Volume 22, Issue 2, Pages 377-390 (February 2012) Eya1-Six1 Interaction Is Sufficient to Induce Hair Cell Fate in the Cochlea by Activating Atoh1 Expression in Cooperation with Sox2  Mohi Ahmed, Elaine Y.M. Wong, Jianbo Sun, Jinshu Xu, Feng Wang, Pin-Xian Xu  Developmental Cell  Volume 22, Issue 2, Pages 377-390 (February 2012) DOI: 10.1016/j.devcel.2011.12.006 Copyright © 2012 Elsevier Inc. Terms and Conditions

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 Six1lacZ/+ cochlea from E13.25 to E16.5. Arrows point to primordial organ of Corti. Arrowheads point 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 Six1lacZ/+ littermate controls as used in (B). The apical limit of Atoh1 expression at E14.5 and in medial region at E15.5 (arrows) or in lateral region at E15.5 (arrowhead) is indicated. (D) Sections of X-gal stained Eya1lacZ/+ cochlea at E13.5–E16.5. Developmental Cell 2012 22, 377-390DOI: (10.1016/j.devcel.2011.12.006) Copyright © 2012 Elsevier Inc. Terms and Conditions

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. Developmental Cell 2012 22, 377-390DOI: (10.1016/j.devcel.2011.12.006) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Coexpression of Eya1/Six1 Induces Atoh1-Dependent and Atoh1-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. Developmental Cell 2012 22, 377-390DOI: (10.1016/j.devcel.2011.12.006) Copyright © 2012 Elsevier Inc. Terms and Conditions

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. Developmental Cell 2012 22, 377-390DOI: (10.1016/j.devcel.2011.12.006) Copyright © 2012 Elsevier Inc. Terms and Conditions

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. Arrow indicates GFP+ hair cells in the organ of Corti. Scale bar, 100 μm. (D) Anterior crista (ac) and cochlea from 1.4 kb Atoh1Soxmt+Sixmt-GFP transgenic embryo at E17.5. Dark-field 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: higher magnification of boxed area. Scale bar, 100 μm. (E) Dissected utricle (u), anterior crista (ac), lateral crista (lc), and cochlea from 1.4 kb Atoh1Soxmt+Sixmt-GFP transgenic embryo at E17.5. Dark-field 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: higher magnification of boxed area. Scale bars, 100 μm. (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 pull-down assay showing in vitro-translated Sox2 can be pulled down by GST-Eya1D or -Six1 fusion protein and coIP assays demonstrating physical interaction between Sox2/Eya1/Six1. Developmental Cell 2012 22, 377-390DOI: (10.1016/j.devcel.2011.12.006) Copyright © 2012 Elsevier Inc. Terms and Conditions

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 anti-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 dashed circle in (E) showing Myo7a+ inner (ihc) and outer hair (ohc) cells. (G) Merged image of bright- and dark-field Eya1Cko/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 dashed circle in (G) showing irregular Myo7a+ cells in the mutant organ of Corti. Dashed semicircle in (E) and (G) indicates the apex of the cochlea. (I) 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. (J) 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), whereas Eya1/Six1 efficiently upregulate Atoh1 via enhancer B. Question mark indicates that the involvement of the factor is unclear. Developmental Cell 2012 22, 377-390DOI: (10.1016/j.devcel.2011.12.006) Copyright © 2012 Elsevier Inc. Terms and Conditions