The zebrafish used in this study were outcrossed from the Ekkwill (EK) background. All transgenic strains were analyzed as hemizygotes. The following published transgenic strains were used: Tg(cmlc2:DsRed2)pd15 (RRID:ZFIN_ZDB-ALT-110210-15) (Kikuchi et al., 2010), Tg(gata5:EGFP)pd25 (RRID:ZFIN_ZDB-ALT-110408-5) (Kikuchi et al., 2011), Tg(tcf21:CreER)pd42 (RRID:ZFIN_ZDB-ALT-110818-7) (Kikuchi et al., 2011), Tg(gata4:EGFP)ae1 (RRID:ZFIN_ZDB-ALT-051123-4) (Heicklen-Klein and Evans, 2004), and Tg(cmlc2:EGFP)f1 (RRID:ZFIN_ZDB-GENO-080403-2) (Burns et al., 2005). Tg(ubb:iCRE-GFP)vcc9 was generated by co-injecting pUbb-iCRE-GFP (Figure 2—figure supplement 1A) with I-SceI into single cell-stage–embryos. Zebrafish were used for regeneration experiments at 4–12 months of age. The heart injury experiments were conducted as previously described (Poss et al., 2002). The animals were maintained at a density of 3–5 fish per liter, and clutch-mates of the appropriate genotypes were used as controls. The zebrafish husbandry procedures and all experiments were conducted in accordance with institutional and national animal ethics guidelines.