Three deaf mice: mouse models for TECTA-based human hereditary deafness reveal domain-specific structural phenotypes in the tectorial membrane

Abstract

Tecta is a modular, non-collagenous protein of the tectorial membrane (TM), an extracellular matrix of the cochlea essential for normal hearing. Missense mutations in Tecta cause dominant forms of non-syndromic deafness and a genotype-phenotype correlation has been reported in humans, with mutations in different Tecta domains causing mid- or high-frequency hearing impairments that are either stable or progressive. Three mutant mice were created as models for human Tecta mutations; the Tecta(L1820F,G1824D/+) mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the Tecta(C1837G/+) mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family and the Tecta(C1619S/+) mouse for a zonadhesin-like (ZA) domain mutation responsible for progressive, high-frequency hearing loss in a French family. Mutations in the ZP and ZA domains generate distinctly different changes in the structure of the TM. Auditory brainstem response thresholds in the 8-40 kHz range are elevated by 30-40 dB in the ZP-domain mutants, whilst those in the ZA-domain mutant are elevated by 20-30 dB. The phenotypes are stable and no evidence has been found for a progressive deterioration in TM structure or auditory function. Despite elevated auditory thresholds, the Tecta mutant mice all exhibit an enhanced tendency to have audiogenic seizures in response to white noise stimuli at low sound pressure levels (≤84 dB SPL), revealing a previously unrecognised consequence of Tecta mutations. These results, together with those from previous studies, establish an allelic series for Tecta unequivocally demonstrating an association between genotype and phenotype.

Figure 1.

Structure of the organ of Corti and Tecta. (A) Schematic drawing depicting structure of the organ of Corti in the basal region of an adult mouse cochlea. The tectorial membrane attaches to the spiral limbus and, via Kimura's membrane, to the stereocilia of the OHC. Other features of the tectorial membrane include Hensen's stripe which lies medial to the inner hair cell (IHC) bundle and the marginal band which is situated at the lateral edge. (B) Domain structure of Tecta and location of deafness-causing missense mutations. Mutations in the entactin-G1 like domain, the vWFD1, vWFD2 and TIL2 repeats of the ZA domain and the ZP domain are associated with a mid-frequency hearing loss, while those in other regions of the ZA domain affect the high frequencies (20).

Figure 2.

Generation of Tecta mutants by homologous recombination. Targeting vectors carrying point mutations in the ZA (A) or ZP domains (B) were composed of left and right arms flanking a floxed neo^R cassette. (C) Targeted ES cell lines were identified by Southern blotting. (D) Founder mice generated by blastocyst injection were mated with a Cre expressing mouse line to delete the selection cassette, leaving the point mutation and a single loxP site. (E) Direct sequencing of RT-PCR products was used to demonstrate the presence of the desired mutations in transcribed RNA. B, BamHI; S, SacI; loxP, loxP site; In A and B positions of 5′ and 3′ probes are marked with blue boxes, and the band sizes, in Kbp, identified by the probes on Southern blots are shown. Mutated exons are shown in red, exon 14 for Tecta^C1619S, and exon 17 for Tecta^{L1820F,G1824D} and Tecta^C1837G. The SacI site between exons 17 and 18 in the targeting construct in B was introduced with a single base mutation to aid screening for recombinants.

Figure 3.

Structure of the organ of Corti in Tecta mutants. Toluidine blue stained sections from the basal coil (∼35 kHz region) of the organ of Corti of wild type and Tecta mutants at early (A–D, P21–P32) and late (E–H, 6–11 months) ages. (A P21, E 11 months) Wild type shows the characteristic tectorial membrane profile with attachment to spiral limbus (SL) and extension over the spiral sulcus (SS) to the hair cells (arrowhead points to IHC, arrows point to OHCs). A prominent Hensen's stripe (HS) is seen in close proximity to the IHC bundle and a marginal band (MB) covers the lateral edge of the tectorial membrane. (B P23, F 11 months) Tecta^C1619S/+ mutant showing distorted cross-sectional profile of the tectorial membrane, with reduced limbal attachment, less distinct Hensen's stripe and fragmented marginal band. (C P21, G 6 months) Tecta^{L1820F,G1824D/+} and (D P32, H 8 months) Tecta^C1837G/+. Both ZP domain mutants display a tectorial membrane with a ‘hump-backed’ cross-sectional profile, much reduced limbal attachment, a displaced marginal band and detached, malformed Hensen's stripe. In addition, Kimura's membrane (KM) appears delaminated and can sometimes be observed as a hook-like extension that curves back along the underside of the tectorial membrane (arrowheads). Collagen fibrils protrude from the upper surface in a region close to the lateral edge of the spiral limbus (arrows). There is not an obvious deterioration in structure as a function of age in any of the three mutants. Scale bar in H = 50 μm and applies to all panels.

