Six1 is essential for differentiation and patterning of the mammalian auditory sensory epithelium

Abstract

The organ of Corti in the cochlea is a two-cell layered epithelium: one cell layer of mechanosensory hair cells that align into one row of inner and three rows of outer hair cells interdigitated with one cell layer of underlying supporting cells along the entire length of the cochlear spiral. These two types of epithelial cells are derived from common precursors in the four- to five-cell layered primordium and acquire functionally important shapes during terminal differentiation through the thinning process and convergent extension. Here, we have examined the role of Six1 in the establishment of the auditory sensory epithelium. Our data show that prior to terminal differentiation of the precursor cells, deletion of Six1 leads to formation of only a few hair cells and defective patterning of the sensory epithelium. Previous studies have suggested that downregulation of Sox2 expression in differentiating hair cells must occur after Atoh1 mRNA activation in order to allow Atoh1 protein accumulation due to antagonistic effects between Atoh1 and Sox2. Our analysis indicates that downregulation of Sox2 in the differentiating hair cells depends on Six1 activity. Furthermore, we found that Six1 is required for the maintenance of Fgf8 expression and dynamic distribution of N-cadherin and E-cadherin in the organ of Corti during differentiation. Together, our analyses uncover essential roles of Six1 in hair cell differentiation and formation of the organ of Corti in the mammalian cochlea.

Fig 1. Deletion of Six1 in the developing cochlea using Eya1^CreER or Sox2^CreER leads to shortened and thickened prosensory primordium.

Cochleae were dissected from E14.5 embryos given tamoxifen at E11.5 (9 am) and E12.5 (9 am) and processed for whole-mount (A-D) or section (E-G) immunostaining with anti-Sox2 (green) and -p27^Kip1 (red). Hoechst was used for nuclear-counter staining. (E-G) Section collected from mid-cochlear duct in wild-type, Eya1^CreER or Sox2^CreER;Six1^fl/fl (Cko/Cko) littermates as indicated by dashed line in A, B, D respectively. Bracket indicates p27^Kip1-positive prosensory domain within the cochlea epithelium and its width on mediolateral axis is comparable between control and mutant littermates. (H) Spatial calibration of Sox2⁺ and p27^Kip1+ width, height and square area and value represents average number (±standard deviations) per section (6 μm) (see Methods for calibration). P-value was measured for +/+ and Cko/Cko using Two-tailed Student’s t-test. Scale bars: 200 μm in A-C and 40 μm in D,E.

Fig 2. Altered cell proliferation in Six1 CKO (Eya1^CreER) cochlea epithelium.

(A) Immunostaining for Sox2 (green) and EdU (red) on sections of basal and middle regions of cochleae from E17.5 embryos given EdU at E14.5 and tamoxifen at E11.5–12.5. Arrows point to high levels of Sox2 in cells within the lumenal layer. (B) Immunostaining for Sox2 (green) and EdU (red) on sections of middle region of cochleae from E14.5 embryos given tamoxifen at E11.5–12.5 and EdU at E11.5. (C) Number of EdU⁺Sox2⁺ cells per section (6 μm) or EdU⁺ cells per cochlea (see Methods for quantification). P-value was measured for +/+ and Cko/Cko using Two-tailed Student’s t-test: p<0.001 for E17.5 (EdU at E14.5) and p = 0.003 for E14.5 (EdU at E11.5). Scale bars: 30 μm in A,B.

Fig 3. Downregulation of Sox2 in differentiating hair cells is disrupted in Six1 CKO.

(A) Immunostaining for Sox2 (green) on whole-mount and (B) Sox2 (green)/Myo7a (red) on sections of cochleae from wild-type or Six1 CKO (Eya1^CreER;Six1^fl/fl) littermate embryos at E18.5 (given tamoxifen at E11.5–12.5). Cochlear section region was indicated by dashed line in A. Arrows point to Sox2 expression in the GER cells flanking the inner hair cells. (C) Atoh1 in situ hybridization showing Atoh1 expression in the organ of Corti in wild-type embryos at E17.5 and absence of Atoh1 in Atoh1^fl/fl;Eya1^CreER littermate embryos (arrow) given tamoxifen at E14.75–15.5. (D) Immunostaining for Myo7a (green) and Sox2 (red) on cochlear sections from E17.5 wild-type or Eya1^CreER;Atoh1^fl/fl littermate embryos given tamoxifen at E14.75–15.5. (E) Immunostaining for Pou4f3 (green) and Six1 (red) on cochlear sections from E17.5 wild-type and Eya1^CreER;Atoh1^fl/fl littermate embryos given tamoxifen at E14.75–15.5. Scale bars: 200 μm in A; 30 μm in B,D,E; 40 μm in C.

Fig 4. Lack of outer hair cell formation in Six1 CKO (Eya1^CreER) cochlea.

(A,B) Immunostaining for Myo7a of cochlea from E18.5 embryos given tamoxifen at E11.5–12.5. (A’,B’) H&E staining of section from middle region of cochlea indicated by dashed line in A or B respectively. (A”,B”) Higher magnification of boxed region in A or B respectively. (C-F) Co-immunostaining on sections for Myo7a (green) and Calretinin (red). Abb.: dc, Deiters’ cells; GER, greater epithelial ridge; ibc, inner border cell; ihc, inner hair cells; iph, inner phalangeal cell; oc, organ of Corti; ohc, outer hair cells; pc, pillar cells; sc, supporting cells. Scale bars: 200 μm in A and B; 50 μm in A’,B’ and C-F; 30 μm in A” and B”.

