We attempted to visualize mutant cells by connecting the TagRFP reporter gene to the splice acceptor site via a bicistronic 2A peptide sequence in Zwitch (Figure 1A and Figure 1—figure supplement 1A and B). We detected TagRFP expression in cells in the ganglion cell layer (GCL) in the retina (arrowheads, Figure 2—figure supplement 3), in which robust shha expression was previously reported (Shkumatava et al., 2004). However, the expression level was variable between GCL cells, compared with the uniform expression of endogenous shha mRNA in this tissue (Figure 2—figure supplement 3). Moreover, TagRFP expression was inconsistent in other tissues known to produce Shha at high levels, such as the floor plate and notochord. Given the transcription of shha-2A-TagRFP mRNA (Figure 2C and Figure 2—figure supplement 2B), the variability in the expression of TagRFP might be attributed, at least in part, to inefficient cleavage of the 2A peptide in the target cells. It is of interest to examine whether TagRFP expression would be enhanced in the target tissues by modifying the 2A sequence of Zwitch to include a glycine-serine-glycine spacer (GSG), which was recently found to significantly improve the cleavage efficiency of the 2A peptide (Wang et al., 2015b). We did not detect TagRFP expression in conditional shha mutant epicardial cells during development and regeneration (data not shown). We suspect that the failure to visualize shha-deficient epicardial cells expressing TagRFP was due to the low expression levels of endogenous shha in the epicardium during heart development and regeneration. Further studies are needed to improve the visualization of mutant cells for conducting mosaic studies using Zwitch.