Figure 4.

Distribution of tectorins and glycoconjugates in the TMs of Tecta mutants. Immunostaining with antibodies to Tecta (A–D) and Tectb (E–H) and lectin staining with SBA (I–L) on cryosections from the cochleae of 3-month-old wild type (A, E, I), Tecta^C1619S/+ (B, F, J), Tecta^{L1820F,G1824D/+} (C, G, K) and Tecta^C1837G/+ (D, H, L) mice. Images were obtained with a wide-field epifluorescence microscope. LZ = limbal zone, CN = covernet, MB = marginal band, indicated in I. Scale bar in L= 50 μm and applies to all panels.

Figure 5.

Covernet and marginal band in Tecta mutants. (A–D) Confocal Z-projections of SBA-labelled 60 μm thick horizontal sections of cochleae showing the pattern of covernet fibrils (longitudinal fibrils indicated by arrows) and marginal band (MB) in the tectorial membranes of wild type (A), Tecta^C1619S/+ (B), Tecta^{L1820F,G1824D/+} (C) and Tecta^C1837G/+ (D) adult mice. (E–H) TEM montages showing detail of the lateral region of basal tectorial membranes from wild type (E), Tecta^C1619S/+ (F) Tecta^{L1820F,G1824D/+} (G) and Tecta^C1837G/+ (H) mice. Arrow in each panel points to marginal band material, which is hollow in Tecta^C1619S/+ mice (F) and is displaced in Tecta^{L1820F,G1824D/+} (G) and Tecta^C1837G/+ (H) mice. Arrowhead in E points to Kimura's membrane, in F points to a hole in Kimura's membrane. In G and H arrowheads point to a delaminated region of Kimura's membrane. Double arrowheads point to Hensen's stripe in wild type (E) and Tecta^C1619S/+ (F) mice. Hensen's stripe is dissociated from lower side of the tectorial membrane in Tecta^{L1820F,G1824D} (G) and Tecta^C1837G/+ (H) mice and is out of the field of view. Scale bar in D = 20 μm and applies to A–D, scale bar in H= 5 μm and applies to E–H.

Figure 6.

Covernet fibrils and striated-sheet matrix in Tecta mutants. (A–D) Transmission electron micrographs depicting covernet fibrils from basal tectorial membrane profiles in wild type (A), Tecta^C1619S/+ (B), Tecta^{L1820F,G1824D/+} (C) and Tecta^C1837G/+ (D) mice. In mice of all genotypes, covernet fibrils appear to be comprised of electron dense, compacted striated-sheet-like material. However, in Tecta^C1619S/+ mice (B), the fibrils are more numerous and are of much smaller diameter than those of wild-type mice (A), whilst those of Tecta^{L1820F,G1824D/+} (C) and Tecta^C1837G/+ (D) mice, although of normal cross-sectional appearance, are often partly separated from the underlying collagenous matrix. (E–H) Transmission electron micrographs depicting striated-sheet matrix (arrows) and collagen fibril bundles (arrowheads) from apical-coil tectorial membrane profiles in wild type (A), Tecta^C1619S/+ (B), Tecta^{L1820F,G1824D/+} (C) and Tecta^C1837G/+ (D) mice. In all genotypes striated-sheet matrix with a normal appearance is present in lateral regions of the tectorial membrane. Scale bar in D = 500 nm and applies to A–D, scale bar in H = 500 nm and applies to E–H.

Figure 7.

