A novel splice site mutation of myosin VI in mice leads to stereociliary fusion caused by disruption of actin networks in the apical region of inner ear hair cells

<div><p>An unconventional myosin encoded by the myosin VI gene (<i>MYO6</i>) contributes to hearing loss in humans. Homozygous mutations of <i>MYO6</i> result in nonsyndromic profound congenital hearing loss, DFNB37. Kumamoto shaker/waltzer (<i>ksv</i>) mice harbor spontaneous mutations, and homozygous mutants exhibit congenital defects in balance and hearing caused by fusion of the stereocilia. We identified a <i>Myo6</i>^c.1381G>A mutation that was found to be a p.E461K mutation leading to alternative splicing errors in <i>Myo6</i> mRNA in <i>ksv</i> mutants. An analysis of the mRNA and protein expression in animals harboring this mutation suggested that most of the abnormal alternatively spliced isoforms of MYO6 are degraded in <i>ksv</i> mice. In the hair cells of <i>ksv</i>/<i>ksv</i> homozygotes, the MYO6 protein levels were significantly decreased in the cytoplasm, including in the cuticular plates. MYO6 and stereociliary taper-specific proteins were mislocalized along the entire length of the stereocilia of <i>ksv</i>/<i>ksv</i> mice, thus suggesting that MYO6 attached to taper-specific proteins at the stereociliary base. Histological analysis of the cochlear hair cells showed that the stereociliary fusion in the <i>ksv</i>/<i>ksv</i> mutants, developed through fusion between stereociliary bundles, raised cuticular plate membranes in the cochlear hair cells and resulted in incorporation of the bundles into the sheaths of the cuticular plates. Interestingly, the expression of the stereociliary rootlet-specific TRIO and F-actin binding protein (TRIOBP) was altered in <i>ksv</i>/<i>ksv</i> mice. The abnormal expression of TRIOBP suggested that the rootlets in the hair cells of <i>ksv</i>/<i>ksv</i> mice had excessive growth. Hence, these data indicated that decreased MYO6 levels in <i>ksv</i>/<i>ksv</i> mutants disrupt actin networks in the apical region of hair cells, thereby maintaining the normal structure of the cuticular plates and rootlets, and additionally provided a cellular basis for stereociliary fusion in <i>Myo6</i> mutants.</p></div

Down-regulation of the αA, αB, β and γ-crystallins caused by the <i>miak</i> mutation of <i>Pitx3</i>.

<p>Confocal images show the double-labeled crystallin proteins (red) and DAPI (blue) in the lenses of wild-type and <i>miak</i>/<i>miak</i> mice at E12.5. Scale bar <i> = </i>100 µm. <b>A.</b> The αA-crystallins labeling of the lens. The expression patterns of αA-crystallins were similar in the lens epithelium (le) and lens fiber (lf) of the wild-type and <i>miak</i> mice; however, the αA-crystallins signals may be slightly reduced in the <i>miak</i> mice. <b>B.</b> The αB-crystallins labeling of the lens. The αB-crystallin signals were not detected in the <i>miak</i>/<i>miak</i>. <b>C.</b> The β-crystallin labeling of the lens. The β-crystallin signals were barely detected in the <i>miak</i>/<i>miak</i> lens. <b>D.</b> The γ-crystallin labeling of the lens. Immunohistochemistry reveals the dramatically reduced γ-crystallin signals in the <i>miak</i> mice as well as ectopic expression in the anterior region of the lens.</p

Elongation of the rootlets in the cochlear hair cells of the <i>ksv</i>/<i>ksv</i> mice.

<p>Localization of a marker for the rootlet, TRIOBP5, in the IHCs (A–F) and OHCs (G–J) of the +/+ (A, C, E, G and I) and <i>ksv</i>/<i>ksv</i> (B, D, F, H and J) mice at P4 (A–D, G and H) and P17 (E, F, I and J). The left and right panels show the localization of TRIOBP5 (red), and the right panels show the merged image with phalloidin (green). Highly magnified images of typical TRIOBP5 signals in IHCs from +/+ (A, yellow box) and <i>ksv</i>/<i>ksv</i> (yellow doted boxes) mice are shown in C and D, respectively. Scale bars = 5 μm.</p

Homozygous <i>ksv</i> mutation in mice causes hearing loss and stereociliary fusion of cochlear hair cells.

