Loss of the tectorial membrane protein CEACAM16 enhances spontaneous, stimulus-frequency, and transiently evoked otoacoustic emissions

Abstract

α-Tectorin (TECTA), β-tectorin (TECTB), and carcinoembryonic antigen-related cell adhesion molecule 16 (CEACAM) are secreted glycoproteins that are present in the tectorial membrane (TM), an extracellular structure overlying the hearing organ of the inner ear, the organ of Corti. Previous studies have shown that TECTA and TECTB are both required for formation of the striated-sheet matrix within which collagen fibrils of the TM are imbedded and that CEACAM16 interacts with TECTA. To learn more about the structural and functional significance of CEACAM16, we created a Ceacam16-null mutant mouse. In the absence of CEACAM16, TECTB levels are reduced, a clearly defined striated-sheet matrix does not develop, and Hensen's stripe, a prominent feature in the basal two-thirds of the TM in WT mice, is absent. CEACAM16 is also shown to interact with TECTB, indicating that it may stabilize interactions between TECTA and TECTB. Although brain-stem evoked responses and distortion product otoacoustic emissions are, for most frequencies, normal in young mice lacking CEACAM16, stimulus-frequency and transiently evoked emissions are larger. We also observed spontaneous otoacoustic emissions (SOAEs) in 70% of the homozygous mice. This incidence is remarkable considering that <3% of WT controls have SOAEs. The predominance of SOAEs >15 kHz correlates with the loss of Hensen's stripe. Results from mice lacking CEACAM16 are consistent with the idea that the organ of Corti evolved to maximize the gain of the cochlear amplifier while preventing large oscillations. Changes in TM structure appear to influence the balance between energy generation and dissipation such that the system becomes unstable.

Keywords: CEACAM16; Hensen's stripe; cochlea; mouse; otoacoustic emissions; tectorial membrane.

Figure 1.

Immunoblotting and immunoprecipitation. A, B, Western blots of TMs from WT, Ceacam16^+/βgal, and Ceacam16^βgal/βgal mice stained with antibodies to CEACAM16 (A) and a mixture of antibodies to TECTA, TECTB, and collagen IXa (B). C, D, Densitometry of CEACAM16 levels in WT and Ceacam16^+/βgal mice (C) and of levels of TECTA and TECTB in Ceacam16^+/βgal and Ceacam16^βgal/βgal mice relative to those in WT controls (D). Multiple comparisons were made by one-way ANOVA, followed by t tests to determine pairwise differences among the groups used in each experiment. Asterisks in C and D indicate values that are significantly different at p < 0.01. E, F, Immunoblots of proteins immunoprecipitated with anti-V5 monoclonal antibody and protein A-Sepharose from lysates prepared from HEK293-EBNA cells that were singly or cotransfected with plasmid DNAs as indicated above each lane. Blots in E were stained with mouse monoclonal anti-myc antibody (clone 9E10); blots in F with polyclonal rabbit anti-GFP antibody. Myc-TECTA (E, right lane) and EFGP-TECTB (F, right lane) are coimmunoprecipitated when coexpressed with CEACAM16–V5-His. IgG heavy chain is indicated (IgG).

Figure 2.

Ceacam16 is expressed by nonsensory cells in the organ of Corti. A, X-Gal staining for β-galactosidase reporter expression in the basal coil (∼20 kHz region) of a Ceacam16^βgal/βgal mouse at P21. X-Gal staining is observed in Deiters' cells (DCs), inner pillar cells (IP), outer pillar (OP) cells, border cells (BCs), and epithelial cells of the inner sulcus (IS) and spiral limbus (SL). OHCs, IHCs, and the TM are also indicated for reference. B, Immunofluorescence. A single confocal slice (0.63 μm optical thickness) of an organ of Corti cryosection from a P21 Ceacam16^βgal/βgal mouse stained with anti-βgal (red) and phalloidin to label F-actin (green). βgal is observed in Deiters' cells (DCs), outer pillar cells (OP) and inner pillar cells (IP), but not in hair cells. Scale bars, 20 μm.

Figure 3.

Loss of striated-sheet matrix and Hensen's stripe in the absence of CEACAM16. A–I, Toluidine-blue-stained semithin sections from WT (A–C), Ceacam16^+/βgal (D–F), and Ceacam16^βgal/βgal (G–I) mice from the ∼8 kHz (A, D, G), ∼20 kHz (B, E, H), and ∼40 kHz (C, F, I) regions of the cochlea are provided at P43. Hensen's stripe, although visible in the ∼40 kHz region of WT (arrowhead in C) and Ceacam16^+/βgal (arrowhead in F) TM is not present in the equivalent region of the Ceacam16^βgal/βgal TM (arrowhead in I). Insets show the Hensen's stripe region at 3× enlargement. Arrows in G–I indicate large holes in the TM matrix of the Ceacam16^βgal/βgal mouse. J, Quantification of holes in the TM. Shown is a bar chart comparing the area occupied by holes in the TMs of WT, Ceacam16^+/βgal, and Ceacam16^βgal/βgal mice from four different regions of the cochlea between P30 and P43. Data from between four and seven TM profiles were used for each region and genotype. TM profiles from Ceacam16^βgal/βgal mice have significantly more space occupied by holes (p < 0.001) than either WT or Ceacam16^+/βgal mice in all regions examined. K–L, Toluidine-blue-stained section of an ∼20 kHz cochlear profile from a P43 Ceacam16^βgal/βgal mouse before (K) and after (L) using Photoshop to threshold adjust the TM to distinguish matrix from holes. M–P, Phase-contrast images of alcian-blue-stained TMs from WT (M), Ceacam16^+/βgal (N, P), and Ceacam16^βgal/βgal (O) mice. Images in M–O are from the ∼40 kHz region; image in P is from the ∼14 kHz region. HS, Hensen's stripe. Scale bars, 50 μm.

