To demonstrate the utility of Zwitch, we investigated the functional consequences of inactivation of shha expression in the epicardium, the mesothelial layer covering the heart. Although the epicardium was reported to produce Shh during heart development in mice (Lavine et al., 2006), the precise function of Shh has not been addressed using an epicardium-specific KO approach. Using fluorescence-activated cell sorting (FACS), we purified epicardial cells and cardiomyocytes from the dissected hearts of transgenic reporter fish (see Flow cytometry, Materials and methods) and confirmed shha expression in epicardial cells and ptch1 expression in both epicardial cells and cardiomyocytes (Figure 4A). In situ hybridization analysis also detected shha expression in the epicardium of the ventricle (Figure 4—figure supplement 1A–C). Next, we crossed shhact/+ fish with shhact/+ fish carrying the epicardium-specific inducible Cre transgene Tg(tcf21:CreER) (tcf21:CreER) (Kikuchi et al., 2011) and obtained tcf21:CreER; shhact/ct embryos after PCR genotyping of individual embryos (Figure 3—figure supplement 1C). We inactivated shha expression in the epicardium by treating 24–72 hr post-fertilization (hpf) embryos with 4-hydroxytamoxifen (4-HT; Figure 4B), which induced a nearly complete inversion of the gene trap cassette (Figure 4—figure supplement 2D). Strikingly, most embryos lacking shha expression in the epicardium (epi-KO) developed severe cardiac edema by 96 hpf (Figure 4C). Cardiac edema was unclear or extremely weak in epi-KO hearts at 72 hpf, suggesting that epicardial inactivation of shha expression leads to heart defects at later developmental stages. We could not determine whether a similar cardiac phenotype was also observed in the global shha mutant embryos, as proper characterization was not possible due to the pleiotropic effects of global inactivation of shha expression at later time points. The epi-KO embryos had a thinner myocardial wall (Figure 4D) and significantly fewer cardiomyocytes (Figure 4E). These findings indicate that epicardium-derived Shha plays a crucial role in zebrafish heart development.10.7554/eLife.24635.012Figure 4.Epicardium-specific inactivation of shha expression during heart development.(A) Semi-qRT-PCR analysis of purified cardiomyocytes (CM) and epicardial cells (Epi) from 96 hpf hearts (see also Flow Cytometry, Materials and methods). Cardiomyocyte (vmhc) and epicardial (tcf21) markers were used to confirm the specificity of cell sorting. (B) Semi-qRT-PCR analysis of shha and ptch1 expression in hearts dissected from tcf21:CreER; shhact/ct embryos treated with the vehicle (−) or 4-HT (+). (C) Phenotype of tcf21:CreER; shhact/ct embryos treated with the vehicle or 4-HT. Severe cardiac edema was observed in 4-HT–treated embryos at 96 hpf (six abnormal in eight analyzed; right, arrowheads) but not in vehicle-treated embryos (zero abnormal in eight analyzed; left, arrowheads; n = 8 each; p<0.01, Fisher’s exact test). (D) Immunofluorescence of heart sections obtained from vehicle- or 4-HT–treated tcf21:CreER; shhact/ct embryos. Insets, single-channel images of Mef2 immunofluorescence. Dotted yellow lines in insets depict the outline of the ventricle. (E) Quantification of Mef2+ nuclei from the sections obtained from the vehicle- or 4-HT–treated tcf21:CreER; shhact/ct embryos in D (n = 13 and 12). The data are presented as the mean ± SEM (***p<0.001, Mann–Whitney U test). (F) Semi-qRT-PCR analysis of epicardial marker gene expression in hearts dissected from tcf21:CreER; shhact/ct embryos treated with the vehicle (−) or 4-HT (+). (G) Immunofluorescence staining of heart sections obtained from vehicle- or 4-HT–treated tcf21:CreER; shhact/ct embryos. Raldh2 immunofluorescence was detected in tcf21:DsRed2+ epicardial cells in vehicle-treated embryos (left, arrowheads) but not in 4-HT-treated embryos (right, arrowheads). Bottom panels, single-channel