In contrast to epicardial cell proliferation, myocardial cell proliferation, defined as cardiomyocytes colabeled with immunofluorescence using anti-Mef2 and anti-proliferating cell nuclear antigen (PCNA) antibodies, was significantly decreased in the subepicardial area of injured epi-KO hearts (Figure 5D and E). However, myocardial cell proliferation was not reduced in the trabecular myocardium, an area distant from the epicardium (Figure 5D and E). To investigate the interplay between the epicardium and subepicardial myocardium, we crossed tcf21:DsRed2 fish with Tg(gata4:EGFP) (gata4:EGFP) fish (Heicklen-Klein and Evans, 2004), as gata4:EGFP labels the subepicardial myocardium during cardiac growth (Gupta et al., 2013) and regeneration (Kikuchi et al., 2010). Immunofluorescence analysis of injured tcf21:DsRed2; gata4:EGFP hearts revealed that epicardial cells and subepicardial cardiomyocytes directly interact (Figure 5F), and semi-qRT-PCR analysis revealed that the expression of Hh target genes was strongly induced in gata4:EGFP+ subepicardial cardiomyocytes but not in control cardiomyocytes purified from uninjured Tg(cmlc2:EGFP) (cmlc2:EGFP) hearts (Figure 5G). These findings support the role of epicardial Shha in transmitting direct, short-range signals that promote the proliferation of adjacent cardiomyocytes during zebrafish heart regeneration.