Morphogenetic processes driving development of the superior and inferior leaflets of the AV valve. (A–F) AV ECs marked by Tg(nfatc1:GAL4); Tg(UAS:EGFP-CAAX) expression. (A′–F′) Superior AV ECs. (A″–F″) Inferior AV ECs. (A) At 48 hpf, single-layered ECs were found in the superior and inferior parts of the AVC. (B) At 55 hpf, leading ECs form protrusions in the superior AVC (B′), whereas inferior AV ECs in the majority of embryos examined (25/44) remained single layered (B″). (C) At 60 hpf, collective migration of superior AV ECs into the ECM in a ventricle-to-atrium direction (C′). Inferior AV ECs extended protrusions into the ECM but remain single layered (C″). (D) At 75 hpf, establishment of a folded multilayered structure in the superior part of the AVC (D′). Variability was observed in inferior leaflet formation, as inferior AV ECs appeared as a folded tissue in most larvae (D″; 18/27) and remained single layered in the others (9/27). (E and F) At 100 and 120 hpf, there was elongation of AV EC layers into the vascular lumen in both the superior (E′–F′) and inferior (E″–F″) parts of the AVC. Overviews are single planes (A–F); magnified images of AV ECs are 5-µm-thick maximum projections of five confocal planes (A′–F″). A, atrium; V, ventricle. In models A–F: blue, AV ECs; red, nonvalve ECs; brown, myocardium; yellow, vascular lumen; purple, ECM. Scale bars: (A–F) 20 µm; (A′–F″) 10 µm.