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First published online 13 March 2008
doi: 10.1242/dev.012922

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A core cochlear phenotype in USH1 mouse mutants implicates fibrous links of the hair bundle in its cohesion, orientation and differential growth

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.

View larger version (44K): [in this window] [in a new window]   Fig. 1. The USH1 proteins and their interactions in vitro. (A) Predicted structures of the different USH1 protein isoforms. Myosin VIIa consists of a motor head, a neck region with five isoleucine-glutamine (IQ) motifs, and a large tail comprising an -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; Weil et al., 1995). There are three classes of harmonin isoforms (a, b and c) (Verpy et al., 2000). Harmonin-a and harmonin-c have three and two PDZ domains, respectively, and harmonin-a also has a coiled-coil (CC) domain. Harmonin-b has the same domains as harmonin-a, plus a second CC domain and a proline, serine and threonine (PST)-rich sequence. Cadherin 23 and protocadherin 15 isoforms are also grouped into three classes (Ahmed et al., 2006; Lagziel et al., 2005). Cadherin 23a, cadherin 23b, protocadherin 15a and protocadherin 15b are transmembrane isoforms, with 27, 7, 11 and 1 extracellular cadherin (EC) repeat, respectively. Cadherin 23c isoforms are cytoplasmic, whereas protocadherin 15si isoforms are secreted. Multiple splice variants have been identified for myosin VIIa, protocadherin 15a, and each isoform class of harmonin and cadherin 23 (alternative exons are indicated) (Ahmed et al., 2006; Ahmed et al., 2003; Chen et al., 1996; Lagziel et al., 2005; Michel et al., 2005; Reiners et al., 2003; Verpy et al., 2000; Weil et al., 1995). Finally, sans has three ankyrin (ANK)-like repeats and a sterile alpha motif (SAM) domain (Kikkawa et al., 2003; Weil et al., 2003). Sans does not have any known splice variants. The respective locations of the mutations of the five USH1 mouse models used in this study are indicated by arrows for point mutations, and by inhibition signs for the deletion (del) of the transcription start site. The resulting stop codons are indicated by asterisks. The immunogenic regions for the antibodies used in this study are indicated by brown boxes. (B) Apical views of the auditory epithelium of a P5 wild-type mouse by scanning electron microscopy (SEM). Sensory inner hair cells (IHCs) and outer hair cells (OHCs) are organized into a single medial-side row and three lateral-side rows, respectively (left panel). A hair bundle that consists of stereocilia and a single transient kinocilium is present on top of every hair cell (right). Scale bar: 1 µm. (C) Schematic representation of known in vitro interactions between the USH1 proteins. The domains involved in each interaction are drawn in close apposition. Harmonin can bind to any of the other USH1 proteins. Cadherin 23a and protocadherin 15a/b cytoplasmic regions interact with harmonin PDZ1 and/or PDZ2 domains (Adato et al., 2005; Boeda et al., 2002; Reiners et al., 2005; Siemens et al., 2002). The presence of a consensus PDZ-binding motif at the C-terminus of cadherin 23a and protocadherin 15a/b isoforms is indicated by a star. Through its cytoplasmic region, protocadherin 15a/b can also bind to the myosin VIIa SH3 domain (Senften et al., 2006). The harmonin PDZ1 domain can interact with the second MyTH4-FERM repeat of the myosin VIIa tail and the SAM domain of sans (Boeda et al., 2002; Weil et al., 2003). Finally, the central region of sans can bind to the first MyTH4-FERM repeat of myosin VIIa (Adato et al., 2005). Harmonin and sans can also form homodimers (not shown) (Adato et al., 2005).

View larger version (133K): [in this window] [in a new window]   Fig. 2. Early fragmentation of Ush1 mutant cochlear hair bundles during development. (A) Hair bundles of sensory cells from the cochlear basal turn of E17.5 wild-type and Ush1 mutant mice. Whole-mounts of the organ of Corti were stained with phalloidin to reveal F-actin in stereocilia. (B) Analysis of P0 cochlear hair cells from wild-type and Ush1 mutant mice by SEM. Note the fragmented aspect of the hair bundles, and sometimes also of the microvillar area, in the mutants. (C) OHC hair bundles in wild-type and Ush1 mutant mice at P0. Lateral links are visible between stereocilia from both wild-type and mutant hair cells (see insets), although they appear sparser and frequently disrupted in the mutants, especially Cdh23v2J/v2J and Pcdh15av3J/av3J. Scale bars: 2 µm in A,B; 1 µm in C.

