Epicardial activation following myocardial infarction was diminished in Tβ4KO hearts (A and B). By immunostaining, large regions of WT epicardium contained WT-1⁺ cells by day 7 (A), compared with only small regions in Tβ4KO hearts (B). This was reflected in whole-heart qRT-PCR, with reduced expression of Wt1 (C), Tbx18 (D) and Aldh1a2 (E) by 2-way ANOVA (n = 3 separate hearts per time point, mean ± SEM indicated). Post-hoc tests with Bonferroni correction confirmed significant reduction of Wt1 at day 7 and Aldh1a2 at day 4. **P ≤ 0.01, ***P < 0.001. Defects in epicardial mobilization were confirmed in explant culture: WT explants outgrew within 7 days (F), whereas KO explants produced fewer epicardial cells, and these failed to spread and form an epithelial layer (G; n = 3 of 8 cultures); moreover, several failed to outgrow altogether (H; n = 5 of 8 cultures). Coincident with expansion, a smooth muscle–lined vascular network extended throughout the WT epicardium (I). Even selecting for regions of greatest epicardial expansion in Tβ4KO hearts, vascular growth was severely diminished and notably lacked smooth muscle support (J). Quantification of epicardial vessel density at day 7 confirmed a significant reduction in KO, compared with WT (K). Delaminating αSMA⁺ cells invaded the myocardium of WT hearts (L), while KO cells failed to migrate inward (M). WT cells extended actin cytoskeleton for migration and downregulated WT-1 (N); in contrast, Tβ4KO cells remained spindle shaped, failed to orientate for invasion, and retained WT-1 expression (arrowheads, O), suggesting incomplete mesenchymal transition. Dotted lines indicate the epicardial-myocardial boundary. Sections are representative of n = 10 hearts per genotype and n = 10 of each quantified in K; each data point represents a separate animal; 2-tailed t test; ***P < 0.001. Scale bars: 500 μm (A and B); 50 μm (F–H, L, and M); 200 μm (I and J); 100 μm (N and O).