Epithelial cell stretching and luminal acidification lead to a retarded development of stria vascularis and deafness in mice lacking pendrin

Abstract

Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4(-/-), are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO(3) (-) transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4(-/-) mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4(+/-) and Slc26a4(-/-) mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4(-/-) mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4(-/-) mice, and possibly in humans, lacking functional pendrin expression.

Figure 1. Onset of pendrin expression in the cochlea.

Pendrin (red) was visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. A: Diagram of the inner ear. B–G: Cross-sections of the cochlear duct in the hook region (B), basal turn (C, E and G) and upper turn (D and F) of Slc26a4^+/− mice at age E14.5 (B and C), E16.5 (D and E) and E17.5 (F and G). Abbreviations: K, Kölliker's organ; RM, Reissner's membrane; SV, stria vascularis; OS, outer sulcus. Pendrin expression is marked by arrows.

Figure 2. Onset of pendrin expression in the ampullae and utricle of the vestibular labyrinth.

Pendrin (red) was visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. A: Diagram of the inner ear. B–D: Cross-sections of the anterior canal ampulla of Slc26a4^+/− mice at age E15.5–E17.5. E–I: Cross-sections of utricle of Slc26a4^+/− mice at age E13.5–E17.5. Abbreviations: A, Ampulla; T, transitional cells; HC, hair cells. Pendrin expression is marked by arrows.

Figure 3. Onset of pendrin expression in the saccule of the vestibular labyrinth.

Pendrin (red) was visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. A: Diagram of the inner ear. B–C: Whole-mount of the saccule from a Slc26a4^+/− mice at age E14.5. D–F: Cross-sections of the saccule from Slc26a4^+/− mice at age E14.5–E17.5. Abbreviations: S, Saccule; T, transitional cells; HC, hair cells. Pendrin expression is marked by arrows.

Figure 4. Onset of pendrin expression in the endolymphatic sac.

Na⁺/K⁺ ATPase or pendrin (red) were visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. A, B, D, F, I and J: Cross-sections of the endolymphatic sac from Slc26a4^+/− mice at ages E11.5-P2. C, E, G and H: Whole-mounts of the endolymphatic sac from Slc26a4^+/− mice at age E12.5–E15.5. Abbreviations: U, utricle; S, saccule; ES, endolymphatic sac. Pendrin expression is marked by arrows.

Figure 5. pH measurements in the embryonic inner ear.

A: Measurements of the endolymphatic and perilymphatic pH in the cochlea of isolated in vitro superfused otocysts from Slc26a4^+/− (HET) and Slc26a4^−/− (KO) mice aged E14.5–E17.5. Data from P10 Slc26a4^+/− mice, obtained by in situ measurements and reported earlier , are shown here for comparison. B: Measurements of the endolymphatic pH in the endolymphatic sac of isolated otocysts from Slc26a4^+/− (HET) and Slc26a4^−/− (KO) mice aged E14.5–E17.5. Numbers next to the bars represent the N number of otocysts. Significant differences between endolymph from Slc26a4^+/− (black) and Slc26a4^−/− (red) mice are marked with a star.

Figure 6. Onset of cell stretching in cochlea and endolymphatic sac.

Pendrin (red) was visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. A: Cross-sections of the basal turn of the cochlea from Slc26a4^+/− (HET) and Slc26a4^−/− (KO) mice at age E14.5. The circumference of the cochlear lumen is marked by a white line. B: Summarized measurements (avg±sem) of the luminal circumference of the cochlea at ages E14.5–E18.5. Numbers next to the bars represent the N number of otocysts. Significant differences between Slc26a4^+/− (black) and Slc26a4^−/− (red) mice are marked with a star. C–D: Whole-mounts of the endolymphatic sac from Slc26a4^+/− (HET) and Slc26a4^−/− (KO) mice at ages E13.5 and E14.5. The width of the endolymphatic sac is marked by a double-arrow. E–F: Summarized measurements (avg±SD, each bar N = 15) of the apical surface area of epithelial cells in the endolymphatic sac and duct at ages E13.5–E14.5. Significant differences between Slc26a4^+/− (black) and Slc26a4^−/− (red) mice are marked with a star. Figures preceded by ‘x’ indicate the factor between measurements in Slc26a4^+/− and Slc26a4^−/− mice.

Figure 7. Epithelial cell stretching in the cochlea and the vestibular labyrinth at P2.