images of Raldh2 immunofluorescence. (H) Semi-qRT-PCR analysis of the expression of myocardial growth factor genes in hearts dissected from tcf21:CreER; shhact/ct embryos treated with the vehicle (−) or 4-HT (+). (I) Semi-qRT-PCR analysis of shha-dependent myocardial growth factor genes in purified cardiomyocytes (CM) and epicardial cells (Epi) obtained from 96 hpf hearts. Single confocal sections are shown in D and G. ve, ventricle. Scale bar, 10 μm.DOI: http://dx.doi.org/10.7554/eLife.24635.01210.7554/eLife.24635.013Figure 4—figure supplement 1.shha expression during heart development and regeneration.(A–C) shha expression during heart development in zebrafish. Single confocal slice images of the rectangles in A are shown in B and C. (D–F) shha expression during heart regeneration in zebrafish. Single confocal slice images of the rectangles in the right panel of D are shown in E and F. at, atrium; ba, bulbus arteriosus; es, esophagus; ve, ventricle. Scale bar, 50 μm.DOI: http://dx.doi.org/10.7554/eLife.24635.01310.7554/eLife.24635.014Figure 4—figure supplement 2.Inversion rate measurement.(A) Schematic of the non-mutagenic and mutagenic allele, primer sites, and BglI recognition site. (B) A gel image showing the PCR products of the non-mutagenic alleles without (left) and with BglI digestion (middle) and the PCR products of the mutagenic alleles with BglI digestion (right). Genomic DNA was prepared from 96 hpf shhact/+ embryos injected with (+) or without (−) Cre mRNA at single-cell stage. Cre mRNA was prepared from linearized pCS2-iCRE-BFP, in which iCRE cDNA is linked to TagBFP via P2A peptide sequence. (C) Analysis of inversion rate and shha expression levels in single embryos. Embryos were prepared from an incross of shhact/+ fish and injected with Cre mRNA at single-cell stage. Embryos without Cre mRNA injection were used as control (Sample 1). The injected embryos were separated based on the expression of TagBFP at moderate (Sample 2) and high levels (Sample 3 and 4). Individual embryos were genotyped at 96 hpf and genomic DNA and total RNA of each embryo using TRIzol following the manufactured protocol. The upper panels are the gel images used for inversion rate measurement. The numbers on the bottom are the obtained inversion rate in each sample. The lower panels are the results of semi-qRT-PCR analysis of shha expression levels of each sample. (D) Analysis of inversion rate in epicardial cells in zebrafish embryos. Embryos were prepared from crosses of tcf21:DsRed2; shhact/+ fish with shhact/+ fish (Sample 1) and tcf21:CreER; shhact/+ fish with tcf21:DsRed2; shhact/+ fish (Sample 2). Embryos were treated with 4-HT and examined for DsRed2 expression to screen tcf21:DsRed2 transgene and lens EGFP expression to screen tcf21:CreER transgene (Kikuchi et al., 2011). Lens EGFP from tcf21:CreER transgene is considerably stronger than that from shhact alleles in embryos, which enables us to sort tcf21:CreER+ embryos with shhact background. PCR genotyping on shhact alleles were not performed; this does not affect the measurement as the inversion rate is quantified based on the PCR products of the shhact alleles. tcf21:DsRed2+ epicardial cells were isolated by FACS and 1,000 DsRed2+ cells were used for analysis. The estimated inversion rate is 100%; note that the bands from the non-mutagenic allele (N1 and N2) are undetectable in Sample 2 after BglI digestion. (E) Analysis of inversion rate in epicardial cells in the adult zebrafish ventricle. Ventricles were prepared from 4-HT–treated adult tcf21:DsRed2; shhact/+ fish (Sample 1) and tcf21:CreER; tcf21:DsRed2; shhact/+ fish (Sample 2). tcf21:DsRed2+ epicardial cells were isolated by FACS and 5000 DsRed2+ cells were used for analysis. The estimated inversion rate is 100%; note that the bands from the non-mutagenic allele (N1 and N2) are undetectable in Sample 2 after BglI digestion.DOI: http://dx.doi.org/10.7554/eLife.24635.014