Fig. 3.

Fig. 3.

Deletion of REN increases cardiomyocyte proliferation during regeneration. (A) Top: images of sectioned amputated ventricles (7 dpa) from wild-type and ΔREN mutant fish. Sections are stained for Mef2c (green) and EdU (red). Bottom: double-positive cells are highlighted in black using a MIPAR software rendition. Scale bar: 100 µm. (B) Quantification of CM proliferation indices (Mef2⁺EdU⁺ cells/total Mef2⁺ cells) in 7 dpa ventricles. Wild type (Wt), dark blue; mutant, light blue. (C) Scatterplot of RNA-seq results comparing wild-type (x-axis) and ΔREN mutant (y-axis) samples. Each dot represents a transcript and is plotted by the log₂ for the ratio of normalized reads from regeneration/normalized reads from the uninjured samples. Red dots are those transcripts that deviate by a linear regression >3-fold and blue dots are those transcripts that deviate by linear regression <−3-fold. Transcripts that are highlighted in the text are additionally marked in black circles. Green, pro-regeneration/proliferation genes; pink, sarcomeric genes; runx1-coregulatory factor cbfb, dark red. (D) Venn diagram comparing chromatin marks at the promoters of genes for which mRNA either increases (left) or decreases (right) in ΔREN mutant hearts during regeneration. (E) ddPCR shows the abundance of runx1 transcripts increasing from uninjured wild-type hearts (dark blue) during heart regeneration (red). In ΔREN mutant fish (light blue, pink), runx1 levels increase less so. The y-axis is the calculated runx1 mRNA numbers normalized to calculated number of mob4 mRNA. (F) Images of sectioned amputated ventricles (3 dpa) from wild-type and ΔREN mutant fish. Sections are stained by RNAscope using a probe for runx1 (green) and muscle was immunostained with an antibody towards myosin heavy chain (MHC; blue). Boxes 1-6 show a magnification of boxed regions around the wounds highlighting runx1 mRNA within the muscle (yellow arrows), epicardial cells (pink arrows) and likely endocardial cells adjacent to muscle that remains in the mutant (white arrowheads). (G) The number of runx1 mRNA foci from images such as F 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. (H) There is little to no basal expression of runx1 in muscle around uninjured cardiac valves. Data are mean±s.e.m. **P<0.01, ****P<0.0001 (Welch's t-test). ns, not significant.