Pendrin (red) was visualized by immunocytochemistry and F-actin (green) was labeled in Slc26a4^+/− (HET) mice and Slc26a4^−/− (KO) mice. A–D: Whole-mounts of epithelial cells from the cochlea. Abbreviations: SP, spiral prominence epithelial cells; OS, outer sulcus epithelial cells. E–H) Whole-mounts of epithelial cells from the vestibular labyrinth and endolymphatic sac. Mitochondria-rich cells in endolymphatic sac from Slc26a4^+/− and Slc26a4^−/− mice are marked by arrows. Abbreviations: T, transitional cells; H, hair cells. I: Summary of apical cell surface area measurements (avg±SD, N = 15) made on cochlea epithelial cells. Abbreviations: MC, marginal cells of stria vascularis; OS, outer sulcus epithelial cells; HC, hair cells; RM, Reissner's membrane epithelial cells. J: Summary of apical cell surface area measurements (avg±SD, N = 15) made on vestibular epithelial cells. Abbreviations: UT, utricular transitional cells; UHC, utricular hair cells; ST, saccular transitional cells; SHC, saccular hair cells; M, mitochondria-rich cells; R, ribosome-rich cells; ED, endolymphatic duct epithelial cells. Significant differences between Slc26a4^+/− (black) and Slc26a4^−/− (red) mice are marked with a star. Figures preceded by ‘x’ indicate the factor between measurements in Slc26a4^+/− and Slc26a4^−/− mice.

Figure 8. Connexin 26 expression in the cochlea of Slc26a4 ^+/− and Slc26a4 ^−/− mice at E14.5 and E16.5.

Connexin 26 (red) was visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. A–B: Cross-sections of the basal turn of the cochlea from Slc26a4^+/− and Slc26a4^−/− mice at age E14.5. C–D: Cross-sections of the basal turn of the cochlea from Slc26a4^+/− and Slc26a4^−/− mice at age E16.5. Abbreviations: K, Kölliker's organ; RM, Reissner's membrane; S, stria vascularis; OS, outer sulcus.

Figure 9. Connexin 26 and Na⁺/K⁺ ATPase expression in the cochlea of Slc26a4 ^+/− and Slc26a4 ^−/− mice at E18.5.

Connexin 26 and Na⁺/K⁺ ATPase (both red) were visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. A–B: Cross-sections of the basal turn of the cochlea from Slc26a4^+/− mice at age E18.5. C: Cross-sections of the basal turn of the cochlea from Slc26a4^−/− mice at age E18.5. Abbreviations: K, Kölliker's organ; RM, Reissner's membrane; SS, fibrocytes in the suprastrial region; S, stria vascularis; SP, spiral prominence; OS, outer sulcus. The arrows in A and C point to basal cells at E18.5 that lack connexin 26 expression in Slc26a4^+/− mice but express connexin 26 prematurely in Slc26a4^−/− mice.

Figure 10. Connexin 26 and Na⁺/K⁺ ATPase expression in the cochlea of Slc26a4 ^+/− and Slc26a4 ^−/− mice at P3.

Connexin 26 and Na⁺/K⁺ ATPase (both red) were visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. A–B: Cross-sections of the basal turn of the cochlea from Slc26a4^+/− mice at age P3. C–D: Cross-sections of the basal turn of the cochlea from Slc26a4^−/− mice at age P3. Abbreviations: K, Kölliker's organ; L, fibrocytes in the spiral limbus; RM, Reissner's membrane; SS, fibrocytes in the suprastrial region; S, stria vascularis; SL, spiral ligament; SP, spiral prominence; OS, outer sulcus.

Figure 11. Retarded development of stria vascularis.

A–C: Cross-sections of stria vascularis of Slc26a4^+/− (HET) and Slc26a4^−/− (KO) mice at E14.5, E16.5 and P3. Na⁺/K⁺ ATPase (red) was visualized by immunocytochemistry. F-actin (green) and nuclei (blue) were labeled. D: Cross-sections of stria vascularis at P7 visualized by laser-scanning microscopy without any stain. E: Cross-sections of stria vascularis at ∼P30. The Na⁺/2Cl⁻/K⁺ cotransporter Slc12a2 was visualized by immunocytochemistry. The ∼P30 data, previously reported , were added here for comparison. Tissue height of stria vascularis is marked by arrow-heads. Cross-sections of individual capillaries are marked with a star.