<p>A. ABR waveforms from +/+ and <i>ksv/ksv</i> mice at P30. The waveforms from five mice of each genotype represent the ABR to tone-pip stimuli at the highest sound pressure level (dB SPL) at 4, 8, 16 and 32 kHz and are shown in different colors. The locations of ABR peaks I–V are indicated with ranges (two direction arrows) of the negative wave apex in each peak of +/+ mice. B and C. SEM images of stereocilia in the hair cells from the apex area of the cochlea in the +/+ (B) and <i>ksv/ksv</i> (C) mice at P30. The highly magnified images in the white dotted boxes of B and C are shown in the bottom panels (B’ and C’). The fused giant stereocilia (open arrow), bulged base (arrow), loss of tapered region (arrowhead) and bulbous tip (open arrowhead) of the stereocilia detected in the IHCs of <i>ksv</i>/<i>ksv</i> mice (C). Scale bars = 5 μm.</p

Deformation of the cuticular plate membranes of IHCs of <i>ksv</i>/<i>ksv</i> mice during the process of stereociliary fusion.

<p>A. SEM image showing stereocilia in IHCs from the middle area of the cochlea in a <i>ksv</i>/<i>ksv</i> mouse at P0. Raised membrane of the cuticular plate (arrows). B. TEM image showing apical regions of the IHCs of a <i>ksv</i>/<i>ksv</i> mouse at P1. Highly magnified image of the stereociliary base shown in B’. C. Highly magnified TEM image showing the stereociliary base of the IHCs of a +/+ mouse at P1. D. Typical phenotypes of the apical surfaces of IHCs of <i>ksv/ksv</i> mice at P2. Highly magnified images in dotted and dashed boxes of D show the bulging of the cuticular plate membrane in D’ and D” (arrows), respectively. Scale bars = 3 μm (D), and 1 μm (A–C, D’ and D”).</p

Misorientation and fusion at the bases of the stereocilia in vestibular hair cells in <i>ksv</i>/<i>ksv</i> mice.

<p>A–D SEM images of stereocilia in the utricles of the +/+ (A and B) and <i>ksv/ksv</i> (C and D) mice at P7. The dashed pink line indicates the virtual line of polarity reversal (LPR) in vestibular hair cells (A). The magnified images of stereociliary bundles from +/+ and <i>ksv</i>/<i>ksv</i> mice are shown in B and D. The elongated stereociliary bundle (open arrow) is noted in the utricle. The highly magnified images in the white dotted boxes of B and D are shown in the right panels. Arrows indicate a slightly bulged base and a loss of taper in stereocilia in <i>ksv</i>/<i>ksv</i> mice. Scale bars = 5 μm (A, B, C and D), and 1 μm (B’ and D’).</p

Alternative splicing errors and decrease in <i>Myo6</i> mRNA caused by the <i>ksv</i> mutation.

<p>A. RT-PCR analysis of <i>Myo6</i> expression in cochlear and vestibular RNA from +/+, +/<i>ksv</i> and <i>ksv/ksv</i> mice at P30. The upper panels show the RT-PCR products obtained from both tissues by using <i>Myo6</i>-specific primers located in exons 10 and 15/16 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183477#pone.0183477.s006" target="_blank">S1 Table</a>). The integrity of the cDNA was confirmed by using a <i>Gapdh</i> control (bottom panels). B. Relative levels of <i>Myo6</i> mRNA in the cochlear and vestibular tissues of +/+ (<i>n</i> = 3), +/<i>ksv</i> (<i>n</i> = 3) and <i>ksv/ksv</i> (<i>n</i> = 3) mice at P30. *<i>P <</i> 0.05. C. Patterns of exon skipping in <i>Myo6</i> alternatively spliced isoforms transcribed in the <i>ksv</i> mutant. The two-direction arrows and arrowhead indicate deletions and the c.1381G>A missense mutation, respectively.</p

Evaluation of the interaction between <i>Foxe3-</i> (A) and <i>Mip-</i> (B) <i>bicoid</i> sites and proteins in wild-type and –<i>miak</i> mice by electrophoretic mobility assay (EMSA).

