Ongoing PE cluster formation. Ventral (A) and lateral (B-D) views of hearts from tcf21:DsRed2 larvae. PE and epicardial cells are marked with immunostaining for DsRed2 (red) and cardiomyocytes are marked with ALCAM (green). Nuclei are stained with DAPI (blue) in panel D. (A) Confocal z-stack of heart at 74 hpf. Epicardial cells have attached to the ventricle and additional PE clusters (arrows) are forming. A PE aggregate (arrowhead) that has been released into the pericardial cavity is located near the atrioventricular (AV) junction (n = 10). (B) Confocal z-stack of a ventricle at 84 hpf. Epicardial cells are established on the ventricle. PE cells clustered on the pericardial wall projecting towards the heart (n = 10). (C) A single optical slice taken from the z-stack, showing the persisting PE cluster (arrow). (C) PE cell aggregates (white arrows) in the pericardial cavity at 120 hpf (n = 7). For all panels, anterior is to the left and V is ventricle. Scale bars = 50 microns.