Follistatin regulates the specification of the apical cochlea responsible for low-frequency hearing in mammals

Abstract

The cochlea's ability to discriminate sound frequencies is facilitated by a special topography along its longitudinal axis known as tonotopy. Auditory hair cells located at the base of the cochlea respond to high-frequency sounds, whereas hair cells at the apex respond to lower frequencies. Gradual changes in morphological and physiological features along the length of the cochlea determine each region's frequency selectivity, but it remains unclear how tonotopy is established during cochlear development. Recently, sonic hedgehog (SHH) was proposed to initiate the establishment of tonotopy by conferring regional identity to the primordial cochlea. Here, using mouse genetics, we provide in vivo evidence that regional identity in the embryonic cochlea acts as a framework upon which tonotopy-specific properties essential for frequency selectivity in the mature cochlea develop. We found that follistatin (FST) is required for the maintenance of apical cochlear identity, but dispensable for its initial induction. In a fate-mapping analysis, we found that FST promotes expansion of apical cochlear cells, contributing to the formation of the apical cochlear domain. SHH, in contrast, is required both for the induction and maintenance of apical identity. In the absence of FST or SHH, mice produce a short cochlea lacking its apical domain. This results in the loss of apex-specific anatomical and molecular properties and low-frequency-specific hearing loss.

Keywords: cochlea; follistatin; frequency discrimination; tonotopy.

Fig. 1.

Specification of apical cochlear regional identity is compromised in Fst^−/− cochleae. (A–P) In situ hybridization analysis with apical (Fst, Msx1, Slitrk3, and Efnb2) and basal (A2m and Inhba) regional markers and readouts of SHH signaling (Ptch1 and Gli1) in Fst^+/− and Fst^−/− embryos at E15.5. Arrows and arrowheads indicate relatively strong and weak expression, respectively. Red asterisks and red arrowheads indicate absence or down-regulation of expression, respectively. (Q–S) Paint-fill analysis of the Fst^−/−inner ear (Q, R) and cochlear lengths of control and Fst^−/−at E15.5(S). (T) Relative in situ hybridization signal intensities along the cochlear duct in Fst^+/− and Fst^−/− embryos. Red arrows indicate the apical end of the cochleae of Fst^−/− embryos. Gray boxes below 0% in each graph indicate background signal. Representative graphs are presented from one Fst^+/− and one Fst^−/− cochlea for each gene. Additional samples are in SI Appendix, Fig. S1. The scale bar in A, 100 μm, also applies to B–P. The scale bar in Q, 100 μm, also applies to R.

Fig. 2.

Failure of apical cochlear expansion caused by reduced cell proliferation in Fst^−/− cochleae. (A–H) Fate-mapping of Msx1-lineage cells using Msx1^CreERT2/+; R26-tdtomato mice in the presence (Fst^+/−) or absence (Fst^−/−) of FST. After tamoxifen was injected into pregnant female mice at E11.5, the embryos were harvested at E18.5 (A). Quantification of tdTomato-fluorescence intensity along the cochlear duct (H). (I–R) Comparing EdU-labeled cells in Fst^+/− and Fst^−/− embryos. After EdU was injected into pregnant female mice at E12.5, EdU-labeled cells (green) were counted among the lateral non-sensory cells (yellow brackets) and MYO7A-positive hair cells (white brackets) (I–P). The number of EdU-positive cells in the lateral compartment (Q) and the percentage of EdU-positive hair cells (R) were plotted against the distance along the cochlear duct divided into five regions from base to apex. (S–T) Comparisons of cochlear length and hair cell number between E18.5 Fst^+/− and Fst^−/− embryos. Data in Q–T are presented as means ± SE. Statistical comparisons were determined via two-way ANOVA for the EdU analysis and unpaired t tests with Bonferroni corrections for the cochlear length measurements (*P < 0.05, **P < 0.01, and ***P < 0.001). The scale bar in C, 200 μm, also applies to B–C, E–F, and I–M. The scale bar in D, 200 μm, also applies to G. The scale bar in J, 10 μm, also applies to K–L and N–P.

Fig. 3.

Loss of apical region stereocilia morphology in 4-wk-old Fst cKO mutants. (A–R) Scanning electron micrographs of the organ of Corti from control (lox) (Fst^lox/lox) and inner ear-specific Fst cKO (Pax2-Cre; Fst^lox/lox) mice. The lengths of the outer hair cell stereocilia were measured at the vertex of the V-shaped hair bundles from the lateral side (G–L). The angle of the V-shaped stereocilia and the number of stereocilia per outer hair cell were measured using a top-down view (M–R). The scale bar in A, 10 μm, also applies to B–F. The scale bar in G, 1 μm, also applies to H–R. (S–U) Quantification of the length, angle, and number of outer hair cell stereocilia along the tonotopic axis in each of the five cochlear regions beginning with the basal end. These represent the base (10 to 18%), mid-base (28 to 36%), mid (46 to 54%), mid-apex (64 to 72%), and apex (82 to 90%) regions. Statistical comparisons were determined via two-way ANOVA with Bonferroni corrections for multiple comparisons (n.s., non-significant, **P < 0.01, and ***P < 0.001).

Fig. 4.

Low-frequency-specific hearing loss in 4-wk-old Fst cKO mutants. (A–C) ABR analyses of 4-wk-old Fst^lox/lox and Pax2-Cre; Fst^lox/lox mice. ABR thresholds of Fst^lox/lox and Pax2-Cre; Fst^lox/lox mice to click stimuli and individual frequencies (A). I/O function analyses of wave I amplitudes from the ABRs to low (6 kHz) and high (18 kHz) frequencies (B, C). (D–F ) DPOAE analyses of 4-wk-old Fst^lox/lox and Pax2-Cre; Fst^lox/lox mice. 2f₁-f₂ DPOAE thresholds of Fst^lox/lox and Pax2-Cre; Fst^lox/lox mice (D). DPOAE I/O function analyses at 6 kHz and 18 kHz (E, F ). Data are presented as means ± SE. Statistical comparisons were determined via two-way ANOVA with Bonferroni corrections for multiple comparisons (n.s., non-significant, *P < 0.05, **P < 0.01, and ***P < 0.001).

Fig. 5.

Summary diagram illustrating tonotopic organization in the mammalian cochlea. (A) The tonotopic organization of the cochlea is established sequentially. First, apical-to-basal regional identity is established. This then provides a framework upon which the basal-to-apical tonotopic characteristics tuned to specific frequencies appear along the mature cochlea. (B–D) Loss or gain of FST or SHH function perturbs this framework in the developing cochlea, resulting in changes in the tonotopic characteristics of the mature cochlea. See the Discussion section for details on each genotype.