Figure 1.
αII-spectrin determines epidermal cell shape. (a) Representative examples of 3D-rendered keratinocytes in the different epidermal layers of newborn epidermal confetti mice using cytoplasmic YFP expression. (b) Schematic illustration and denotation of the epidermal layers and the distance of the border of each layer relative to the stratum corneum surface. (c) Quantification of cell shape parameters from rendered cells. 71 rendered cells from three mice have been analyzed. R-squared values for the linear regression analysis of the accumulated values are shown. (d) Newborn epidermal whole-mount immunofluorescence analysis for phalloidin (F-actin) revealing top-view cell shapes in the spinous (SS cells) and granular layer (SG cells) within tissue (in vivo). Right column: Phalloidin staining of single cells isolated from the spinous or granular layer showing deformation upon isolation only in spinous cells. (e) Quantification of cell top-view area/shape of granular and spinous cells in the tissue and after isolation using stainings as shown in d. *P < 0.05, ****P < 0.0001; >100 cells from three mice (isolated) or six mice (in tissue) with Kolmogorov–Smirnov per layer. (f) Newborn epidermal whole-mount immunofluorescence analysis for phalloidin (F-actin) and αII-spectrin. Max. projections of the spinous (SS) and granular layer 3 (SG3) are shown. d and f representative images of N ≥ 3 biological replicates. (g) Immunofluorescence analysis for phalloidin (F-actin) and αII-spectrin on newborn skin cryosections. Representative image of n > 3 biological replicates. (h and i) Quantification of cortical αII-Spectrin and F-actin intensity across the epidermal layers. Mean values (lines) of pooled cells (dots) from three mice. ****P < 0.0001, ***P = 0.0001, **P < 0.007; n ≥ 157 cells with Kruskal–Wallis, Dunn’s multiple comparison test. (j) Skin sections from shCtr and shSptan1 0595-transduced E17.5 embryos immunolabeled for E-cadherin. Upper Insets show transduced cells (H2B−GFP+). Dashed lines indicate the dermal-epidermal border. (k) Quantification of cell sagittal area and cell shape (perimeter/√area) from data shown in j. Mean (lines) ± SD from ∼200 individual cells (dots) from n = 3 embryos per condition. ****P < 0.0001 by Kolmogorov–Smirnov test for sagittal cell area. ***P = 0.0002 basal shSptan1 0595; *P = 0.0181 for basal shSptan1 9753, ****P < 0.0001 by Kolmogorov–Smirnov test for cell shape index.

αII-spectrin determines epidermal cell shape. (a) Representative examples of 3D-rendered keratinocytes in the different epidermal layers of newborn epidermal confetti mice using cytoplasmic YFP expression. (b) Schematic illustration and denotation of the epidermal layers and the distance of the border of each layer relative to the stratum corneum surface. (c) Quantification of cell shape parameters from rendered cells. 71 rendered cells from three mice have been analyzed. R-squared values for the linear regression analysis of the accumulated values are shown. (d) Newborn epidermal whole-mount immunofluorescence analysis for phalloidin (F-actin) revealing top-view cell shapes in the spinous (SS cells) and granular layer (SG cells) within tissue (in vivo). Right column: Phalloidin staining of single cells isolated from the spinous or granular layer showing deformation upon isolation only in spinous cells. (e) Quantification of cell top-view area/shape of granular and spinous cells in the tissue and after isolation using stainings as shown in d. *P < 0.05, ****P < 0.0001; >100 cells from three mice (isolated) or six mice (in tissue) with Kolmogorov–Smirnov per layer. (f) Newborn epidermal whole-mount immunofluorescence analysis for phalloidin (F-actin) and αII-spectrin. Max. projections of the spinous (SS) and granular layer 3 (SG3) are shown. d and f representative images of N ≥ 3 biological replicates. (g) Immunofluorescence analysis for phalloidin (F-actin) and αII-spectrin on newborn skin cryosections. Representative image of n > 3 biological replicates. (h and i) Quantification of cortical αII-Spectrin and F-actin intensity across the epidermal layers. Mean values (lines) of pooled cells (dots) from three mice. ****P < 0.0001, ***P = 0.0001, **P < 0.007; n ≥ 157 cells with Kruskal–Wallis, Dunn’s multiple comparison test. (j) Skin sections from shCtr and shSptan1 0595-transduced E17.5 embryos immunolabeled for E-cadherin. Upper Insets show transduced cells (H2B−GFP+). Dashed lines indicate the dermal-epidermal border. (k) Quantification of cell sagittal area and cell shape (perimeter/√area) from data shown in j. Mean (lines) ± SD from ∼200 individual cells (dots) from n = 3 embryos per condition. ****P < 0.0001 by Kolmogorov–Smirnov test for sagittal cell area. ***P = 0.0002 basal shSptan1 0595; *P = 0.0181 for basal shSptan1 9753, ****P < 0.0001 by Kolmogorov–Smirnov test for cell shape index.

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