The zebrafish has been used for regeneration research for more than a decade, but the cKO approach has yet to be applied to these studies. To illustrate the utility of our cKO approach in zebrafish regeneration research, we inactivated epicardial shha expression in injured hearts and investigated its effects on regeneration (Figure 5A). Studies using cyclopamine, which globally inhibits Hh signaling, indicated that epicardial Shha promotes myocardial proliferation (Choi et al., 2013) and epicardial regeneration in cultured injured hearts in vitro (Wang et al., 2015a). Consistent with the observation that the shha promoter is activated in regenerating epicardium (Choi et al., 2013), we detected shha expression in epicardial cells via in situ hybridization (Figure 4—figure supplement 1D–F) and semi-qRT-PCR in purified epicardial cells from tcf21:DsRed2 hearts at 7 days postinjury (dpi) (Figure 5B). Next, we analyzed adult tcf21:DsRed2; tcf21:CreER; shhact/ct fish treated with 4-HT, which induced a nearly complete inversion of the gene trap cassette (Figure 4—figure supplement 2E). We used tcf21:DsRed2; shhact/ct fish treated with 4-HT as a negative control (CreER−) because a low level of cassette inversion was detected in tcf21:CreER; shhact/ct fish in adults, but not in embryos, in the absence of 4-HT (Figure 5—figure supplement 1A–C). Semi-qRT-PCR confirmed that shha expression was depleted in epicardial cells purified from injured hearts (Figure 5C). Interestingly, unlike the embryonic epi-KO heart (Figure 4B and F), ptch1 and raldh2 expression was unchanged in shha mutant adult hearts (Figure 5C and Figure 5—figure supplement 2A and B). Moreover, epicardial cells normally infiltrated the injury site (Figure 5—figure supplement 2A–C) and incorporated 5-ethynyl-2′-deoxyuridine (EdU), a DNA synthesis marker, in epi-KO hearts (Figure 5—figure supplement 2C and D). Consistent with the previous report (Wang et al., 2015a), we detected the expression of other Hh ligand genes, namely dhh and ihhb, in epicardial cells purified from injured hearts (Figure 5B) and epi-KO hearts (Figure 5C). These results suggest that redundant Hh proteins or non-epicardium-derived Shha plays a role in RA synthesis and epicardial regeneration in the adult zebrafish heart.10.7554/eLife.24635.015Figure 5.Epicardial shha expression promotes subepicardial cardiomyocyte proliferation during heart regeneration.(A) Schematic of the experiment. (B) Semi-qRT-PCR analysis of shha in purified tcf21:DsRed2+ epicardial cells obtained from uninjured and injured (7 dpi) tcf21:DsRed hearts. Injury was confirmed by the induction of raldh2 expression. (C) Semi-qRT-PCR analysis of Hh pathway genes using purified tcf21:DsRed2+ epicardial cells obtained from 4-HT–treated 7 dpi tcf21:DsRed; shhact/ct (control, left) and tcf21:DsRed; tcf21:CreER; shhact/ct hearts (right). (D) Immunofluorescence images of the subepicardial (top) and trabecular areas (bottom) of heart sections obtained from 4-HT–treated 7 dpi shhact/ct (control) or tcf21:CreER; shhact/ct hearts. Brackets, subepicardial areas. Dotted lines, approximate amputation plane. Arrows indicate proliferating cardiomyocytes. (E) Quantification of cardiomyocyte proliferation in the subepicardial and trabecular areas of the heart sections obtained from 4-HT–treated 7 dpi shhact/ct (control) or tcf21:CreER; shhact/ct hearts shown in D (n = 6 each). The data are presented as the mean ± SEM (**p<0.01, Mann–Whitney U test). N.S., not significant (p=0.3367). (F) Image of heart sections obtained from 7 dpi gata4:EGFP; tcf21;DsRed2 fish. Subepicardial cardiomyocytes (green, arrows) associate with epicardial cells (magenta, arrowheads). (G) Semi-qRT-PCR analysis of shha pathway genes using purified subepicardial cardiomyocytes obtained from 7 dpi gata4:EGFP ventricles. Cardiomyocytes purified from uninjured cmlc2:EGFP ventricles were used as negative controls. Scale bar, 50 μm.DOI: http://dx.doi.org/10.7554/eLife.24635.01510.7554/eLife.24635.016Figure 5—figure supplement 1.Assessment of spontaneous cassette inversion.(A) Schematic of the inverted shhact allele and primers sites. (B) The inverted allele was not detected in untreated tcf21:CreER; shhact/ct embryos using 40 cycles of PCR. (C) The inverted allele was not detected in untreated adult tcf21:CreER; shhact/ct fish using 30 cycles of PCR, but it was detected when the cycle number was increased to 35. Each PCR sample used genomic DNA isolated from five pooled hearts as a template.DOI: http://dx.doi.org/10.7554/eLife.24635.01610.7554/eLife.24635.017Figure 5—figure supplement 2.A redundant role for epicardial shha in epicardial migration and proliferation during heart regeneration.(A) In situ hybridization analysis of raldh2 expression in sections obtained from 4-HT-treated 7 dpi shhact/ct (control) and tcf21:CreER; shhact/ct hearts. Brackets, injury site. (B) Immunofluorescence staining of Raldh2 and DsRed2 using sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:DsRed; tcf21:CreER; shhact/ct hearts (right). Single-channel images of the rectangle are shown at the bottom. Dotted line, approximate amputation plane. (C) Immunofluorescence staining of DsRed2 and EdU in sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:CreER; tcf21:DsRed; shhact/ct hearts. Inset, non-injured area. Arrows indicate proliferating tcf21+ epicardial cells, which were defined as epicardial cells colabeled DsRed2 and EdU. (D) Quantification of epicardial cell proliferation in the sections obtained from 4-HT-treated 7 dpi tcf21:DsRed; shhact/ct (control) and tcf21:CreER; tcf21:DsRed; shhact/ct hearts shown in C (n = 5 each). The data represent the mean ± SEM (wound, p=0.754; remote, p=0.602; Mann–Whitney U test). N.S., not significant. Single confocal slice images are shown in B and C. Scale bar, 50 μm.DOI: http://dx.doi.org/10.7554/eLife.24635.017