Figure 7. Wg/Wnt4 inhibit intercellular Fz recruitment of Vang to the cell membrane.

(a-a”) S2+R cells co-transfected with Fz-YFP (green) and DE-cadherin were mixed with S2+R cells co-transfected with Vang-Flag (red) and DE-cad. Vang (red) is recruited to cell membrane (arrow) in areas that are in contact with neighboring Fz expressing cells. Anti-GFP and anti-Flag were used to detect Fz-YFP and Vang-Flag proteins, respectively.

(b) Vang-Flag, E-cad transfected cells alone (not mixed with Fz transfected cells), display Vang protein in vesicular structures, but not at the membrane.

(c-c'”) S2+R cells co-transfected with Fz-YFP (green), DE-cad (blue), and Unpaired (Upd) were mixed with S2+ cells co-transfected with Vang-Flag (red) and DE-cad (blue). Upd was used as a control secreted ligand; it did not affect intercellular Vang recruitment by Fz (arrow). Cells in contact expressed DE-cad localized to their contact sites (blue; monochrome in c'”).

(d-d'”) S2+R cells co-transfected with Fz-YFP (green), DE-cad (blue), and Wg were mixed with S2+ cells co-transfected with Vang-Flag (red) and DE-cad (blue). In the presence of Wg (0.8 μg), Fz largely failed to recruit Vang to the contacting cell membranes. Fz membrane localization was not affected.

(e) Quantification of Wg effect on intercellular Fz-Vang recruitment. Increasing amounts of Wg DNA (as indicated) were co-transfected with Fz/DE-cad cells, which were subsequently mixed with Vang/DE-cad transfected cells. Vang recruitment shown as % of total cell pairs analyzed (n=number of contacting pairs; 3 independent experiments were used to score these). Statistical analysis: Fischer's exact test (two-tailed) and p values are as indicated. Inhibitory effect of Wg on Fz-Vang recruitment is dosage dependent and highly reproducible (Suppl. Fig. S4 for additional examples).

(f) Secreted extracellular Wnt4 and Wg affect Fz-vang interaction. Conditioned media (CM) with Wnt4 or Wg affect Fz-Vang recruitment, Fz/DE-cad and Vang/DE-cad transfected S2+R cells were incubated in Wnt4, Wg or Upd-V5 (as negative control) CM. Recruitment quantified as percentage of Fz/DE-cad and Vang/DE-cad cell-cell contacts resulting in Vang localization at contact membranes. Vang recruitment was reduced in presence of Wnt4 or Wg CM, n=number of contacting cell pairs (statistical analysis as in [e]; confirmed by 3 independent experiments; Suppl. Fig. S4 for experimental samples).