Figure 4.

Ultrastructural examination of the TM. A–D, Electron micrographs of the covernet (CN) (A, B) and central region (C, D) of apical-coil TMs from P30 Ceacam16^+/βgal (A, C) and Ceacam16^βgal/βgal (B, D) animals. Striated-sheet matrix (arrow in A) emanates from the densely packed fibrils of the covernet in Ceacam16^+/βgal mice, whereas in Ceacam16^βgal/βgal mice, covernet fibrils are embedded in a dense amorphous matrix (arrow in B). The central core of the TM from Ceacam16^+/βgal mice contains striated-sheet material (arrow in C) intermingled with collagen fibrils (arrowhead in C), whereas in Ceacam16^βgal/βgal mice, the striated-sheet matrix is replaced by a tangled meshwork of filaments (arrow in D) interspersed between collagen fibrils of typical appearance (arrowhead in D). E–F, Electron micrographs of Hensen's stripe in basal-coil TMs from P43 Ceacam16^+/βgal (E) and Ceacam16^βgal/βgal (F) animals. Dense material on the underside of the TM (arrow in E) forms a distinctive, V-shaped Hensen's stripe in Ceacam16^+/βgal mice (E). In Ceacam16^βgal/βgal mice, however, this material (arrow in F) forms a flattened band on the underside of the TM (F). Scale bars: D, 200 nm and applies to A–D; F, 1 μm and applies to E–F.

Figure 5.

Developmental expression of Ceacam16. A–F, Cryosections from the basal (A–C) and apical (D–F) regions of X-Gal-stained cochleae from Ceacam16^βgal/βgal mice at P12 (A, D), P14 (B, E), and P16 (C, F). Large and small arrowheads point to X-Gal staining in the spiral limbus and border cells, respectively. Arrows indicate X-Gal staining in Deiters' cells. Scales bars in A–F, 50 μm. G–I, Transmission electron micrographs of the central region of the TM from WT animals in the ∼8 kHz region of the apical coil at P12 (G), P14 (H), and P16 (I). Arrows and arrowheads point to developing striated-sheet matrix and collagen fibrils, respectively. Scale bars in G–I, 200 nm.

Figure 6.

DPOAEs. A, B, DP-grams for 2F1–F2 at moderate (L1 = 50 dB; L2–35 dB, bottom) and high (L1=L2 = 70 dB, top) stimulus levels. WT control data are plotted in black, Ceacam16^+/βgal heterozygotes in blue, and Ceacam16^βgal/βgal homozygotes in red. The asterisks indicate statistically significant differences between homozygotes and their WT controls. Differences were not significant between heterozygotes and controls. In A, the p-value at f2 = 40.9 kHz was 0.0169; that for f2 = 15.5 kHz 0.0081, and that for f2 = 13.5 kHz 0.0309. In B, the p-value for f2 = 47 kHz was 0.0444). DPOAE input–output functions collected when the level for F1 is 10 dB higher than that for F2 (F2/F1 = 1.2) are plotted in C–F. The cubic difference tone is plotted for F2 = 12 kHz (C) and F2 = 27 kHz (D). In addition, DPOAE input–output functions for cubic (E) and quadratic (F) difference tones are also provided for F2 = 23 kHz and F2/F1 = 1.4.

Figure 7.

SOAEs. SOAEs are plotted in A for Ceacam16 ^βgal/βgal homozygotes in red and the only Ceacam16^βgal WT mouse with a SOAE in black. The bar graph in B shows the incidence of SOAEs per kHz bin across frequency for all homozygous mice showing this activity, as well as the sole control WT mouse, in black. C, Distribution of SOAEs, which peaks at ∼17 dB for mice lacking CEACAM16. The amplitude of the sole SOAE recorded from a WT mouse is plotted in black.

Figure 8.

SFOAEs and TEOAEs. Emission magnitude is plotted across frequency in A for an individual mouse lacking CEACAM16. The two curves in blue at the top show the pressure measured in the ear canal without the suppressor (solid line) and during presentation of the suppressor (dashed line), which removes the SFOAE generated in the cochlea and reflected back into the ear canal. The curve in black shows the SFOAE for a 30 dB probe, whereas the red curve provides the SOAEs. SFOAEs are plotted across frequency for homozygous mice (red) and their WT controls (black) in B. The noise floor is indicated in gray. C, Input–output functions for the SFOAE producing the largest response at 30 dB. The p-values for the input–output function were 0.0457 at 15 dB, 0.0103 at 20 dB, 0.0205 at 25 dB, 0.0055 at 30 dB, 0.0052 at 35 dB, 0.0020 at 40 dB, 0.0009 at 45 dB, 0.0012 at 50 dB, 0.0026 at 55 dB, and 0.0051 at 60 dB). D demonstrates that TEOAEs are larger in Ceacam16^βgal/βgal mice. Shown is the sound-equivalent level in dB for TEOAEs produced at 50 dB and for a 100 μs tone pip at the same frequency that produced the largest SFOAE and for which the SFOAE input–output function was collected.