To investigate the mechanism underlying the developmental defects in epi-KO hearts, we analyzed the expression of the pan-epicardium markers tcf21 and pard3 using semi-qRT-PCR (Plavicki et al., 2014; Serluca, 2008) and detected the normal expression of these marker genes in epi-KO hearts (Figure 4F). This result suggests that epicardium-specific loss of shha does not directly affect the development of the epicardium, consistent with the observation that epicardial development is normal in mutant mouse hearts lacking the Shh receptor smoothened (Rudat et al., 2013). However, the expression of the well-characterized epicardial marker raldh2/aldh1a2, which encodes an enzyme that plays a key role in retinoic acid (RA) synthesis (Begemann et al., 2001; Cunningham and Duester, 2015; Niederreither et al., 1999; Sucov and Evans, 1995), was strongly decreased (Figure 4F). To analyze epicardial cell development and raldh2 expression in epi-KO hearts, we crossed tcf21:CreER; shhact/+ with shhact/+ fish carrying the epicardium-specific DsRed2 reporter transgene Tg(tcf21;DsRed2) (tcf21:DsRed2) (Kikuchi et al., 2011) and obtained tcf21:DsRed2; tcf21:CreER; shhact/ct embryos after PCR genotyping of individual embryos. We induced epicardium-specific inactivation of shha expression in these embryos and found that DsRed2+ epicardial cells were normally distributed throughout the epi-KO heart (Figure 4G). Consistent with the PCR results (Figure 4F), Raldh2 protein expression was barely detectable in mutant DsRed2+ cells (Figure 4G). These findings suggest that defects in RA synthesis might underlie the developmental defect in epi-KO hearts.