Fig. 5.

REN regulates different genes in uninjured hearts and during regeneration. (A) Images of uninjured valves from wild-type and ΔREN mutant fish. Sections are stained by RNAscope using a probe for adamts1 (green) and muscle was immuno-stained with an antibody towards myosin heavy chain (MHC, blue). Yellow arrows, likely epicardial signal; blue arrows, CM signal; white dashed lines, REN expression domain; red dotted lines, canal to OFT; white asterisks, signal from endocardial interstitial cells that compromise the valve itself. (B) The number of adamts1 mRNA foci from images like A were counted using MIPAR. Quantification of foci that colocalized with muscle (MHC) is shown on the left. Quantification of foci that are not muscle is shown on the right. Wild type, dark blue; mutant, light blue. (C) The number of adamts1 mRNA foci from the sectioned amputated ventricles (3 dpa) analyzed for runx1 in Fig. 3F. Quantification of foci that colocalized with muscle is shown on the left. Quantification of foci that are not muscle is shown on the right. (D,E) AFOG staining of uninjured wild-type and uninjured ΔREN mutant hearts (D) and uninjured wild-type and uninjured ΔREN/Δrunx1 double heterozygote hearts (E). Valve leaflets are shown on the right in a magnification of boxed areas. (F) Valves around the outflow tract in uninjured wild-type and ΔREN mutant hearts are immunostained with anti-Collagen I (green) and anti-Mef2c (red) to mark CM nuclei. (G) Quantification of Collagen I stain intensity. Data are mean±s.e.m. **P=0.01 (Mann Whitney). ns, not significant.