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. 2020 Oct 29;8:577201. doi: 10.3389/fcell.2020.577201

FIGURE 6.

FIGURE 6

Reduced YAP signaling in embryonic Vangl2Lp airways and WNT5A rescues cell migration defects in Vangl2Lp/+. (A,B) Representative images show immunostaining for YAP in E18.5 wildtype, heterozygous Vangl2Lp/+, and homozygous Vangl2Lp/Lp mouse lungs. Sections were counterstained with hematoxylin. Negative controls with primary antibody omitted are shown. Insets (B) show the morphology of airway lumen. Arrows indicate nuclear YAP staining. (C) Percentage of nuclear YAP-positive cells in the airways. n = 3 mice per group; eight airways were quantified per mouse; a total of 1389 cells (wildtype), 1381 cells (Vangl2Lp/+), and 995 cells (Vangl2Lp/Lp) were analyzed (presented as mean ± SEM); one-way ANOVA with Tukey’s post hoc test, ***p < 0.001. Histograms show transcript levels for YAP target genes: Ctgf, Cyr61, and Ankrd1 in E18.5 wildtype vs Vangl2Lp/Lp (D) and adult wildtype vs Vangl2Lp/+ lungs (E). n = 3 independent experiments; three mice per genotype; each experiment was run in triplicate. Data are presented as mean ± SEM; Mann–Whitney U-test, *p < 0.05, **p < 0.01, ***p < 0.001. (F) Representative images showing untreated and WNT5A-treated Vangl2Lp/+ TECs at 0 and 20 h post-scratch. Wound edges are indicated by green lines. Percentage of wound healed 20 h after wound scratch in untreated Vangl2Lp/+ TECs and Vangl2Lp/+ TECs treated with WNT5A (G) and wound-healing rate in WNT5A-treated Vangl2Lp/+ TECs in relative to untreated Vangl2Lp/+ TEC controls (H). Data are presented as mean ± SEM. n = 4 independent experiments; TECs from three mice were pooled for each group per experiment; three technical replicates for each group per experiment; each dot represents mean percentage of wound healed per experiment. Mann–Whitney U-test, *p < 0.05. (I) Immunostaining for Golgi marker (GM130; red) in fixed untreated and WNT5A-treated Vangl2Lp/+ TECs 20 h post-scratch to determine cell polarity at leading edge. Nuclei were labeled with DAPI (blue) and dotted line indicates the wound edge. (J) Percentage of polarized cells 20 h post-scratch in untreated and WNT5A-treated Vangl2Lp/+ TECs (presented as mean ± SEM). n = 4 independent experiments; TECs from three mice were pooled for each group per experiment; three technical replicates for each group per experiment. Mann–Whitney U-test.