Individual USH2 proteins make distinct contributions to the ankle link complex during development of the mouse cochlear stereociliary bundle

Abstract

Usher syndrome (USH) is the leading cause of inherited deaf-blindness, with type 2 (USH2) being the most common clinical form. Studies suggest that proteins encoded by USH2 causative genes assemble into the ankle link complex (ALC) at the hair cell stereociliary bundle; however, little is known about the in vivo assembly and function of this complex. Using various USH2 mutant mice, we showed by immunofluorescence that USH2 proteins play different roles in cochlear ALC assembly, with G protein-coupled receptor 98 being the most important protein. Complex assembly likely occurs at the stereociliary bundle but not along the protein transport route in the cell body. Stereociliary morphological defects in USH2 mutant mice suggest roles for the ALC in regulating inner hair cell stereociliary growth and differentiation as well as outer hair cell stereociliary rigidity and organization during development. These roles are unique from the bundle cohesion role of Usher syndrome type 1 protein complexes. Loss of individual USH2 gene expressions leads to variable morphological and functional consequences, correlating with the severity of ALC disruption. This finding suggests a potential genotype-phenotype correlation in USH2 patients. In summary, this study provides novel insights into the molecular mechanism underlying cochlear stereociliary bundle development and hearing loss pathogenesis of various USH2 subtypes. Our thorough phenotypical characterization of USH2 mouse models is essential for future use of these animal models in therapeutic development.

Figure 1.

Localization of USH2 and PDZD7 proteins at the ALC of developing cochlear hair cells. Double immunofluorescence demonstrates that GPR98 (A), usherin (B), whirlin (C) and PDZD7 (D) are localized at the ALC, i.e. the stereociliary base, while whirlin (C) is also present at the stereociliary tip in P4 wild-type (WT) mouse IHCs and OHCs. Absence of USH2 and PDZD7 immunoreactivities in their corresponding mutant cochlear hair cells verifies loss of protein expression and the specificity of our antibodies. For each panel, two WT IHC bundles, viewed from the medial side of hair cells (front) at slightly different angles, show USH2 or PDZD7 signals in either one or multiple rows of stereocilia. Amplified views of several IHC stereocilia are shown on the left of original images. The Adgrv1^−/−, Ush2a^−/− and Pdzd7^−/− OHC bundles are viewed from the back side (the lateral side of hair cells), while other stereociliary bundles are shown from either front or side views. Arrows point to stereociliary bases. Magenta signals in stereociliary bundles are specific signals labeled from USH2 and PDZD7 antibodies. Magenta signals outside stereociliary bundles are non-specific. Green signals are from phalloidin staining. Scale bars, 1 μm.

Figure 2.

GPR98 is required for localization of usherin, whirlin and PDZD7 at the ALC of cochlear hair cells. (A) Usherin immunoreactivity is undetectable in the stereociliary bundle of P4 Adgrv1^−/− IHCs and OHCs. (B) Whirlin immunofluorescence is detectable only at stereociliary tips but not at ALCs in P4 Adgrv1^−/− cochlear hair cells. (C) PDZD7 immunoreactivity is distributed along the entire stereocilia, except the tips, in P4 Adgrv1^−/− IHCs and OHCs. Images (a), (d) and (g) are low-magnification views of Adgrv1^−/− IHC and OHC stereociliary bundles; (b) (front view), (e) (back view) and (h) (front view) are single Adgrv1^−/− IHC stereociliary bundles; (c), (f) and (j) are back views of single Adgrv1^−/− OHC stereociliary bundles; (i) is an amplified view of several stereocilia within an Adgrv1^−/− IHC bundle. Arrows point to stereociliary bases. Magenta signals in stereociliary bundles are from staining of USH2 and PDZD7 proteins, and magenta signals outside stereociliary bundles are non-specific. Phalloidin signals are green. Scale bars, 5 μm (a, d and g) and 1 μm (b and c, e and f, h and j).

Figure 3.

Subtilisin treatment disrupts ankle links but not the ALC in cochlear stereociliary bundles. Subtilisin treatment eliminates ankle links in P4 cochlear hair cells, shown by a lack of immunoreactivity with an antibody directed against the GPR98 extracellular region (A). However, this treatment does not affect distribution of the GPR98 intracellular fragment at the P4 stereociliary base, shown by immunofluorescence using an antibody against the GPR98 cytoplasmic region (B). Localization of usherin (C), PDZD7 (D) and whirlin (E) at P4 stereociliary bases appears normal after subtilisin treatment (right column) compared with PBS-treated negative controls (left column). Stereociliary bundles are viewed from the front and side except the PBS-treated IHC bundle in (B), which is a back view. Arrows point to stereociliary bases. Green signals are from phalloidin staining. Magenta signals in stereociliary bundles are specific signals of USH2 and PDZD7 proteins, while magenta signals outside stereociliary bundles are non-specific. Scale bars, 1 µm.