Hair-cell numbers in Tecta mutants. (A) An entire organ of Corti dissected from a decalcified P28 wild-type mouse cochlea by iterative trimming. Organ of Corti pieces were stained with phalloidin (red) and antibodies to Myosin VIIA (green) in order to visualise hair bundles and entire hair cells, respectively. The image was reconstructed from confocal stacks using Photoshop, and the organ of Corti trimmed out from the surround. Numbers 0–696 label IHCs, starting at the apical end. Values in kHz represent estimated best frequencies along the cochlea. (B–M) selected regions from the organ of Corti of P28 wild type (B, C, D), P28 Tecta^C1619S/+ (E, F, G), 11-month-old wild type (H, I, J) and 11-month-old Tecta^C1619S/+ (K, L, M) mice illustrating myosin VIIA staining. Little hair-cell loss is seen at P28 in any of the mice, whilst at 11 months, significant hair-cell loss is seen in extreme low-frequency and high-frequency (>45 kHz) regions of the cochlea in all mice. Scale bar in A= 100 μm, bar in M= 50 μm and applies to B–M.

Figure 8.

Hair-bundle imprints in TMs of Tecta mutants. Confocal Z-projections of stereocilin-labelled tectorial membranes from adult wild type (A, B), Tecta^C1619S/+ (C, D) and Tecta^C1837G/+ (E, F) mice showing location of individual rows of hair-bundle imprints (arrowheads). Three rows of hair-bundle imprints are visible in both the apical (A) and basal (B) coils of wild-type tectorial membranes. Imprints are not visible in the apical region of Tecta^C1619S/+ tectorial membranes (C) whilst in basal regions (D) imprints are indistinct and are restricted to a maximum of two clear rows. In Tecta^C1837G/+ tectorial membranes, imprints are visible but weakly stained in apical regions (E) and are not detected more basally (F). Scale bar = 10 μm and applies to all panels. (G) RT-qPCR measurement of prestin expression levels in wild type and Tecta^C1619S/+, Tecta^{L1820F,G1824D/+} and Tecta^C1837G/+ littermates. Mean prestin expression levels in heterozygous mutant mice are expressed as a percentage of the levels in wild-type littermates (n = 3 for each genotype). Error bars represent standard deviations.

Figure 9.

Auditory thresholds in Tecta mutants. ABR thresholds in Tecta^C1619S, Tecta^{L1820F,G1824D} and Tecta^C1837G mutant mice were tested at 2 to 11 months of age. ABR thresholds in the 8-40 kHz range are elevated by 20–30 dB in Tecta^C1619S/+ heterozygotes compared with wild-type controls, whilst those in the Tecta^{L1820F,G1824D/+} and Tecta^C1837G/+ heterozygotes are elevated by 30–40 dB.

Figure 10.

Seizure sensitivity of Tecta mutants. Percentage of wild-type and heterozygous Tecta mutant mice exhibiting wild running behaviour in response to 8–16 kHz white noise at a SPL ≤ 84 dB.

Figure 11.

Structure of the organ of Corti in homozygous Tecta mutants. Toluidine blue stained resin sections (A–C) and anti-Tecta (red) and phalloidin (green) labelled cryosections (D–F) from basal cochlear coil of homozygous Tecta^{C1619S/C1619S} (A, D) Tecta^{L1820F,G1824D/L1820F,G1824D} (B, E) and Tecta^{C1837G/C1837G} (C, F) mutant mice. The tectorial membrane in the Tecta^{C1619S/C1619S} mouse (A, D) has reduced attachment to the spiral limbus (SL), a highly fragmented marginal band (arrow) but retains strong labelling for Tecta (D). The tectorial membranes of Tecta^{L1820F,G1824D/L1820F,G1824D} and Tecta^{C1837G/C1837G} mice are completely detached (arrows in B–C, E–F) from the spiral limbus and instead are associated with Reissner's membrane (arrowhead in B and C). Although the matrix of the tectorial membrane in the Tecta^{L1820,G1824D/L1820F,G1824D} mouse stains positively for Tecta (E) that in Tecta^{C1837G/C1837G} mutant does not (F). This may be reflected in the relative density of the tectorial membranes in the Toluidine blue sections (B, C). Scale bar in C= 50 μm and applies to A–C, scale bar in F = 50 μm and applies to D–F.