
complex (red arrowhead in D). Many microvilli remain on the surface
of the hair cell, but taller stereocilia can be seen oriented in graded
heights that are tallest on the side closest the kinocilia (see B). The
tight junctions are becoming more substantial (arrows in C) and a
slight increase in density at the central, surface portion of the cell indicates the initial development of the cuticular plate (* in C). B: Scanning
electron microscopy image of the outer hair cell surface in a control
(1/3J) showing lateralized stereocilia with the taller stereocilia oriented
toward the top of the image. C,D: Transmission electron microscopy
images of tangentially sectioned outer hair cells from control animals.
C: Higher magnification image of an outer hair cell sectioned just beneath
the surface of the cell. Arrows 5 tight junction; red arrowhead 5 basal
body associated with the kinocilia, * 5 beginning of the cuticular plate.
D: Lower magnification image of an outer hair cell sectioned at the
cell surface. Yellow arrow 5 radially oriented microtubules; red arrowhead 5 basal body are the surface of the cell. E–K: Laser confocal
images of the apical portions of outer hair cells labeled with phalloidin
for F-actin (green) and/or anti-acetylated tubulin (red). E,F: Outer hair
cell cuticular plates from control (E, 1/3J) and homozygous (F, 3J/3J)
animals. The regions of denser F-actin staining (green) are associated
with the stereocilia and the tight junctions. The area in the cell devoid
of actin stain (asterisks) is the fonticulus, where the kinocilia would be
located. There are clear differences in stain density between the control and homozygous tissue, as well as size and orientation of the fonticulus. G–I: Control outer hair cell, focusing just beneath the cuticular
plate. F-actin stained the cell junctions and cuticular plate (green,
G,H), and stained microtubules (red, H,I) can be seen in the area of
the fonticulus (asterisk) and microtubules radiating from the fonticulus
toward the cell wall (red lines indicated by arrow in H,I), which are in
close proximity to the lower portion of the cuticular plate (green, arrow
in G pointed at same position as in H,I). J,K: Apical portions of outer
hair cells from 3J/3J mice demonstrating the near-normal (J) and
altered (K) positioning of the fonticulus and organization of microtubules (yellow arrows) in the 3J/3J mouse. Scale bar 5 5 mm in
B,E,F,G–I, 1 mm in J,K.
are embedded and from which stereocilia extend in a
specific pattern. This is thicker at P2 than at P0 and
extends farther across the cell surface. However, a space
between the cell membrane and the cuticular plate is
present, which is filled with transport vesicles (Fig. 4C,
yellow arrows). Vesicles can also be seen within the fonticulus, surrounding and beneath the basal bodies.
At P2, av mutants showed abnormally positioned stereocilia bundle (asterisk in Fig. 4D). The actin mesh in
the cuticular plate is also disrupted (green arrowhead in
4D), however, stereocilia-to-stereocilia distance in a bundle appears to be normal in mutants and no stereocilia
fusion is observed. The density of transport vesicles in
the fonticulus (arrow in 4D) seems similar between controls and mutants.
OHC Anatomy, 5 Days After Birth (P5)
At P5, the cuticular plate and actin mesh (green
arrowheads in Fig. 5C) are thicker than at P0 or P2.
Fig. 4. Maturation of the normal outer hair cell continues 2 days after birth (P2). A: Diagram of the normal outer hair cell at P2 illustrating
the continued presence of the kinocilia, with very few microvilli
remaining on the surface of the control outer hair cell (B). The stereocilia are also taller, the cell junctions are more substantial at P2 than
at P0. The cuticular plate is getting thicker and the actin mesh at the
surface of the cuticular plate is becoming more substantial, but some
microtubules still run radially just beneath it (C arrows). B: Scanning
electron microscopy image of the outer hair surface in a control animal. All of the kinocilia are still present, but a large portion of the surface of the hair cell is now void of stereocilia or microvilli. C: Transmission electron microscopy (TEM) image of tangential section through
the lower portion of the cuticular plate of an outer hair cell from a control (C, 1/3J) animal. Microtubules (yellow arrows in A,C) can be seen
running radially just beneath the cuticular plate. D: TEM image of tangential section through the apical surfaces of hair cells from a homozygous (D, 3J/3J) animal oriented with the lateral-most side of the cell
to the upper left. The cross-sectioned stereocilia bundle (*) does not
have the standard wedge shape. However, there is no obvious
increase in distance between the stereocilia here and no stereocilia
fusion is seen. The basal body and associated vesicles (white arrow)
are out of position, at the right of the cell. The mesh within the cuticular plate (green arrowheads in A, D) is disrupted. Scale bar 5 5 mm in
B, 500 nm in D.
