Blocking FA signaling through DN Vinculin overexpression severely affects AV EC migration and valve tissue establishment. (A) Schematics of the UAS-driven DN Vinculin transgene, expressing the head domain of Vinculin fused to GFP, Tg(UAS:DN-Vcl-EGFP). (A–C′) 60 hpf: AV EC migration stage. (B) In WT embryos, AV ECs extended protrusions and formed a multilayered tissue. (C) In embryos overexpressing DN Vinculin, AV ECs remained single layered and extended microvilli-like protrusions. (D) Categorization of migration stages of superior AV ECs in WT (n = 12) and DN Vinculin–overexpressing (n = 21) embryos. (E) Number of protruding ECs in superior and inferior AVC was significantly reduced when DN Vinculin was overexpressed (averages of 2.25 and 1.32 cells per embryo in WT and DN Vinculin, respectively; P = 3.1^e−3, unpaired Student’s t test). WT, n = 47 embryos; DN Vinculin, n = 34 embryos. Error bars represent standard deviation; asterisks indicate statistical significance. (F–G′) 72 hpf: establishment of prevalvular structure. (F) WT AV ECs completed migration and established a folded multilayered prevalvular structure. (G) DN Vinculin–overexpressing AV ECs appeared disorganized and did not clearly establish a multilayered tissue. (H–I′) 96 hpf: establishment of functional AV valve leaflets. (H) In WT larvae, both superior and inferior leaflets formed and extensively elongated into the vascular lumen. (I) In DN Vinculin–overexpressing larvae, AV endocardial tissue remained single layered, without established leaflet structures. Scale bars: (B and C, and F–I) 20 µm; (B′ and C′ and E′–H′) 10 µm.