Eya1-Six1 interaction is sufficient to induce hair cell fate in the cochlea by activating Atoh1 expression in cooperation with Sox2

Abstract

Inner-ear hair cell differentiation requires Atoh1 function, while Eya1, Six1, and Sox2 are coexpressed in sensory progenitors and mutations in these genes cause sensorineural hearing loss. However, how these genes are linked functionally and the transcriptional networks controlling hair cell induction remain unclear. Here, we show (1) that Eya1/Six1 are necessary for hair cell development, and their coexpression in mouse cochlear explants is sufficient to induce hair cell fate in the nonsensory epithelium expressing low-level Sox2 by activating not only Atoh1-dependent but also Atoh1-independent pathways and (2) that both pathways induce Pou4f3 to promote hair cell differentiation. Sox2 cooperates with Eya1/Six1 to synergistically activate Atoh1 transcription via direct binding to the conserved Sox- and Six-binding sites in Atoh1 enhancers, and these proteins physically interact. Our findings demonstrate that direct and cooperative interactions between the Sox2, Six1, and Eya1 proteins coordinate Atoh1 expression to specify hair cell fate.

Figure 1. Expression of Sox2, Six1 and Eya1 in the organ of Corti

(A) Cochlear sections at E13.25–E16.5 stained with anti-Sox2 antibody. Arrowheads point to border cells within the GER. Arrow points to the prosensory domain (ps). (B) Whole-mount and sections of X-gal stained Six1^lacZ/+ cochlea from E13.25–E16.5. Arrows point to primordial organ of Corti. Arrowhead points to Six1⁺ cells in the medial border of the prosensory domain where inner hair cells (ihc) develop near the basal cochlea duct. Open arrowheads point to second column of Six1⁺ cells where innermost outer hair cells (ohc) appear. Dashed lines point to the planes of sections shown for E13.5 and E14.5. (C) Atoh1 ISH of cochleae from Six1^lacZ/+ littermate controls as used in panel B. The apical limit of Atoh1 expression at E14.5 and in medial region at E15.5 (arrow) or in lateral region at E15.5 (arrowhead) are indicated. (D) Sections of X-gal stained Eya1^lacZ/+ cochlea at E13.5–E16.5.

Figure 2. Coexpression of Eya1/Six1 induces ectopic hair cells in GER cells

E13.5 cochlea electroporated with Eya1.GFP (A), Six1.GFP (B) or both (C) stained with anti-Myo7a antibody after 6 DIV. Transfected cells are identified as GFP⁺ (green) and hair cells are identified as Myo7a⁺ (red). oc, organ of Corti. Arrows point to Myo7a⁺GFP⁺ cells.

Figure 3. Coexpression of Eya1/Six1 induces Atoh1-dependent and – independent pathways, and Sox2 acts cooperatively with Eya1/Six1 to ectopically activate endogenous Atoh1 gene

Explants transfected with the indicated constructs stained with Atoh1 or Pou4f3 probe, anti-Myo7a antibody (red) or anti-GFP antibody (green). Arrows point to Atoh1⁺Myo7a⁺ or Atoh1⁺ and arrowheads point to Atoh1⁺Myo7a⁻ cells.

Figure 4. Synergistic actions of Sox2/Eya1/Six1 on transcription via Atoh1 enhancers

(A) Schematic of Atoh1 enhancers driving GFP reporter under control of β-globin minimal promoter. (B) Explants electroporated with the 1.4 kb Atoh1-GFP and different combinations of Sox2, Eya1 and Six1. A control plasmid expressing RFP was cotransfected to identify transfected cells. (C) The 561 bp enhancer A or 271 bp ΔA2 shows similar responsiveness to Sox2-mediated transcription. (D) The 405 bp enhancer B or 267 bp ΔB1 shows similar responsiveness to Six1-mediated transcription.

Figure 5. Interactions between Eya1/Six1/Sox2 and Atoh1 in inducing hair cell specification

(A) Multiple DNA sequence alignment of vertebrate Atoh1 enhancer A containing the conserved Sox-binding site (red) and enhancer B containing the conserved Six-binding site (red). Point mutations of the Sox- or Six-binding site (Soxmt or Sixmt) are listed. (B) Responsiveness of different Soxmt- or Sixmt-GFP reporters to Sox2- or Six1-mediated transcription in explants. (C) Bright- and dark-field images of cochlea duct (cd) from 1.4 kb Atoh1-GFP transgenic embryo at E16.5 expressing GFP in the hair cells. (D) Anterior crista (ac) and cochlea from 1.4 kb Atoh1Soxmt+Sixmt-GFP transgenic embryo at E17.5. Darkfield image showing GFP⁺ cells in anterior crista. Merged bright- and dark-field image showing very few GFP⁺ cells in the organ of Corti (oc). Right panel is a higher magnification of boxed area. (E) Dissected utricle (u), anterior (ac), lateral crista (lc) and cochlea from 1.4 kb Atoh1Soxmt+Sixmt-GFP transgenic embryo at E17.5. Darkfield image showing GFP expression in vestibular hair cells. Bright- and dark-field images showing no GFP⁺ cells in the organ of Corti. Arrow indicates autofluorescence. Right panel is a higher magnification of boxed area. Scale bars: 100μm. (F) ChIP analysis. Anti-Sox2 or –Six1 antibody is used for IP. Normal rabbit or goat IgG for ChIP and water for PCR were used as negative control. (G) GST pulldown assay showing in vitro translated Sox2 can be pulled-down by GST-Eya1D or –Six1 fusion protein and co-IP assays demonstrating physical interaction between Sox2/Eya1/Six1.

Figure 6. Coexpression of shSox2 with Eya1/Six1 and temporal and cell-specific deletion of Eya1 blocks hair cell induction

(A–D) Explants transfected with the indicated constructs stained with Atoh1 probe, anti-Myo7a (red) or –GFP (green) antibody after 2 or 6 DIV. (E) Merged image of bright- and dark-field wild-type cochlea at E18.5 stained with anti-Myo7a (red). (F) Higher magnification of circled area in E showing Myo7a⁺ inner (ihc) and outer hair (ohc) cells. (G) Merged image of bright- and dark-field Eya1^Cko/Cko cochlea at E18.5 stained with anti-Myo7a. Arrow points to irregular organization of hair cells, especially outer hair cells from medial-to-apical direction (from arrow-to-arrowhead). (H) Higher magnification of circled area in G showing irregular Myo7a⁺ cells in the mutant organ of Corti. (J) Molecular relationships among the key transcription factors for hair cell differentiation. This study demonstrates that a direct interaction between Eya1/Six1/Sox2 proteins coordinately regulates Atoh1 expression, and that Pou4f3 is a common downstream factor of the Atoh1-dependent and –independent pathways. Dashed lines indicate that Sox2 may repress Pou4f3 or downstream factors of Pou4f3. (K) Possible mechanisms for Atoh1 activation by Sox2/Eya1/Six1. Eya1/Six1 in collaboration with Sox2 activity in prosensory progenitors can induce Atoh1 activation via direct binding to the Sox- and Six-binding sites within enhancer A and B respectively. These three factors may directly interact (model a) or Eya1 may bridge Six1 and Sox2 (model b). These three factors may also form an active complex to regulate Atoh1 activation via enhancer A (model c), while Eya1/Six1 efficiently upregulate Atoh1 via enhancer B. Question mark indicates that the involvement of the factor is unclear.