Figure 4.

Usherin plays differential roles in localization of GPR98, whirlin and PDZD7 at the cochlear ALC. (A) GPR98 (magenta) distribution at the ALC is altered in most IHCs and OHCs of P4 Ush2a^−/− mice. GPR98 punctate signals are localized predominantly at both the tip and base of Ush2a^−/− IHC stereocilia (a–c). (a) Back view of four Ush2a^−/− IHC stereociliary bundles showing abnormal GPR98 distribution; (b) front view of an Ush2a^−/− IHC bundle showing normal GPR98 distribution; (c) side view of an Ush2a^−/− IHC bundle with abnormal GPR98 signal (left) and front view of another Ush2a^−/− IHC bundle with near-normal GPR98 signal (right); (d) GPR98 punctate signals are randomly distributed along Ush2a^−/− OHC stereocilia. (B) Whirlin (magenta) is completely absent at the stereociliary base but detectable at the stereociliary tip in both P4 Ush2a^−/− IHCs and OHCs. (e and f) Front and back views of Ush2a^−/− IHC bundles, respectively; (g) back view of an Ush2a^−/− OHC bundle. (C) PDZD7 (magenta) distribution in Ush2a^−/− IHC (h and i) and OHC (j and k) stereociliary bundles is similar to that in Adgrv1^−/− IHC and OHC bundles at P4. Insets (h′ and i′) are amplified views and shown in original images for context. Images (h), (j) and (k): back view; (i): side view. Arrows point to stereociliary bases. Green signals are from phalloidin. Magenta signals outside stereociliary bundles are non-specific. Scale bars, 1 μm.

Figure 5.

Minor mislocalization of GPR98 and normal localization of usherin and PDZD7 in Dfnb31^neo/neo cochlear hair cells. (A) GPR98 (magenta) is localized normally at stereociliary bases of most P4 Dfnb31^neo/neo IHC (a, b, back and front views, respectively) and OHC (d) bundles, while GPR98 is partially mislocalized to stereociliary tips in a small number of Dfnb31^neo/neo IHCs (c) and OHCs (e). (f) Single magenta channel image of (e). (B) Usherin signals (magenta) remain at stereociliary bases of P4 Dfnb31^neo/neo cochlear hair cells. (g and h) Back and front views of Dfnb31^neo/neo IHC bundles, respectively; (i and j) back and front views of Dfnb31^neo/neo OHC bundles, respectively. (C) PDZD7 (magenta) localization appears normal in P8 Dfnb31^neo/neo IHC (m) and OHC (n) relative to age-matched wild-type (WT) IHC (k) and OHC (l). Arrows point to stereociliary bases. Green signals are from phalloidin labeling. Magenta signals outside stereociliary bundles are non-specific. Scale bars, 1 μm.

Figure 6.

Minor mislocalization of GPR98 and normal localization of usherin and PDZD7 in Dfnb31^wi/wi cochlear hair cells. (A) GPR98 (magenta) is localized at the stereociliary base in most P4 Dfnb31^wi/wi IHCs (a and b, front views) and OHCs (c, front view). GPR98 is sometimes present at both the stereociliary tip and base in P4 Dfnb31^wi/wi OHC stereociliary bundles (d and e). (B) Usherin (magenta) distribution is normal in P4 Dfnb31^wi/wi IHC (f and g) and OHC (h and i) stereociliary bundles. (C) PDZD7 (magenta) is localized normally at stereociliary bases in Dfnb31^wi/wi IHCs (j and k) and OHCs (l and m) at P4. Arrows point to stereociliary bases. Green signals are from phalloidin staining. Magenta signals outside stereociliary bundles are non-specific. Scale bars, 1 µm.

Figure 7.

