Figure 1.
Phenotypic observations of Myo7a mutant strains.
(A) Hearing profile of Myo7a+/+, Myo7aI487N/I487N ewaso and Myo7aF947I/F947I dumbo strains at 4 weeks (*p = 2.2×10−25, **p = 4.5×10−10) and 24 weeks (*p = 3.7×10−29, **p = 7.2×10−20). (B–G) Video surveillance and middle ear morphology in Myo7a strains. Observations highlighted an increased number of turns in Myo7aI487N/I487N ewaso mice (C), when compared to wild-type (B). No such behaviour was seen in Myo7aF947I/F947I dumbo mutants (D). Middle ear bones appear largely normal in Myo7aI487N/I487N ewaso (F) and Myo7aF947I/F947I dumbo (G) mutants, comparable to normal morphology of the malleus, incus and stapes (E). M; manubrium of malleus, A; articulation surfaces of malleus and incus joint, T; tubercle, G; gonial angle, LI; attachment points of suspensory ligaments of incus, LP; lenticular process, C; capitulum of stapes, V; arched ventral crus, F; footplate. Scale bar; 1 mm (E–G).
Figure 2.
Myo7a mutations identified in Myo7aI487N/I487N ewaso and Myo7aF947I/F947I dumbo strains.
(A) Direct sequencing identified a 1460T>A (I487N) missense mutation in Myo7aI487N/I487N ewaso and a missense mutation 2839T>A (F947I) in the Myo7aF947I/F947I dumbo mouse strain (B). Wildtype and heterozygote sequences are shown for comparison. (C) Sequence conservations of Myo7aI487N/I487N ewaso (red) and Myo7aF947I/F947I dumbo (blue) mutations, and the closely positioned Myo7ash-1 shaker mutation (green) showing high evolutionary conservation of our two mutant strains.
Figure 3.
Molecular modelling of the Myo7aI487N/I487N ewaso mutation.
(A) Partial ribbon representation of a 3D model of myosin V 2DFS highlighting the Ile487 amino acid residue, which is localised to the hinge region between the head and tail domains of the protein. (B) Schematic diagram showing superimposed images of the hinge region (Myo7a+/+ in red and the Myo7aI487N/I487N ewaso mutation in blue) after 4 ns of molecular dynamics simulation showing a conformational change in the hinge region close to the Ile487 mutation site, showing the involvement of residues 670–673 around the hinge region causing the distortion.
Figure 4.
Haematoxylin and Eosin (H&E) staining of cochlear sections from Myo7a mutant strains at 8 and 12 weeks old.
(A–F) Myo7a+/+, (G–L) Myo7aI487N/I487N ewaso and (M–R) Myo7aF947I/F947I dumbo mice at the apical, middle and basal levels. Normal cochlear morphology shows an intact organ of Corti and the presence of a tunnel containing inner and outer hair cells and intact spiral ganglion and stria vascularis (B). Early signs of cochlea degeneration are evident in the basal region of Myo7aI487N/I487N ewaso mutant cochleae by 8 weeks of age (I), where the OC has collapsed (arrow in I). Complete lack of cellular architecture along the basilar membrane at the mid level is evident in this strain by 12 weeks (arrowhead in K). The cellular architecture in Myo7aF947I/F947I dumbo mutants is largely normal at both 8 and 12 weeks. In both mutant strains the the spiral ganglion and the stria vascularis were normal at 8 and 12 weeks of age. RM, Reisner's membrane; SG, spiral ganglion; OHC, outer hair cells; IHC, inner hair cells; OC, organ of Corti; BM, basilar membrane; SV, stria vascularis. Scale bar; 100 µM.
Figure 5.
Scanning electron micrographs of cochlear sensory epithelium from Myo7a mutant strains at 8 weeks old.