View larger version (86K): [in this window] [in a new window]   Fig. 3. Mispositioning of the kinocilium in Ush1 mutant hair cells. (A) Basal view of E18.5 cochlear whole-mounts from wild-type, Ush1c-/- and Cdh23v2J/v2J mice were stained with an antibody that labels the kinocilia (green), and with phalloidin to reveal F-actin in stereocilia (red). (B) Schematic of the method used to measure the angular deviation of the kinocilium with respect to the PCP axis on SEM images. A P0 wild-type OHC is taken as an example. (C) Evaluation of the kinocilium position on P0 wild-type and Ush1 mutant hair cells. For each mouse strain, kinocilia were falsely labeled orange (using Adobe Photoshop) to facilitate their visualization on SEM images of control and mutant OHCs. Mean absolute positions (±s.e.m.) of the kinocilia for the total population of hair cells (orange) and per hair cell row (blue to purple) are represented on the bar charts. P-values, which were determined with respect to the positions of kinocilia in wild-type mice using the Welch's t-test, are indicated by asterisks (*P0.05, **P0.01, ***P0.001). The pie charts present the percentage of hair cells in which the kinocilium is mispositioned by 0-14° (white), 15-44° (light-gray), 45-89° (medium-gray), or 90-180° (dark-gray) from its expected location at the lateral-most edge of the cell surface. Scale bars: 2 µm in A; 1 µm in C.

View larger version (157K): [in this window] [in a new window]   Fig. 4. Distribution of the mouse Ush1 proteins in wild-type hair bundles during development. Hair bundles from E16.5, E18.5, P1 and P5 wild-type mouse cochlear sensory cells stained with phalloidin (red) and antibodies to myosin VIIa (Myo7a-F1, A-F), harmonin-b (harmonin-H1b, G-L), cadherin 23a (Cdh23-N1, M-P), or protocadherin 15a/b (Pcdh15-cter, Q-T) (green). Unless otherwise stated, views are from the basal cochlear turn. At E16.5, the four Ush1 proteins are detected in the actin-rich protrusions that grow on top of the newly differentiated hair cells, with a particular concentration at their actin-free distal end (A,G,M,Q). Similar distribution patterns are also observed later, at E18.5, in the stereocilia and surrounding hair cell microvilli, which have become distinguishable by their different lengths (B,H,N,R). Note in M and N the strong Cdh23-N1 signals at the tip of the tallest protrusions adjacent to the kinocilia (arrowheads), which are stained for acetylated tubulin (blue) in M and Q. During postnatal stages, both Cdh23-N1 and Pcdh15-cter signals become restricted to stereocilia tips (O,P,S,T), whereas the harmonin-H1b signal is relocalized from the tip to the tip link upper insertion region in tall and medium stereocilia (arrowheads in J-L). A new Myo7a-F1 signal is detected near stereocilia bases during this period of hair bundle development (C-F), and the shafts of the growing stereocilia also stain positive for myosin VIIa (not shown). Scale bars: 2 µm.

