NMHC IIB ablated epicardial cells do not show migration defects in vitro.

A: Cells cultured from E11.5 Myh10∆/+ heterozygote and B: Myh10∆ homozygous mutant heart explants form an epicardial monolayer. Epicardial status was confirmed by positive staining for the epithelial marker ZO-1 (left panels), and the epicardial marker, Wt1 (centre panels). Rhodamine-phalloidin staining of the actin cytoskeleton also revealed characteristic ‘cobblestone’ morphology, indicative of an epithelial cell population (right panels). C: Control and D: mutant epicardial monolayers were scratched at T0 (left panels) and imaged at 10-minute intervals for 20 hours. The migration of ten cells per field of view was tracked using ImageJ (right panels, coloured lines). E: Graph showing the subsequent comparison of mean cell migration speed (control = 0.3099μm/min, mutant = 0.3098μm/min, Mann-Whitney U-test p = 0.6717). F: Graph showing the comparison of cell migration directional persistence (control = 0.7342, mutant = 0.7411, Mann-Whitney U-test p = 0.2494). Total tracked cells = 240 control and 270 mutant. Cultures were generated from at least four hearts for each genotype. Control refers to data compiled from both wild type and ∆/+ genotypes. Scale bars: 250 μm. Abbreviations: ∆: Myh10∆.