Figure 3

Dkk1 Reduces Cell Adhesion

(A) Single-plane confocal imaging, dorsal view, of tail bud stage embryos (n = 6 of each condition) ubiquitously expressing EGFP-tagged myosin light chain (MyosinEGFP, green) stained with phalloidin (red) labeling filamentous actin. Scale bar, 20 μm.

(B) Single-plane confocal images of axial cells in Tg(gsc:GFP) embryos (green axis) acquired at the tail bud (TB) stage, showing lack of organized cell-cell adhesions by E-cadherin antibody staining (red) in Dkk1-expressing embryos (n = 8 for each condition). Scale bar, 20 μm.

(C) Distribution of endogenous β-catenin (red) in Tg(gsc:GFP) embryos (green axis). Dorsal view maximum projections of embryos were acquired at 80% epiboly (EB). Dashed squares indicate the area of higher magnification shown on the right panels in single-plane confocal images. Single-plane higher magnification confocal images reveal loss of cell-cell adhesion integrity in axial cells of Dkk1-RNA-injected embryos. Control (Ctrl) n = 14 and Dkk1 RNA n = 16. Scale bar, 20 μm.

(D) Tg(gsc:GFP) embryos stained with anti-E-cadherin (red). Dashed squares show one example of six neighboring cells in control and Dkk1-expressing embryos, used for quantification of E-cadherin distribution across boundaries. Dkk1-expressing embryos exhibit a diffuse distribution of E-cadherin (red) compared to the control, indicating a loss of cell-cell adhesion (n = 5 for each condition). Scale bar, 20 μm.

(E) Quantification of E-cadherin distribution across boundaries between neighboring axial (green) cells. Normalized intensity distributions across cell-cell boundaries show that Dkk1 expression reduces E-cadherin intensity at cell boundaries and broadens lateral distribution of E-cadherin intensity. Normalized intensity distributions were calculated for boundaries between six neighboring cells in five embryos for both control and Dkk1-expressing embryos. ^∗∗∗p < 0.001. p value was calculated using the Kolmogorov-Smirnov test.