Most of the kinocilia are shriveled (large arrowhead in
Fig. 5B), but remain at the cell surface, with the basal
body. The indentation in the V-shaped rows of stereocilia
is more pronounced and the majority of the microvilli
have disappeared. Microtubules still run crosswise
beneath the cuticular plate (arrow in Fig. 5C).
At this stage, the difference between control and 3J/
3J mutant animals is more obvious. In mutants kinoci-
Fig. 5. At 5 days after birth (P5), changes in architecture continue
to occur at the apical portion of the cell. A: Diagram of the normal
outer hair cell at P5 illustrating the first sign of breakdown of the kinocilia (large arrowhead in B) and very few microvilli remain on the hair
cell surface. The cuticular plate is thicker and the actin mesh is more
substantial (C green arrowheads) than at P2, running between the
stereocilia rootlets (C black arrowhead). The tight junctions are one
continuous junction at this stage (see C). Radial orientation of microtubules is also still present at this stage, (black arrow in C) and the basal
body is still near the cell surface. B: Scanning electron microscopy
(SEM) image of the outer hair cell surface of a control animal. Arrowhead 5 shriveling kinocilia. C: Transmission electron microscopy
image of a longitudinally sectioned outer hair cell from a control animal. A stereocilia rootlet can be seen extending into the cuticular plate
(black arrowhead), and fibers of the actin mesh can be seen running
just under the cell surface, parallel with the surface. Black arrow
points to a microtubule running parallel with the cell surface. D: SEM
image of outer hair cells from a homozygous (3J/3J) animal. Some
kinocilia (large arrowhead) remain, and are positioned randomly on the
cell surface. The disorganization of the stereocilia is more obvious
with clumps of stereocilia scattered across the surface of the cell,
with large areas of the cell surface void of stereocilia or microvilli.
Scale bar 5 1 mm in B, 500 nm in C, 1 mm in D.
lia are positioned randomly on the hair cell surface (arrowhead in Fig. 5D) and the disorganization
of the stereocilia bundle becomes more evident,
sometimes to the extent that the stereocilia bundles
are broken into clumps scattered across the surface
of the cell. In contrast, no dramatic shortening or
splayed appearance of stereocilia can be seen in the
This study used SEM, TEM, and confocal microscopy
to chronicle development of the apical portion of the
outer hair cell during the time of stereociliogenesis in av
mice. In our mouse model, stereocilia formation and organization occurs between E16.5 and P5. This is just
before final physiological development of cochlear auditory transduction (P10–P12) (Kros et al., 1998). The
OHC alteration first to be described in av mice was an
alteration in stereocilia organization (Alagramam et al.,
2001a), and it has been suggested that this phenotype
may be associated with disruption in stereocilia side
links (Adato et al., 2005). However, the sidelinks appear
to be intact in the clumps of stereocilia seen in av
mutants (Pawlowski, et al., 2006).
Stereocilia organization is thought to be closely associated with positioning of the kinocilium and basal body
and also with microtubule formation (Sobkowicz et al.,
1995). Our finding that most components of the apical
cytoskeletal system appear to be disrupted in the av
mutants at the earliest stages of stereocilia bundle development would suggest that Pcdh15 is involved more
globally in organization of the hair cell at this stage of
Usher Syndrome Mice and Pcdh15 Function
Usher’s syndrome type 1 (USH1) is a genetically heterogeneous group of recessive disorders that causes deafness in humans at or near birth and blindness by adolescence. Mutations in Usher syndrome proteins have
been shown in mice to disrupt the arrangement of stereocilia on the surface of OHCs (Reiners et al., 2006).