Fig 5. Reduced Fgf8 expression in Six1^Cko/Cko mutant (Eya1^CreER;Six1^fl/fl given tamoxifen from E11.5–12.5).

(A) Immunostaining for S100A (red) on sections of cochleae from E18.5 wild-type or Six1^Cko/Cko littermate embryos. (B) Whole-mount in situ hybridization with Fgf8 ribo-probe showing Fgf8 expression in inner hair cells (IHC) in wild-type control littermates and decreased expression in remaining hair cells in Six1^Cko/Cko littermates at E16.5. Lower panels are higher magnification of boxed areas. (C) Whole-mount in situ hybridization of E15.5 cochleae showing Atoh1 mRNA expression in the hair cells in Six1^Cko/Cko cochlea. Arrow in B,C points to lack of Fgf8 or Atoh1 expression in the basal end of cochlea in Six1^Cko/Cko. Lower panels are higher magnification of boxed areas. (D) Fgf8 and Atoh1 mRNA expression were examined by qRT-PCR in Eya1^CreER and Six1 CKO littermates at E15.5 and E17.5 (given tamoxifen at E11.5–12.5). Two-tailed Student’s t-test was used for statistical analysis. Scale bars: 20 μm in A,B, 100 μm in upper panels of C,D and 50 μm in lower panels of C,D.

Fig 6. Temporal deletion of Six1 between E12.75–13.5 blocks hair cell induction.

(A) Whole-mount Myo7a staining of E18.5 cochleae showing four rows of hair cells along the entire cochlea in Eya1^CreER mice but extra inner hair cells present from middle toward apex (arrows). In Six1 CKO cochlea, the organ of Corti appears narrower and hair cells show abnormal morphology with gradually decreased outer rows of hair cells from four in the base and one row in the apex. (B) Co-immunostaining for Myo7a and Sox2 on sections from P0 cochleae treated with tamoxifen between E13.5–14.5 showing one inner and three outer hair cells in the base, one inner and two outer hair cells in the middle and only one hair cells in the apex in Six1 CKO. All Myo7a⁺ hair cells are also Sox2⁺ in the CKO mutant. Scale bars: 30 μm.

Fig 7. Differentiation and misalignment of supporting cell subtypes in the absence of Six1.

Antibody labeling for Myo7a for hair cells and p27^Kip1 (A-C), Prox1 (D-F) and GLAST (G,H) for supporting cells in the organ of Corti in wild-type and Six1 CKO (Eya1^CreER;Six1^fl/fl) littermates given tamoxifen from E11.5–12.5. A’,B’,C’ are A.B,C counter-stained with hematoxylin respectively. Arrow in B,C points to expansion of p27^Kip1 expression in the GER cells. Asterisks in B,B’,C,C’ point to remaining hair cells and dots point to supporting cells. Asterisks in E,F,H point to supporting cells. Abb.: D, Deiters’ cells; H, Hensen’s cells; IB, inner border cell; IHC and OHC, inner and outer hair cells; IPh, inner phalangeal cells; IP and OP, inner and outer pillar cells. Scale bars: 30 μm.

Fig 8. Alteration in cellular morphology and distribution of N- and E-cadherin in Six1 CKO organ of Corti (tamoxifen at E11.5–12.5, Eya1^CreER).

(A,B) Inner pillar cells labeled by p75^NTR in wild-type cochlea and Six1 CKO littermate cochleae at E17.5. Arrow in A points to dynamic remodeling of cellular contacts toward the less differentiated apical region. Arrows and arrowhead in B point to three or more inner pillar cells in contact and cellular contact shrinkage respectively in Six1 CKO organ of Corti. (C) F-actin (phalloidin) and acetylated tubulin staining showing alteration in cell orientation and alignment in Six1 CKO at P0. (D-F) Lumenal surface views of the cochleae from wild-type, Eya1^CreER, and Six1 CKO (tamoxifen at E11.5–12.5, Eya1^CreER) (D,E) embryos at E15.5 (D), E16.0 (E) or E18.5 (F). Samples were stained for phalloidin and N-cadherin (D) or E-cadherin (E,F). Arrowheads mark the separation between inner hair cells and outer hair cells. The developing organ of Corti is identified by cellular morphology and cortical actin enrichment in the nascent hair cells. Scale bars: 30 μm A,B, 20 μm C, and 30 μm E-F.

Fig 9. Model illustrating the role of Six1 in regulating auditory sensory cell development.

This study demonstrates that Six1 regulates proliferation of sensory progenitors in the cochlea epithelium. While a direct interaction between Eya1/Six1/Sox2 proteins coordinately regulates Atoh1 expression in cochlear explant, this study provides in vivo evidence supporting a role for Six1 in hair cell fate specification and Atoh1 expression. In differentiating hair cells, our data show that Six1 activity is necessary for downregulation of Sox2 and maintenance of Fgf8 expression.