Fig 1.
Schematic view of the gene structure and assembly of collagen IV.
The COL4A6/Col4a6 gene is located on the X chromosome and paired with COL4A5/Col4a5 in a head-to-head manner. COL4A1/Col4a1 and COL4A2/Col4a2, and COL4A3/Col4a3 and COL4A4/Col4a4 pairs are located in a head-to-head manner on their respective chromosomes (not shown). Each chain comprises a 400 nm long triple-helical domain, which contains many interruptions in the Gly-X-Y repeated sequence. The globular noncollagenous domain (NC1) is located at the C-terminal end, while the 7S domain is at the N-terminal end. Three α chains assemble the triple-helical molecule (triple-helix). The heterotrimers extracellularly form a hexamer. α1α2α1(IV) and α5α6α5(IV) bind to each other through the NC1 domain [α1α2α1-α5α6α5(IV)] and the 7S domains (not shown), which facilitates their assembly to a higher-ordered supra structure. α1α2α1(IV) is widely distributed, whereas α3α4α5(IV) and α5α6α5(IV) distribution is tissue specific [7–9, 12]. In Col4a6 KO mice, the loss of α6(IV) chain expression inhibits the formation of the α5α6α5(IV) heterotrimer. This study showed that the phenotypes of the auditory system in Col4a6 KO mice were distinct from those of individuals with the COL4A6 missense mutation c.1771G>A, p.Gly591Ser. The presence of the mutant protein α6(IV) chain and/or α5α6α5(IV) may induce deleterious effects on the cochlea. The pathogenesis involved in congenital hearing loss warrants further investigations.
Fig 2.
Distribution of collagen α6(IV) chain in the adult mouse cochlea.
Sections were immunostained using antibodies against collagen α2(IV) (A, I–K: green) and α6(IV) (B–H: green; I–K: red) chains, and CD31 (G, H: red) for the detection of endothelial cells. Subepithelial BMs and perivascular BMs are indicated by open arrowheads and solid arrowheads in the insets, respectively (G, J, K). (I) The superficial plate and invaginated region of the interdental cells network are indicated by open arrows and solid arrows in insets, respectively. (L) Schematic diagram of the distribution of collagen α6(IV) chain [yellow line: subepithelial BMs; pink line: perivascular BMs]. The negative control was established by immunostaining with only the secondary antibody and no specific reaction was observed (S2 Fig). Nuclei were counterstained with Hoechst 33258 (blue). SLim: spiral limbus; B, basilar membrane; SLig, spiral ligament; SV, stria vascularis; RM, Reissner’s membrane; NF, neural fasciculus; SG, spiral ganglion; IDC, interdental cells; ISC, inner sulcus cells; OSC, outer sulcus cells; RC, root cells; Cap, capillary. Scale bars = 50 μm. Scale bars in inset = 10 μm.
Fig 3.
Distribution of collagen α5(IV) chain in the cochlea of WT and Col4a6 KO mice.
(A, D–F) The collagen α5(IV) chain located in the subepithelial BMs, perivascular BMs, and perineural BMs in WT mice (green). (B, G–I) In Col4a6 KO mice, the α5(IV) chain is widely distributed (green). However, weak signals were detected in the perivascular BMs of the spiral limbus (SLim), spiral ligament (SLig), and stria vascularis (SV) (dashed arrows). (C) No immunopositive signal of the collagen α6(IV) chain was present in Col4a6 KO mouse cochlea (green). Nuclei were counterstained with Hoechst 33258 (blue). B, basilar membrane; RM, Reissner’s membrane; NF, neural fasciculus; SG, spiral ganglion; IDC, interdental cells; ISC, inner sulcus cells; OSC, outer sulcus cells; RC, root cells; Cap, capillary. Scale bars = 50 μm.
Table 1.
Qualitative assessment using immunohistochemistry: Distribution of collagen α1(IV), α2(IV), α5(IV), and α6(IV) chains and the predicted molecular composition of the collagen IV network in mouse cochlea.
Fig 4.
Hearing threshold assessed by click-evoked auditory brainstem responses in WT and Col4a6 KO mice.
No significant difference observed between WT mice (mean 40 ± 3.5 dB SPL, n = 5) and Col4a6 KO mice (mean 43 ± 2.7 dB SPL, n = 5). P = 0.172 by Student’s unpaired t-tests. The values are presented as mean ± SD.
Fig 5.
Comparison of the cochlea bone structure of WT and Col4a6 KO mice by micro-CT analysis.
(A) Representative three-dimensional images of the petrous portion of the left temporal bone in WT and Col4a6 KO mice. The top shows the apex of petrous in the superior view. (B–G) The three orthogonal plane images were automatically obtained as a best-matched plane from the Col4a6 KO data set compared to the images manually selected from the cochlea structure of the WT mice data set. (H–J) The overlay images demonstrated that no differences in the size or shape are apparent in the cochlear bone between WT and Col4a6 KO mice. Blue squares represent the Volume of Interest. (K) Light blue color indicates a highly matched degree based on the color map.
Fig 6.
Representative images of HE-stained midmodiolar cochlear sections.
Histological comparison between WT (A, C, E) and Col4a6 KO (B, D, F) mice. (C-F) Images with higher magnification of cochlear basal turn are shown. No differences are observed between WT and Col4a6 KO mice. ⁎, basal turn; #, apical turn; SG, spiral ganglion; SLim, spiral limbus; TM, tectorial membrane; B, Basilar membrane; Co, organ of Corti; SLig, spiral ligament; SV, stria vascularis; RM, Reissner’s membrane; closed arrows, OHC (outer hair cells); arrows, IHC (inner hair cells); open arrowheads, Dieter’s cells.
Fig 7.
Distribution of major basement membrane components in the cochlea of WT and Col4a6 KO mice.
Cryosections were immunostained by antibodies against collagen α1(IV) chain (A, B), perlecan (C, D), laminin α1 (E, F), laminin α2 (G, H), laminin γ1 (I, J), and nidogen-1 (K, L). Nuclei were counterstained with Hoechst 33258 (blue). No differences are apparent between WT and Col4a6 KO mice. Scale bars = 50 μm.