USH1 proteins (myosin VIIA, harmonin, cadherin 23,
Pcdh15, and SANS) interact with each other and these
proteins are believed to be associated with cytoskeletal
proteins such as actin and microtubules (Adato et al.,
2005). The av mice used in this study harbor a mutation
in the Pcdh15 gene, a transmembrane protein belonging
to the cadherin superfamily. This mutant serves as a
model for inner ear dysfunction in USH1F and DFNB23
Our work over the years has demonstrated that mutation in Pcdh15 affects stereocilia morphogenesis and polarity (Alagramam et al., 2000, 2001a,b, 2005). More
recently, we showed that misorientation of the bundle in
av mice was associated with changes in the actin meshwork within the cuticular plate (Pawlowski et al., 2006).
Subsequent reports by Senften et al. (2006) have shown
that mutation in Pcdh15 affects bundle morphogenesis
and orientation. More recently, Ahmed et al. (2006)
reported that several isoforms of Pcdh15 are expressed
in the hair cells and some of these isoforms localize to
various parts of the stereocilia including the tip.
Although Pcdh15 may have a role in extracellular links,
our investigation (including the one described here)
shows that Pcdh15 has a rudimentary role in establishing bundle orientation and this function is linked to subapical cytoskeletal structures in the hair cell.
How could mutation in Pcdh15 lead to defects in bundle positioning? An interesting observation was reported
with shaker-1 mice, which is a myosin VIIA mutant
(USH1B model; Self et al., 1998). Shaker-1 hair cells
show disorganized hair bundles similar to that of av
mutants, and on TEM study, its cuticular plate is inter-
rupted by areas of vesicle-rich cytoplasm. We observed
an abnormally positioned and enlarged fonticulus in av
mutants, which was full of vesicles, suggestive of
increased vesicle accumulation in this area (Fig. 3E,F).
We proposed earlier that the proline-rich sequences of
the cytoplasmic domain of Pcdh15 could interact with SH3 domain-containing proteins (Alagramam et al.,
2001). Recently, it was reported that the proline-rich
sequence in the cytoplasmic domain of Pcdh15 interacts
with Myosin VIIA by means of the SH3 domain (Senften
et al., 2006). Myosin VIIA was linked to vesicular trafficking (El-Amraoui et al., 2005), and therefore, it is
tempting to suggest that loss of Pcdh15 function leads to
abnormal vesicle trafficking during hair bundle development and maturation due to impaired interaction with
Myosin VIIA. This impaired interaction could then lead
to a misorientation of the bundle including the kinocilium.
Reports in the literature suggest that the Pcdh15 protein in the mouse could play other roles in hair cell development and differentiation. In Drosophila, a loss of
Cad99C (a Pcdh15 orthologue) function leads to shorter
microvilli in ovarian follicle cells (D’Alterio et al., 2005).
At this point, we do not know whether mammalian
Pcdh15 is involved in stereocilia elongation. Experiments are under way to determine whether mutation in
mouse Pcdh15 affects stereocilia length. The mouse
Pcdh15 was also reported to interact with other proteins
associated with inner ear dysfunction, namely Harmonin
(USH1C protein; Adato et al., 2005; Senften et al.,
2006). More recently, mouse Pcdh15 protein was
reported to be a component of the of the tip-link complex
(Ahmed et al., 2006, Kazmierczak et al., 2007). These
reports combined with data from our work suggest multiple roles for Pcdh15 in mammalian hair cells. The
observations reported here indicate that Pcdh15 plays a
role in cellular architecture very early in stereocilia bundle development that has an effect on the organization/
polarization of the kinocilia, basal bodies, fonticulus and
its associated vesicles, and the microtubule network at
the time of stereociliogenesis.
We thank Dr. William Snell (UT Southwestern Medical Center, Department of Cell Biology) for kindly providing valuable discussions and anti-acetylated tubulin
antibody; Dr. Chris Gilpin, Mr. George Lawton, and Mr.
Tom Januszewski (UTSW, Molecular and Cellular Imaging Facility) for technical assistance and use of the scanning and transmission electron microscopes; Dr. Kate
Luby-Phelps (UTSW, Live Cell Imaging Core Facility)
for confocal microscope usage. This work was supported
in part by funds from NIDCD to K.A., and from Deafness Research Foundations to Y.S.K.
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development, protocadherinx, associates, micemutation, affects, ames, hair, plato, cells, structure, waltzer, outer, cuticular
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