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. 2016 Nov 29;5:e19593. doi: 10.7554/eLife.19593

Figure 1. Multi-dimensional visualization of epidermal TJs and TJ-bearing cells in mouse-ear skin.

(A) 3D structure of the epidermis. (B) En face image of ZO-1-positive honeycomb in mouse-ear epidermis showing double-edged polygons (*) and single-edged polygons (#). (C) Regularity in the size of the ZO-1-positive polygons represented in (B) and Figure 1—figure supplement 1, shown by the mean ± SEM [error bars] (one-way ANOVA multiple comparison test). (D) 3D image of a ZO-1-positive double-edged polygon in en face view (top) and 90°-rotated side view of the yellow-dotted rectangle (bottom). Upper exterior polygon, yellow arrowheads; lower interior polygon, white arrows. See Video 1. (E) Regularity of relative Z-axis position. Boxplots show the median, minimum, maximum, and interquartile range (one-way ANOVA multiple comparison test) for the ZO-1-positive polygons represented in Figure 1—figure supplement 2. (F) In vivo live images of Venus in the ear of ZO-1-Venus mice (left column) and their schematics (right column). Yellow arrowheads and green edges, edges of a Venus-positive polygon; white arrows and purple edges, edges of a newly appearing Venus-positive polygon; black arrows, Venus-positive edges connecting each vertex of the two polygons. See Video 4. Scale bars, 10 µm. TJ, tight junction; SC, stratum corneum.

DOI: http://dx.doi.org/10.7554/eLife.19593.003

Figure 1—source data 1. Percentage of double-edged polygons in ZO-1-positive honeycomb.
The number of single- and double-edged polygons in 20 square en face images of 15376 µm2 from five independent assays. A represented image is shown in Figure 1B.
DOI: http://dx.doi.org/10.7554/eLife.19593.004
elife-19593-fig1-data1.xlsx (36.1KB, xlsx)
DOI: 10.7554/eLife.19593.004
Figure 1—source data 2. Size of the ZO-1-positive polygons.
The size of single- and double-edged polygons in ZO-1-positive honeycomb statistically analyzed in Figure 1C. How to define areas of polygons are shown in Figure 1—figure supplement 1.
DOI: http://dx.doi.org/10.7554/eLife.19593.005
elife-19593-fig1-data2.xlsx (40KB, xlsx)
DOI: 10.7554/eLife.19593.005
Figure 1—source data 3. Z-axis position of the ZO-1-positive polygons.
The Z-axis position of single- and double-edged polygons in ZO-1-positive honeycomb statistically analyzed in Figure 1E.
DOI: http://dx.doi.org/10.7554/eLife.19593.006
elife-19593-fig1-data3.xlsx (58.8KB, xlsx)
DOI: 10.7554/eLife.19593.006

Figure 1.

Figure 1—figure supplement 1. Areas of exterior, interior and single-edged polygons.

Figure 1—figure supplement 1.

(A–C) Drawings of the ZO-1-positive edges shown in Figure 1B. The areas of polygons evaluated in Figure 1C are depicted for the exterior [pink in (A)] and interior [yellow in (B)] polygons of double-edged polygons (*), and for the single-edged polygon [green in (C)] adjacent to the double-edged polygons. The area of the single-edged polygon includes the overlapping area with the adjacent exterior polygons (red arrows).
DOI: http://dx.doi.org/10.7554/eLife.19593.007

Figure 1—figure supplement 2. Relative Z-axis position of TJ polygons in TJ honeycomb evaluated in vivo.

Figure 1—figure supplement 2.

A representative image of a ZO-1-positive double-edged polygon surrounded by six single-edged polygons evaluated in Figure 1E. The Z-axis position of each polygon was defined by an average of the Z-axis positions (numbers) of its vertices, analyzed by Imaris software (purple arrowheads, external polygon; yellow arrowheads, internal polygon; green arrowheads, adjacent single-edged polygons). Scale bar, 10 µm. TJ, tight junction.
DOI: http://dx.doi.org/10.7554/eLife.19593.008

Figure 1—figure supplement 3. Epidermal TJ in ZO-1-Venus transgenic mice.

Figure 1—figure supplement 3.

(A) Colocalization of occludin, a transmembrane protein located at the TJs, with Venus in whole-mounted epidermal sheet from the ear skin of a ZO-1-Venus transgenic mouse. No morphological changes were observed in the TJ honeycomb in ZO-1-Venus transgenic mice. (B) Skin section staining of ZO-1-Venus transgenic mice after intradermal injection of Sulfo-NHS-LC-biotin as a tracer (Furuse et al., 2002; Yokouchi et al., 2015). Venus-positive junctions were observed to limit the inside-out permeation of the tracer (yellow arrows), indicating that Venus successfully labeled the TJs without any apparent change in their occlusive function. Scale bars, 10 µm. TJ, tight junction.
DOI: http://dx.doi.org/10.7554/eLife.19593.009