Reduced <i>Fgf8</i> expression in <i>Six1</i>^{<i>Cko/Cko</i>} mutant (<i>Eya1</i>^<i>CreER</i><i>;Six1</i>^<i>fl/fl</i> given tamoxifen from E11.5–12.5).

<p>(A) Immunostaining for S100A (red) on sections of cochleae from E18.5 wild-type or <i>Six1</i>^{<i>Cko/Cko</i>} littermate embryos. (B) Whole-mount in situ hybridization with <i>Fgf8</i> ribo-probe showing <i>Fgf8</i> expression in inner hair cells (IHC) in wild-type control littermates and decreased expression in remaining hair cells in <i>Six1</i>^{<i>Cko/Cko</i>} littermates at E16.5. Lower panels are higher magnification of boxed areas. (C) Whole-mount in situ hybridization of E15.5 cochleae showing <i>Atoh1</i> mRNA expression in the hair cells in <i>Six1</i>^{<i>Cko/Cko</i>} cochlea. Arrow in B,C points to lack of <i>Fgf8</i> or <i>Atoh1</i> expression in the basal end of cochlea in <i>Six1</i>^{<i>Cko/Cko</i>}. Lower panels are higher magnification of boxed areas. (D) <i>Fgf8</i> and <i>Atoh1</i> mRNA expression were examined by qRT-PCR in <i>Eya1</i>^<i>CreER</i> and <i>Six1</i> 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.</p

Lack of outer hair cell formation in <i>Six1</i> CKO (<i>Eya1</i>^<i>CreER</i>) cochlea.

<p>(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”.</p

Temporal deletion of <i>Six1</i> between E12.75–13.5 blocks hair cell induction.

<p>(A) Whole-mount Myo7a staining of E18.5 cochleae showing four rows of hair cells along the entire cochlea in <i>Eya1</i>^<i>CreER</i> mice but extra inner hair cells present from middle toward apex (arrows). In <i>Six1</i> 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 <i>Six1</i> CKO. All Myo7a⁺ hair cells are also Sox2⁺ in the CKO mutant. Scale bars: 30 μm.</p

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

<p>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 <i>Atoh1</i> expression. In differentiating hair cells, our data show that Six1 activity is necessary for downregulation of Sox2 and maintenance of <i>Fgf8</i> expression.</p

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

<p>(A,B) Inner pillar cells labeled by p75^NTR in wild-type cochlea and <i>Six1</i> 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 <i>Six1</i> CKO organ of Corti. (C) F-actin (phalloidin) and acetylated tubulin staining showing alteration in cell orientation and alignment in <i>Six1</i> CKO at P0. (D-F) Lumenal surface views of the cochleae from wild-type, <i>Eya1</i>^<i>CreER</i>, and <i>Six1</i> CKO (tamoxifen at E11.5–12.5, <i>Eya1</i>^<i>CreER</i>) (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.</p

Differentiation and misalignment of supporting cell subtypes in the absence of <i>Six1</i>.

<p>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 <i>Six1</i> CKO (<i>Eya1</i>^<i>CreER</i><i>;Six1</i>^<i>fl/fl</i>) 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.</p

Deletion of <i>Six1</i> in the developing cochlea using <i>Eya1</i>^<i>CreER</i> or <i>Sox2</i>^<i>CreER</i> leads to shortened and thickened prosensory primordium.

<p>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, <i>Eya1</i>^<i>CreER</i> or <i>Sox2</i>^<i>CreER</i><i>;Six1</i>^<i>fl/fl</i> (<i>Cko/Cko</i>) 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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006967#sec008" target="_blank">Methods</a> for calibration). <i>P-</i>value was measured for +/+ and <i>Cko/Cko</i> using Two-tailed Student’s t-test. Scale bars: 200 μm in A-C and 40 μm in D,E.</p

Altered cell proliferation in <i>Six1</i> CKO (<i>Eya1</i>^<i>CreER</i>) cochlea epithelium.

<p>(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 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006967#sec008" target="_blank">Methods</a> for quantification). <i>P</i>-value was measured for +/+ and <i>Cko/Cko</i> using Two-tailed Student’s t-test: <i>p</i><0.001 for E17.5 (EdU at E14.5) and <i>p</i> = 0.003 for E14.5 (EdU at E11.5). Scale bars: 30 μm in A,B.</p

The expression of fast-type genes and <i>linc-MYH</i> is impaired in <i>cSix1 KO</i> mice during postnatal development.

<p>mRNA expression level of <i>Six1</i>, <i>linc-MYH</i>, <i>Myh3</i>, slow-type genes (red) and fast-type genes (blue) in back muscles of <i>cSix1 KO</i> mice at E18.5, P2W, P4W and P8W, as determined by qPCR experiments, (n = 3 to 6 for each point). *P<0.05, **P<0.01, ***P<0.001.</p

<i>linc-MYH</i> is expressed in adult fast-type skeletal muscles and accumulates in their nuclei.

<p>(A) Tissue distribution of <i>Six1</i>, <i>Myh4</i> and <i>linc-MYH</i> RNAs. BAT, brown adipose tissue; WAT, white adipose tissue. (n = 4). (B) Luciferase assays of adult TA electroporated with <i>linc-MYH</i> promoter luciferase vectors (indicated) and a TK-Renilla luciferase vector (allowing normalization). *P<0.05. (C) FISH of isolated EDL myofiber with a <i>linc-MYH</i> antisense RNA fluorescent-labeled probe (green) and Dapi staining (blue).</p