Previously inaccessible aspects of organ morphogenesis can be addressed by combining conditional genetic analysis with established embryological, pharmacological, and transgenic manipulations in the zebrafish system. Moreover, the cKO approach will enhance the utility of zebrafish in elucidating the mechanisms underlying biological phenomena in adults that cannot be adequately investigated using a global KO approach. As an example, we used the shha gene trap line with an epicardium-specific inducible Cre driver and provided the first direct evidence that shha expression in the epicardium is required for the synthesis of RA and cardiomyocyte mitogens in the epicardium during heart development (Figure 4G and H). We further provided evidence that Shha may transmit a short-range signal to induce subepicardial muscle cell proliferation in the regenerating heart (Figure 5D and E), a mechanism that was not identified in studies of cyclopamine-mediated global Hh signaling inhibition (Choi et al., 2013). In situ hybridization analysis of embryo and adult hearts detected the expression of shha in tcf21:DsRed-negative cells (Figure 4—figure supplement 1A–F), suggesting that non-epicardial cells also produce Shha. Moreover, semi-qRT-PCR analysis detected the expression of dhh and ihhb in epicardial cells purified from epi-KO hearts (Figure 5C). Investigations of shha function using broader Cre driver lines and conditional inactivation of other Hh ligand genes will be needed in the future to definitively clarify the function of Shha during heart development and regeneration. However, the results described in this study demonstrate that conditional genetic analysis is a feasible, effective approach to elucidate developmental and regenerative mechanisms in zebrafish, and it might also be useful for deciphering other complex phenomena such as immune responses, metabolism, and behavior.