Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea

Abstract

Sox2 is a high-mobility transcription factor that is one of the earliest markers of developing inner ear prosensory domains. In humans, mutations in SOX2 cause sensorineural hearing loss and a loss of function study in mice showed that Sox2 is required for prosensory formation in the cochlea. However, the specific roles of Sox2 have not been determined. Here we illustrate a dynamic role of Sox2 as an early permissive factor in prosensory domain formation followed by a mutually antagonistic relationship with Atoh1, a bHLH protein necessary for hair cell development. We demonstrate that decreased levels of Sox2 result in precocious hair cell differentiation and an over production of inner hair cells and that these effects are likely mediated through an antagonistic interaction between Sox2 and the bHLH molecule Atoh1. Using gain- and loss-of-function experiments we provide evidence for the molecular pathway responsible for the formation of the cochlear prosensory domain. Sox2 expression is promoted by Notch signaling and Prox1, a homeobox transcription factor, is a downstream target of Sox2. These results demonstrate crucial and diverse roles for Sox2 in the development, specification, and maintenance of sensory cells within the cochlea.

Fig. 1.

Sox2 is expressed in the prosensory domain and inhibits hair cell formation. (A-D) Immunohistochemistry using anti-Sox2 (red in A,B, and D and brown in C) and actin (green in A, B, and D) on mid-modiolar cross-sections of WT cochleae shows Sox2 expression at E12.5, E16, and P0. Sox2 is broadly expressed in the prosensory cells at E12.5 (A); however, by E16 Sox2 levels are downregulated in cells that will subsequently acquire a hair cell fate (B, arrows). (C) Co-localization of Sox2 (brown) and Atoh1 (β-gal staining in blue) in the organ of Corti of Atoh1^LacZ/+ mice at E16.5 demonstrates downregulation of Sox2 in Atoh1-positive hair cells. (D) By P0 the expression of Sox2 is restricted to supporting cells (Inset: organ of Corti) with weak expression in a subset of cells within KO. Sox2 overexpression inhibits hair cell formation. (E) Low-magnification confocal image of a cochlear explant culture transfected with Sox2.nucEGFP. Explants are comprised of three regions, KO, the SE, and the LER, see Experimental Procedures for details. Transfected cells are typically present in all three regions (arrows in KO and LER, and arrowhead in SE). (F) High-magnification confocal image of a cochlear prosensory cell transfected with Sox2.nucEGFP (arrowhead). The transfected cell is negative for the hair cell marker Myo6 (red); (Inset) Z-stack confocal cross-section of the cell illustrated in F. Although the cell is located in the hair cell layer, it is negative for Myo6. KO, Kolliker's organ; LER, lesser epithelial ridge; SE, sensory epithelium; OC, organ of Corti; IHC, inner hair cell; O1-O3, outer hair cells; IPh, inner phalangeal cell; IP, inner pillar cell; OP, outer pillar cell; D1-D3, Deiters' cells; HeC, Hensens' cells. (Scale bars, A, B, D, 20 μm; C, 10 μm; E, 50 μm; F, 10 μm.)

Fig. 2.

An antagonistic relationship between Sox2 and Atoh1. (A-D) P19 cells, labeled with DAPI (blue in A), endogenously express Sox2 (red in B). However, the cell transfected with Atoh1.EGFP (asterisk in A-D) is negative for Sox2 expression (B-D). Sox2 antagonizes Atoh1 in vitro. (E-G) Cells within KO or the LER transfected with Atoh1.EGFP alone are positive for Myo6 (red) indicating development as hair cells (E-F) while Sox2-transfected cells never develop into hair cells (G). (H-J) Cells within the LER that are co-transfected with Sox2.nucEGFP and Atoh1.EGFP. One cell is positive for Myo6 (red), while the other is not, illustrating the antagonistic interaction between Sox2 and Atoh1. Early hair cell differentiation and formation of extra hair cells in Sox2 hypomorphic cochlea. (K-O) Cross-sections of the basal turn of the cochlea from Sox2^+/+ and Sox2^EGFP/LP mice at E15.5. (K) Anti-Myo6 labeling shows the presence of a single inner hair cell in the Sox2^+/+ cochlea (arrowhead). (L) In contrast, the Sox2^EGFP/LP cochlea contains a single inner hair cell (arrowhead) and two outer hair cells (arrows). (M and N) Analysis of hair cells (labeled with anti-Myo6) in cochleae from E18 littermates. Progressive decrease in the level of Sox2 activity result in an increase in the number of extra inner hair cells (stars in N). (O) Quantification of extra inner hair cells in Sox2^+/+ (n = 5), Sox2^EGFP/+ (n = 5), and Sox2^EGFP/LP (n = 3 from 2 mice) littermate cochleae indicates a significant increase in extra inner hair cells in Sox2^EGFP/LP cochlea (P < 0.001). Error bars are S.E.M. IHC, inner hair cell; O1-O3, outer hair cells. (Scale bars, A-J, 10 μm; K-N, 20 μm.)