(A–C) Myo7a+/+, (D–F) Myo7aI487N/I487N ewaso and (G–I) Myo7aF947I/F947I dumbo mice at the apical, middle and basal cochlear level. Signs of degeneration and/or mis-orientation of OHC bundles is evident in both Myo7aI487N/I487N ewaso (D–F) and Myo7aF947I/F947I dumbo (G–I) mice at all levels of the cochlea. This appears to be more severe in Myo7aI487N/I487N ewaso mutants, where many bundles are missing in the mid and basal regions (E and F). IHC bundles are also affected, appearing disorganised and/or showing signs of fusion in the basal levels of Myo7aI487N/I487N ewaso cochleae (asterisk in F), and conversely in the apical region of Myo7aF947I/F947I dumbo mutants (asterisk in G). Scale bar; 10 µM (A–I).
Figure 6.
Scanning electron micrographs of inner (IHC) and outer (OHC) hair cells from 8 week old mice.
(A–D) Myo7a+/+, (E–L) Myo7aI487N/I487N ewaso and (M–T) Myo7aF947I/F947I dumbo mice at the middle and basal cochlear level. OHC bundles of Myo7aI487N/I487N ewaso mutants appear to have a regular V shaped array, however are missing the majority of their inner-row stereocilia at the middle level (arrow head in E). This is more severe at the basal level where many OHC bundles are missing (G). This phenotype is more severe in Myo7aF947I/F947I dumbo mutants, where OHC bundles are disoriented, forming an array of OHC-type bundles (M, Q, O and S). Many Myo7aI487N/I487N ewaso IHC bundles are affected at this age, with less numbers of stereocilia within the bundle and those remaining often showing signs of fusion (asterisk in J). The IHC bundles in Myo7aF947I/F947I dumbo contain additional rows of stereocilia, however do still maintain a staircase-like structure (N, R, P and T). Scale bar; 2 µM.
Figure 7.
Scanning electron micrographs of P2–5 vestibular epithelia.
(A–C and J) Myo7a+/+, (D–F and K) Myo7aI487N/I487N ewaso and (G–I and L) Myo7aF947I/F947I dumbo. A clear line of polarity reversal can be identified in Myo7a+/+ saccular maculae (A; dashed line), and normal hair bundle morphology is apparent, with a staircase arrangement of stereocilia and kinocilium located with the tallest stereocilia (B and C). Hair cell bundles are affected in Myo7aI487N/I487N ewaso saccules, and no clear zone of polarity is evident (D). These bundles do contain stereocilia of various lengths, however are generally disorientated and unstructured (E and F). A zone of polarity can be identified in Myo7aF947I/F947I dumbo saccules (G), however hair bundles do appear to have a mild phenotype, with bundles missing some of the tallest (H) and/or shortest stereocilia (I). Scale bar; 10 µM (A, D and G), 2 µM (B, C, E, F, H and I).
Figure 8.
Immunohistochemistry of P5 sensory epithelia.
Images of phalloidin (green) and Myo7a (red) stained cochlear sensory epithelium from P5 Myo7a+/+ (A and B), Myo7aI487N/I487N ewaso (C and D) and Myo7aF947I/F947I dumbo (E and F) mice at the basal cochlear level. No Myo7a expression is evident in Myo7aI487N/I487N ewaso mutant tissue (D), and may be slightly reduced in Myo7aF947I/F947I dumbo mutants (F). No difference in protein localisation was observed between wilt-type and Myo7aF947I/F947I dumbo tissue. Phalloidin staining highlights abnormal IHC structure in Myo7aI487N/I487N ewaso mutants (C) and OHC hair bundles appear misorientated and/or fragmented in Myo7aF947I/F947I dumbo (arrowheads in E). Scale bar; 8 µM (A–H).
Figure 9.
Schematic diagram of Myo7a protein structure showing the location of Myo7aI487N/I487N ewaso and Myo7aF947I/F947I dumbo mutations (in red) in relation to reported shaker mutations.
DFNA11/DFNB2/USH1B human mutations within close proximity to a reported shaker mutation are shown in parentheses [14], [56], [57]. Details of these mouse mutations are included in Table 1. IQ, isoleucine-glutamine motif; CC1, Coiled Coil domain; MyTH4, Myosin Tail Homology 4; SH3, SRC Homology 3 domain.
Table 1.
Reported Myo7a mouse models.