Development of Outer Hair Cells in Ames Waltzer MiceMutation in Protocadherin 15 Affects Development of Cuticular Plate and Associated Structures.код для вставкиСкачать
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 YAYOI S. KIKKAWA,1,2 KAREN S. PAWLOWSKI,1,3,4* CHARLES G. WRIGHT,1,4 5 AND KUMAR N. ALAGRAMAM 1 Department of Otolaryngology-Head and Neck Surgery, University of Texas Southwestern Medical Center, Dallas, Texas 2 Department of Otolaryngology-Head and Neck Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan 3 Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, Dallas, Texas 4 Callier Center, Behavior and Brain Sciences, University of Texas at Dallas, Dallas, Texas 5 Department of Otolaryngology-Head and Neck Surgery, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, Ohio ABSTRACT 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: firstname.lastname@example.org Ó 2007 WILEY-LISS, INC. Received 19 July 2007; Accepted 5 November 2007 DOI 10.1002/ar.20632 Published online 18 December 2007 in Wiley InterScience (www. interscience.wiley.com). OUTER HAIR CELL DEVELOPMENT IN av MICE 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 225 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. MATERIALS AND METHODS Mice 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 226 KIKKAWA ET AL. 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. RESULTS 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 OUTER HAIR CELL DEVELOPMENT IN av MICE 227 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 G,H. 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, arrowhead). 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 Materials). 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 228 KIKKAWA ET AL. 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. OUTER HAIR CELL DEVELOPMENT IN av MICE 229 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- 230 KIKKAWA ET AL. 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 mutants. OUTER HAIR CELL DEVELOPMENT IN av MICE DISCUSSION 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 development. 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 patients. 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- 231 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. ACKNOWLEDGMENTS 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. LITERATURE CITED Adato A, Michel V, Kikkawa Y, Reiners J, Alagramam KN, Weil D, Yonekawa H, Wolfrum U, El-Amraoui A, Petit C. 2005. Interactions in the network of Usher syndrome type 1 proteins. Hum Mol Genet 14:347–356. Ahmed ZM, Riazuddin S, Bernstein SL, Ahmed Z, Khan S, Grifﬁth AJ, Morell RJ, Friedman TB, Riazuddin S, Wilcox ER. 2001. Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F. 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