Whirler mutant hair cells have less severe pathology than shaker 2 or double mutants

Abstract

MYOSIN XV is a motor protein that interacts with the PDZ domain-containing protein WHIRLIN and transports WHIRLIN to the tips of the stereocilia. Shaker 2 (sh2) mice have a mutation in the motor domain of MYOSIN XV and exhibit congenital deafness and circling behavior, probably because of abnormally short stereocilia. Whirler (wi) mice have a similar phenotype caused by a deletion in the third PDZ domain of WHIRLIN. We compared the morphology of Whrn (wi/wi) and Myo15 (sh2/sh2) sensory hair cells and found that Myo15 (sh2/sh2) have more frequent pathology at the base of inner hair cells than Whrn (wi/wi), and shorter outer hair cell stereocilia. Considering the functional and morphologic similarities in the phenotypes caused by mutations in Myo15 and Whrn, and the physical interaction between their encoded proteins, we used a genetic approach to test for functional overlap. Double heterozygotes (Myo15 (sh2/+), Whrn (wi/+)) have normal hearing and no increase in hearing loss compared to normal littermates. Single and double mutants (Myo15 (sh2/sh2), Whrn (wi/wi)) exhibit abnormal persistence of kinocilia and microvilli, and develop abnormal cytoskeletal architecture. Double mutants are also similar to the single mutants in viability, circling behavior, and lack of a Preyer reflex. The morphology of cochlear hair cell stereocilia in double mutants reflects a dominance of the more severe Myo15 (sh2/sh2) phenotype over the Whrn (wi/wi) phenotype. This suggests that MYOSIN XV may interact with other proteins besides WHIRLIN that are important for hair cell maturation.

FIG. 1

Myo15^sh2 and Whrn^wi double heterozygotes hear as well as single heterozygotes. ABR tests were performed on 2- and 6-month-old doubly heterozygous mice (filled circles) and single heterozygote controls (open circles) at frequencies of 4, 10, and 20 kHz. The sound pressure level thresholds, measured in decibels (dB), are indistinguishable between double heterozygotes and controls.

FIG. 2

Double mutants and sh2 mutants have shorter stereocilia than wi mutants. SEM was used to visualize the apical surface of cochlear hair cells in the mid turn region in mice at age P21. OHC stereocilia were analyzed in cochlear whole mounts from double heterozygotes (a), both single mutants, Myo15^sh2/sh2 (b) and Whrn^wi/wi (c), and Myo15^sh2/sh2, Whrn^wi/wi double mutants (d). The stereocilia on OHCs of double heterozygotes are indistinguishable from those of wild-type mice. OHC stereocilia in the Myo15 mutant are extremely short with abnormally persistent microvilli (arrows). Some stereocilia are arranged in a U shape (asterisk) instead of the normal V shape. The OHC stereocilia of Whrn mutants are shorter than normal, and there are persistent microvilli. Some cells have stereocilia arranged in an abnormal U shape. Myo15, Whrn double mutants have an OHC stereocilia phenotype that looks like the Myo15 single mutant. Scale bars: a–d 5 μm, e–h 1 μm, i–l 1 μm. The length of the OHC stereocilia of double heterozygotes (e) is longer than single mutants of Myo15 (f) or Myo15, Whrn double mutants (h), but subtly longer than Whrn single mutants (g). Scale bars 1 μm. The lengths of the IHC stereocilia are obviously normal in double heterozygotes and obviously longer (i) than in single mutants for Myo15 (j) or Whrn (k), or for double mutants (l), which were extremely short. Scale bars 1 μm.

FIG. 3

Quantification of mean stereocilia length confirms differences in stereociliar length. The length of the stereocilia was measured in micrometers from SEM photographs of IHC and OHC for the four genotype classes. Means ± SE of the mean of stereocilia length are shown for IHCs and OHCs of each genotype. Brackets indicate consecutive means shown to be significantly different by t test, after Bonferroni adjustment for multiple tests.

FIG. 4

Differences in stereocilia lengths are apparent by confocal microscopy of the actin cytoskeleton. Confocal images of phalloidin labeled organ of Corti show that stereocilia of inner hairs cells are long in wild types (a) and double heterozygotes (b) and distinctly shorter in mutants (c–e). Scale bars 10 μm.

FIG. 5

Kinocilium regression is impaired in Myo15 and Whrn mutants. SEM analysis of IHCs in the apical turn of cochlea revealed abnormalities in apical surface morphology of mutants relative to controls. Cochlear whole mounts from P21 animals revealed abnormal persistence of kinocilia (arrows) in IHCs of single mutants of Myo15 (b) and Whrn (c), and Myo15, Whrn double mutants (d), relative to double heterozygotes in which the kinocilia have regressed (a). Scale bars 5 μm.

FIG. 6

Cytocaud pathology is more severe and appears earlier in mutants with the shortest stereocilia. Epifluorescence images of phalloidin-labeled whole mounts of the organ of Corti of P21 control mice (a), single mutants of Myo15 (b) and Whrn (c), and Myo15, Whrn double mutants (d) reveal cytoskeletal actin organization abnormalities in mutants relative to controls (arrows). At the focal plane near the base of the cuticular plate, an abnormal actin-positive stain, known as a cytocaud, is detected in the IHCs of single mutants of Myo15 (b) and Whrn (c), and Myo15, Whrn double mutants (d).

FIG. 7

Vestibular hair cells of Myo15 and Whrn single and double mutants exhibit cytocaud pathology at birth. Epifluorescence images of whole mounts of the ampulla of P21 control mice (a), single mutants of Myo15 (b) and Whrn (c), and Myo15, Whrn double mutants (d) labeled with phalloidin. At the focal plane just beneath the cuticular plate, an actin-positive stain is detected in most of the vestibular hair cells of Myo15 (b) and Whrn (c) single mutants and Myo15, Whrn (d) double mutants (arrows), but not in control mice.