Fig. 3.

Sox2 regulates Prox1 expression. (A and A′) Labeling of Prox1 (red) and actin (green) on a mid-modiolar cross-section from a WT cochlea at P0 shows expression of Prox1 in a subset of support cell nuclei; IP, OP, and D1-D3. (B) Double-immunolabeling using anti-Prox1 (red) and anti-Sox2 (green) indicates overlap of Prox1 and Sox2 expression in the lateral region including PC and DC nuclei. (C and D) Prox1 is absent in cochleae from Sox2^Lcc/Lcc mice. (C) Anti-Prox1 (red) staining labels nuclei of prosensory cells (circle) at E15.5. (D) In contrast with WT, Prox1 staining is absent in Sox2^Lcc/Lcc mutant cochlea at E15.5. DAPI (blue) staining in C and D shows cell nuclei. Sox2 induces ectopic expression of Prox1. (E-J) Sox2.nucEGFP transfected in cells within KO (E-G), or the LER (H-J) are positive for Prox1 (red). (K and L) Forced expression of Prox1 inhibits hair cell formation. Cochlear prosensory cells were transfected with Prox1.nucEGFP. (K) Low-magnification view of confocal image of explant culture illustrating Prox1.nucEGFP transfection (green) in KO and SE (arrowheads). (L) High-magnification view of the SE demonstrating that Prox1.nucEGFP expressing cells (arrowheads) are negative for the hair cell marker Myo6 (red). (M) Co-transfection with Prox1.nucEGFP and Atoh1.EGFP results in inhibition of Atoh1. Despite expression of Atoh1, the cell is negative for Myo6 (red). IHC, inner hair cell; IP, inner pillar cell; OP, outer pillar cell; D1-D3, Deiters' cell; O1-O3, outer hair cells; IPh, inner phalangeal cell; HeC, Hensens' cell; KO, Kolliker's organ; LER, lesser epithelial ridge; SE, sensory epithelium. (Scale bars, A, 20 μm; A′-B, 10 μm; C-K, 20 μm, L, 10 μm, M, 20 μm.)

Fig. 4.

Notch signaling is required for Sox2 expression. (A-F) Inhibition of Notch signaling using the γ-secretase inhibitor, DAPT on cochlear explant cultures beginning at E13 results in the reduction of Sox2 (A and B) and Prox1 (C and D) expression. (E and F) In contrast, Myo6 labeling indicates an increase in the number of inner and outer hair cells in DAPT-treated explant cultures. (G) Forced expression of NICD.EGFP in cells within the LER induces Sox2 (yellowish red nuclei) expression. (H) Notch1 expression (brown) is present in E15.5 Sox2^Lcc/Lcc mutant cochleae. (Scale bars, 20 μm.)

Fig. 5.

The prosensory domain is present in Atoh1 mutants. Sox2, Jagged1, and Prox1 were used as markers for the prosensory domain. Immunolabeling was done on apical cochlear sections at E16.5. (A and B) Sox2 immunolabeling (brown) in Atoh1^LacZ/+ and Atoh1^LacZ/LacZ (mutant) cochleae shows no change in Sox2 expression. The X-gal staining (blue) illustrates reporter activity for Atoh1. (C-F) Expression of Jagged1 (red in C and D) and Prox1 (red in E and F) are also present in the Atoh1 mutants. OC, organ of Corti; SG, spiral ganglion. (Scale bars, 20 μm.)