View larger version (123K): [in this window] [in a new window]   Fig. 5. Ush1 protein localization in early developing Ush1 mutant hair bundles. (A) Hair bundles of sensory cells from the cochlear basal turn of E18.5 Myo7a4626SB/4626SB, Cdh23v2J/v2J, Pcdh15av3J/av3J and Ush1gjs/js mice stained with the harmonin-H1b antibody (green) and phalloidin (red). Note the absence of signal in the hair bundles of Ush1gjs/js and Myo7a4626SB/4626SB mice, and the presence of immunoreactive spots in the cuticular plate of the latter (arrowheads). (B) Hair bundles of sensory cells from the cochlear basal turn of E17.5 wild-type mice and Ush1c-/- mice that lack harmonin. Whole-mount cochleas were labeled with phalloidin (red) and antibodies to myosin VIIa (Myo7a-F1), harmonin-b (harmonin-H1b), cadherin 23a (Cdh23-N1) or protocadherin 15a/b (Pcdh15-cter) (green). Cdh23-a and Pcdh15-a/b are detected along the stereocilia shafts both in wild-type and Ush1c-/- hair bundles. Note the absence of a Myo7a-F1 signal at stereocilia tips in Ush1c-/- hair cells (arrowheads). Scale bars: 2 µm.

View larger version (77K): [in this window] [in a new window]   Fig. 6. Elongation and tip defects of stereocilia in Ush1 mutants during postnatal stages. (A) Hair bundle maturation in P0 to P15 wild-type and Ush1c-/- OHCs from the end of the apical cochlear turn. At P0, mutant OHC bundles are composed of four to six stereocilia rows almost equal in height, resembling their wild-type counterparts. From this stage onwards, wild-type stereocilia undergo differential growth, depending on the row they belong to. In the absence of harmonin, small and medium stereocilia rows show marked elongation defects from P2 onward, and most of the stereocilia from these rows have disappeared by P15. By contrast, stereocilia from the tall row are of normal length at all stages examined. Note that some small and medium row stereocilia located near clump vertices show some elongation at first (see black, green and red lines running along the tall, medium and small rows of stereocilia, respectively, in P2 and P5 wild-type and mutant OHC bundles). (B) OHC bundles of P5 Myo7a4626SB/4626SB, Cdh23v2J/v2J, Pcdh15av3J/av3J and Ush1gjs/js mice (view from the end of the apical cochlear turn). Note the abnormal height of many stereocilia of the medium row and the frequent absence of small stereocilia in mutant hair bundles. (C) Mid-cochlear IHC bundles of P5 wild-type, Myo7a4626SB/4626SB, Ush1c-/-, Cdh23v2J/v2J, Pcdh15av3J/av3J and Ush1gjs/js mice. As differential elongation occurs, stereocilia of the medium row in wild-type hair bundles acquire a particular prolate shape (see gray lines in the wild-type IHC inset), which could result from tension forces applied to their tip membrane (Rzadzinska et al., 2004). Medium stereocilia of Ush1 mutant IHCs, however, never display prolate tips (see insets). In addition, apical links (arrowheads) are either absent (Cdh23v2J/v2J and Pcdh15av3J/av3J), or appear sparser (Myo7a4626SB/4626SB, Ush1c-/- and Ush1gjs/js) in the Ush1 mutants, compared with the controls. Scale bars: 1 µm.

View larger version (126K): [in this window] [in a new window]   Fig. 7. Mouse Ush1 protein localization in late developing Ush1 mutant hair bundles. (A) Hair bundles of sensory cells from the cochlear basal turn of P5 Myo7a4626SB/4626SB, Cdh23v2J/v2J, Pcdh15av3J/av3J and Ush1gjs/js mice were stained with the harmonin-H1b antibody (green) and phalloidin (red). Note again the absence of harmonin-b labeling in hair bundles of Ush1gjs/js and Myo7a4626SB/4626SB mice, and the presence of immunoreactive spots that have increased in number and size since E18.5, in the cuticular plate of the latter mutant (arrowheads). In Cdh23v2J/v2J and Pcdh15av3J/av3J mice, harmonin-b is also mislocated, as it is still detected at stereocilia tips (arrowheads) instead of near the tip link side attachment point (see wild-type control in B). (B) Cochleas of P5 wild-type and Ush1c-/- mice were stained with phalloidin (red) and antibodies to myosin VIIa (Myo7a-F1), harmonin-b (harmonin-H1b), cadherin 23a (Cdh23-N1) or protocadherin 15a/b (Pcdh15-cter) (green). The stainings for myosin VIIa, Cdh23-a and Pcdh15-a/b are the same in Ush1c-/- and wild-type hair bundles. Scale bars: 2 µm.

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