Real-time imaging shows WT AV ECs migrating and establishing a multilayered tissue, whereas DN Vinculin–overexpressing ECs failed to extend protrusions. (A–L) Maximum projections of the entire heart. (A–F) At the AVC, WT ECs originated as a single layer of cells (A), extended long protrusions, moved into the overlying ECM (B–D), and established a multilayered tissue (E and F; no more protrusions were observed at these stages). n = 4 animals. (G–L) DN Vinculin–overexpressing ECs did not extend prominent protrusions and remained single layered until 60 hpf (G–I). By 72 hpf, they appeared disorganized and did not adopt the folded tissue arrangement, and the AVC appeared collapsed (J–L). n = 2 animals. Scale bars: 20 µm.