<p>EMSA performed with <i>Foxe3</i>- <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432-Ahmad1" target="_blank">[16]</a> and <i>Mip-</i> <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111432#pone.0111432-Sorokina1" target="_blank">[21]</a> <i>bicoid</i> oligonucleotides (oligo probe) and nuclear extracts (NE) from wild-type and <i>miak</i> eyes at E17.5. Although the formation of the specific EMSA complex occurred by combining oligo probes and NEs from wild-type and <i>miak</i> mice, the binding ability was increased with <i>miak</i>-NE and both <i>Foxe3</i>- and <i>Mip-bicoid</i> oligo probes. The binding ability of both oligo probes was inhibited by 10-fold excess unlabeled competitive probes (competitor).</p

Decreased expression levels and/or mislocalization of MYO6 and stereociliary taper-specific proteins in the hair cells of <i>ksv</i>/<i>ksv</i> mice.

<p>A–G. Double staining for MYO6 (red: C-ter, A–E and G; N-ter, F) and phalloidin (green) in the cochlear (A–D, F and G) and vestibular (E) hair cells of +/+ (A and C) and <i>ksv/ksv</i> homozygous (B and D–G) mice. MYO6 immunoreactivity was detected in the cuticular plates, pericuticular necklaces, and cytoplasm of the OHCs and IHCs of +/+ mice but was not observed in the stereocilia (A). In <i>ksv/ksv</i> mice (B and D–G), MYO6 signals were not observed in the cytoplasm (arrows) but were observed along the length (arrowheads) of the stereocilia in the cochlea (B, F and G). Highly magnified images of representative expression patterns of the OHCs (boxes) and IHCs (dotted boxes) in A and B are shown in the left panels of C and D. The intensity of the white lines in the left panels indicates the degree of decrease in the MYO6 signals in the cuticular plates in <i>ksv/ksv</i> mice. A similar decrease in the cuticular plates was observed in the utricle hair cells of <i>ksv/ksv</i> mice (E) and in IHCs stained with the anti-MYO6 (N-ter) antibody (F). A bulbous signal (yellow arrowhead) of MYO6 was detected at the tips of the fused stereocilia in <i>ksv/ksv</i> mice (G). H–K. Double staining for stereociliary taper-specific proteins (red: PTPRQ, H and I; TPRN, J and K) with phalloidin (green) in the IHCs of +/+ (H and J) and <i>ksv/ksv</i> (I and K) mice. Highly magnified images of dotted boxes in the middle panels of H–K are shown in each right panel. Arrowheads indicate the mislocalization along the length of the stereocilia of each protein in <i>ksv</i>/<i>ksv</i> mice (I and K). Scale bars = 5 μm.</p

Rapidly progressive stereociliary fusion of the cochlear hair cells in <i>ksv</i>/<i>ksv</i> mice during early postnatal stages.

<p>A–D. Comparison of stereociliary phenotypes between the +/<i>ksv</i> heterozygous (A and C) and <i>ksv</i>/<i>ksv</i> homozygous (B and D) mice at P0 (A and B) and P4 (C and D). SEM images showing stereocilia in the hair cells from the middle area of the cochlea. Highly magnified images of the OHCs (middle panels) and IHCs (bottom panels) are shown. The arrowheads and arrows indicate the kinocilia and bulging basal region of the stereocilia, respectively. The bundling of the stereocilia in both the OHCs and IHCs of <i>ksv</i>/<i>ksv</i> mice at P0 are magnified (white boxes). E. Stereociliary phenotypes in the hair cells from the middle area of the cochlea in <i>ksv</i>/<i>ksv</i> mouse at P7. The highly magnified image (right panel) in the white dotted box in the left panel shows the bulging basal region (arrow) in the fused stereocilia in IHCs. F. Stereociliary phenotypes in the hair cells from the apex area of the cochlea in a <i>ksv</i>/<i>ksv</i> mouse at P14. The highly magnified image (right panel) in the white dotted box in the left panel shows the bulging basal region (arrow) and bulbous tip (open arrowhead) in the elongated giant stereocilia (open arrows) of the IHCs. Scale bars = 5 μm.</p