Fig. 2

Molecular dissection of cLEN to identify the fragment containing injury-responsive elements. (A) Transgene constructs examined for regeneration-dependent expression in response to cardiac injury. Endocardial expression results are summarized on the right. EC, endocardial cell. Uninj. and Reg. correspond to uninjured and 3 dpa regenerating hearts, respectively. (B) Images of sections of 3 dpa hearts from transgenic fish carrying cLEN fragments. Numbers of animals are shown in Table S3. The arrows indicate endocardial EGFP induction. Note that the P2 promoter directs basal expression in CMs in response to injury (see Fig. S3; Kang et al., 2016). MHC, myosin heavy chain, a CM marker. (C) Transgenic constructs used to examine enhancer activity in the F₀ mosaic injured hearts. Two AP-1 binding sites were mutated in cLEN^AP-1m (black circles). AFNEGx3 is a synthetic cardiac regeneration enhancer consisting of three tandem copies of the AP-1-FOX-NFAT-ETS-GATA motifs. (D) Schematic of enhancer assays in the F0 mosaic hearts. (E) Images of injured hearts of larvae injected with P2, cLEN, cLEN^AP-1m and AFNEGx3. Note that larvae carrying transgenic constructs were selected by mCherry expression in the lens, without a cardiac EGFP signal. The arrows indicate injury-responsive EGFP induction in the injured hearts. Numbers at the bottom corners indicate the total ratio of embryos showing the corresponding expression pattern. Scale bars: 100 µm in B; 50 µm in E.