Figure 4.
Role of Vangl1 in the formation of planar polarized apical MTs. (A) Representative immunofluorescence images of α-tubulin (green; a marker of apical MTs) and Daple (magenta) in MCCs of Vangl1-KO MTECs grown on the ALI for 12–14 d. (B) Color map of the signal for α-tubulin corresponding to the images shown in A. (C) Intensity profiles of immunofluorescence for α-tubulin in cell-1 (Fzd6 asymmetrical) and cell-2 (Fzd6 symmetrical) in tracheal MCCs of the Vangl1-KO. Intensity profiles were measured along linear ROIs (white) in B, which were drawn from the opposite side of the Fzd side of the AJC (distance, 0.00) to the Fzd6 side of the AJC (distance, 1.00) in cell-1 and arbitrarily between one side to the opposite side of the AJC in cell-2. (D) Ratios of the numbers of MCCs with MT accumulation (gray) to MT nonaccumulation (white) in the Fzd sides of the AJCs in WT, Daple-KO, and Vangl1-KO total MCCs (n = 39–55 cells). MCCs were visually divided into MT accumulation and MT nonaccumulation. Fisher’s exact test with the Benjamini-Hochberg multiple-comparison test; **, P < 0.01; *, P < 0.05. (E) Representative images of triple-immunofluorescence for Odf2 (green) and centriolin (Centrl; magenta) as markers of BBs and BFs, respectively, and Fzd6 (blue) in Vangl1-KO adult tracheas. Yellow arrow indicates the asymmetric localization of Fzd6. Red arrows indicate the abnormal localization of Fzd6 in MCCs. Note the asymmetrical localization of Fzd6 in cell-1 and the symmetrical localization of Fzd6 in cell-2 of Vangl1-KO MCCs. (F) Angle histograms of BB directions for representative cell-1 and cell-2 of Vangl1-KO tracheal MCCs corresponding to the images shown in E. Cell-level indices of orientation are in cell-1 (n = 4 cells) and cell-2 (n = 4 cells) of Vangl1-KO MCCs. (G) Boxplots of the indices of orientation in WT adult tracheal MCCs (n = 8 cells), in cell-1 (n = 4 cells), and in cell-2 (n = 4 cells) of Vangl1-KO adult tracheal MCCs. Kruskal-Wallis tests with the Steel-Dwass multiple-comparison tests; **, P < 0.01; n.s., P ≥ 0.05. Scale bars represent 5 µm.

Role of Vangl1 in the formation of planar polarized apical MTs. (A) Representative immunofluorescence images of α-tubulin (green; a marker of apical MTs) and Daple (magenta) in MCCs of Vangl1-KO MTECs grown on the ALI for 12–14 d. (B) Color map of the signal for α-tubulin corresponding to the images shown in A. (C) Intensity profiles of immunofluorescence for α-tubulin in cell-1 (Fzd6 asymmetrical) and cell-2 (Fzd6 symmetrical) in tracheal MCCs of the Vangl1-KO. Intensity profiles were measured along linear ROIs (white) in B, which were drawn from the opposite side of the Fzd side of the AJC (distance, 0.00) to the Fzd6 side of the AJC (distance, 1.00) in cell-1 and arbitrarily between one side to the opposite side of the AJC in cell-2. (D) Ratios of the numbers of MCCs with MT accumulation (gray) to MT nonaccumulation (white) in the Fzd sides of the AJCs in WT, Daple-KO, and Vangl1-KO total MCCs (n = 39–55 cells). MCCs were visually divided into MT accumulation and MT nonaccumulation. Fisher’s exact test with the Benjamini-Hochberg multiple-comparison test; **, P < 0.01; *, P < 0.05. (E) Representative images of triple-immunofluorescence for Odf2 (green) and centriolin (Centrl; magenta) as markers of BBs and BFs, respectively, and Fzd6 (blue) in Vangl1-KO adult tracheas. Yellow arrow indicates the asymmetric localization of Fzd6. Red arrows indicate the abnormal localization of Fzd6 in MCCs. Note the asymmetrical localization of Fzd6 in cell-1 and the symmetrical localization of Fzd6 in cell-2 of Vangl1-KO MCCs. (F) Angle histograms of BB directions for representative cell-1 and cell-2 of Vangl1-KO tracheal MCCs corresponding to the images shown in E. Cell-level indices of orientation are in cell-1 (n = 4 cells) and cell-2 (n = 4 cells) of Vangl1-KO MCCs. (G) Boxplots of the indices of orientation in WT adult tracheal MCCs (n = 8 cells), in cell-1 (n = 4 cells), and in cell-2 (n = 4 cells) of Vangl1-KO adult tracheal MCCs. Kruskal-Wallis tests with the Steel-Dwass multiple-comparison tests; **, P < 0.01; n.s., P ≥ 0.05. Scale bars represent 5 µm.

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