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Supplementary Material

DEV0675326 Supplementary Material

Files in this Data Supplement:

  • Supplemental Table S1 -
  • Supplemental Figure S1 -

    Fig. S1. The levels of E-cadherin are significantly reduced in the developing p120cko/cko cochleae. (A-H) Cochleae from control (A-D) and p120CKO/CKO (E-H) embryos at E15 (A,B,E,F) and E18 (C,D,G,H) were stained with phalloidin (green) and an antibody against E-cadherin (red). The levels of E-cadherin are significantly reduced. The brackets, arrowheads and asterisks indicate the outer hair cell region, the pillar cell region that separates inner from outer hair cells, and the Hensen cell region immediately lateral to the outer hair cells, respectively.

  • Supplemental Figure S2 -

    Fig. S2. The levels of N-cadherin are significantly reduced in developing p120cko/cko cochleae. (A-H) Cochleae from control (A,C,E,G) and p120CKO/CKO (B,D,F,H) embryos at E14 (A-D) and E18 (E-H) were stained with Phalloidin (green) and an antibody against N-cadherin (red). The levels of N-cadherin are significantly reduced. The brackets, arrowheads and asterisks indicate the outer hair cell region, the pillar cell region that separates inner from outer hair cells, and the Hensen cell region immediately lateral to the outer hair cells, respectively.

  • Supplemental Figure S3 -

    Fig. S3. Localization of Vangl2 relative to adherens junctions and tight junctions in the organ of Corti. (A-L) Confocal optical scans (A-E,G-K) and the orthogonal images (F,L) of z-scans of whole mounts of the organ of Corti at E18.5. Z1 to Z5 represent images taken from the apical-most toward the basal domain of the cells in the organ of Corti. Phalloidin (blue), antibodies against aPKCζ (green) and E-cadherin (red) were used to visualize F-actin enrichment, the tight-junctions and the adherens junctions, respectively. One of the samples (A-F) contains Vangl2-GFP (green). Note that Vangl2 and E-cadherin appear in the similar z scans, whereas the aPKCζ signal is more apical than Vangl2 and E-cadherin.

  • Supplemental Figure S4 -

    Fig. S4. Pcdh15 is required for hair cell polarity while dispensable for cochlear extension. (A-D) Surface views of the organ of Corti from E18.5 control (A,C) and Pcdh153J/3J mutant (B,D) animals stained for F-actin (A,B; Phalloidin) or Fz3 (C,D) to visualize the hair bundle morphology and the location of the fonticulus where the kinocilium is projected from, and to examine the distribution of core PCP proteins, respectively. White and yellow arrowheads mark the inner pillar cell region and the hair cells with a centrally localized fonticulus. (E-H) Surface views of E14.5 cochlear epithelia from control (E,G) and Pcdh153J/3J mutant (F,H) animals stained for F-actin (green) and N-cadherin (red). Asterisks mark the nascent inner hair cells identified by enriched cortical F-actin accumulation. The dotted lines outline the clear border of N-cadherin-expression domain in both the control and the mutant animals. (I,J) Dissected cochleae from E18.5 control (I) and Pcdh153J/3J (J) animals. (K) Oriana3 plots for the orientation of each row of hair cells (IHCs and OHC1-OHC3). For Pcdh153J/3J samples, the angles measured were formed between the medial-to-lateral axis of the cochlea and the line drawn from the center of the apical surface to the fonticulus. Hair cells with centrally positioned fonticulus were considered to have lost the intrinsic polarity and the angle was designated as 180°. The orientation is divided into 15° sections and each bar represents the number of cells oriented within that 15° section.

  • Supplemental Figure S5 -

    Fig. S5. Pcdh15 interacts genetically with Polaris in the formation of polar hair bundles in inner hair cells. Confocal micrographs of the organ of Corti from control (A), Pcdh153J/+ (B), PolarisCKO/CKO (C) and PolarisCKO/CKO;Pcdh153J/+ (D) animals at E18.5. The images were taken at the apical surface of the cells with the stereocilia visualized by Phalloidin (green). Note that the compound mutant PolarisCKO/CKO;Pcdh153J/+ has an apparent defect in the morphology of the stereociliary bundles in the inner hair cells not seen in the PolarisCKO/CKO mutant. The crescent form of hair bundles oriented toward the lateral (L) side in the inner hair cells was not seen. Instead, clusters of stereocilia were present at the medial (M) side of the inner hair cell surface. Arrowheads indicate the inner hair cells with an abnormal hair bundle in the compound mutant. IHC, inner hair cells; OHC, outer hair cells. Scale bars: 5µm

  • Supplemental Figure S6 -

    Fig. S6. The epithelial integrity in the utricle is affected by p120-catenin inactivation. The utricles were dissected from control (A) and p120CKO/CKO (B) animals at E18, and stained with Phalloidin (green) and an antibody against Spectrin (red) to visualize the hair bundle and hair cell apical surface. The utricle from the mutant animal appears to have a large hole (indicated by an asterisk), which is an indented area with very few hair cells present.