Adgrv1^−/−, Ush2a^−/− and Dfnb31^neo/neo mice exhibit various morphological defects in cochlear stereociliary bundles. (A) Low-magnification SEM images showing abnormal hair cell bundles in Adgrv1^−/−, Ush2a^−/− and Dfnb31^neo/neo mice at P4. (B) SEM images of individual Adgrv1^−/−, Ush2a^−/− and Dfnb31^neo/neo OHC bundles. Compared with the WT OHC bundle (e), Adgrv1^−/− OHC bundles show stereocilia tilted at stereociliary bases (f and g) and missing stereocilia in the shortest stereociliary row (h and i, arrows); Ush2a^−/− OHC bundles have distorted shapes (j–l) and mislocalized kinocilia (k and l, arrows); and Dfnb31^neo/neo OHC bundles usually have a blunt U- rather than a sharp V-shape (m and o). (C) SEM images of individual Adgrv1^−/−, Ush2a^−/− and Dfnb31^neo/neo IHC bundles. Compared with WT IHCs (p), Adgrv1^−/− IHCs have thick stereocilia (q and r) in the bundle and ectopic stereocilia at the neural edge outside the bundle (r, arrows); many Ush2a^−/− IHCs have morphological defects similar to those observed in Adgrv1^−/− IHCs (arrows, ectopic stereocilia, s and t) but with normal stereociliary thickness; Dfnb31^neo/neo IHC bundles have thick stereocilia (u and v) and sometimes more than three rows of stereocilia (u). Scale bars, 5 µm (A) and 1 μm (B and C).

Figure 8.

Quantification of stereociliary thickness in Adgrv1^−/−, Ush2a^−/− and Dfnb31^neo/neo IHCs. Stereociliary thickness was measured using ImageJ on high-magnification SEM images as described in the Materials and Methods section. Student's t-tests were conducted to analyze the significance of differences among genotype groups. Numbers of cells and animals examined in each genotype group are indicated before and after the comma, respectively. *P < 0.05, **P < 0.01; error bars, standard error of the mean.

Figure 9.

Adgrv1^−/−, Ush2a^−/− and Dfnb31^neo/neo mice have different degrees of hearing loss. Hearing function of 1-month-old Adgrv1^−/−, Ush2a^−/− and Dfnb31^neo/neo mice was assessed by ABR tests at frequencies of 4, 5.6, 8.0, 11.3, 16.0, 22.6, 32.0 and 45.2 kHz. Compared with wild-type controls, these mutant mice have significantly elevated ABR thresholds across all the frequencies tested (P < 0.01, 2-way ANOVA with post hoc tests). Threshold differences between Adgrv1^−/− and Ush2a^−/−, Adgrv1^−/− and Dfnb31^neo/neo, and Ush2a^−/− and Dfnb31^neo/neo mice are statistically significant (P < 0.01) at 5.6, 8.0, 11.3 and 16.0 kHz. Adgrv1^−/− and Dfnb31^neo/neo mice have the most and least severe hearing loss, respectively. For comparison, ABR results on Pdzd7^−/− mice at P21–P28 are also shown here (24). Threshold differences between Ush2a^−/− and Pdzd7^−/− mice are not statistically significant at any tested frequency. Numbers in parentheses indicate numbers of animals tested. Error bars, standard error of the mean.

Figure 10.

Original Ush2a^−/− mouse colony has highly variable ABR and DPOAE responses. Three male littermates from the original Ush2a^−/− colony were tested by ABR (left) and DPOAE (right) at 1 month of age. Responses from individual mice are shown. These mice have consistently high thresholds at high frequencies but variable thresholds at low frequencies in both ABR and DPOAE tests.

Figure 11.

Roles of individual USH2 and PDZD7 proteins in the assembly and function of the ALC in cochlear hair cells. This summary includes data from Pdzd7^−/− mice (24). (A) Interdependence of USH2 and PDZD7 proteins for their normal localization in cochlear stereociliary bundles. Proteins pointed by arrows are those whose localization is determined by the proteins on the other end of lines. Continuous and dashed lines indicate complete and partial dependence, respectively. (B) Hypothesis on the assembly of the ALC in hair cells. Arrows represent the recruitment directions of proteins. Dashed arrow, indirect recruitment. G, GPR98; U, usherin; P, PDZD7; and W, whirlin. (C and D) USH2 and PDZD7 protein distribution and stereociliary bundle morphological defects in various mutant IHCs (C) and OHCs (D). Dashed circles denote the normal or disrupted ALCALC. Symbol color matches the color of USH2 and PDZD7 proteins shown in (A). Straight line arrangement of symbols along stereociliary diagrams indicates even distribution of their represented proteins. Random distribution of symbols in the stereocilia indicates random punctate distribution of the represented proteins.