Fig. 4

ncam1a-E2 directs injury-induced epicardial gene expression. (A) The ncam1a-E2:EGFP reporter construct. (B) Whole-mount in situ hybridization showing ncam1a expression in a 2 dpf embryo. Adapted, with permission, from ZFIN (Ruzicka et al., 2019; Thisse and Thisse, 2004; Thisse et al., 2008). (C) Larval expression of ncam1a-E2:EGFP lines at 3 dpf. Higher magnification views of the outlined regions are shown on the right. Scale bar: 500 µm. Arrows indicate representative EGFP signals. (D) Whole-mount images of 6 dpf hearts showing EGFP expression in the outflow tract (OFT) and the atrioventricular valves (arrows). tcf21:H2A-mCherry (red) labels the epicardial cells. V, ventricle. Arrows indicate EGFP signals in the atrioventricular valve. Scale bar: 20 µm. (E) In situ hybridization results showing ncam1a expression in presumed epicardial cells around the injury site at 3 dpa (arrows). Arrows indicate representative in situ signals. Scale bar: 100 µm. (F) Whole-mount images (maximum projections of z-stacks) of the ventricular surface showing expressions of ncam1a-E2:EGFP line 1 (green) in uninjured (Ctrl) and 3, 7, 14 and 30 dpa samples. tcf21:H2A-mCherry (magenta) labels the epicardial cells. White dashed lines indicate the injury sites. The regions outlined in yellow are enlarged in G. Scale bar: 100 µm. (G) Single optical slice of confocal images of the outlined regions in F. Arrows and arrowheads indicate representative cells that express both EGFP (green) and tcf21:H2A-mCherry (magenta). Arrowheads in a,c,e,f also indicate possible tcf21+ perivascular cells. Scale bar: 100 µm. (H) Whole-mount images (maximum projections) of the ventricular surface showing expression of ncam1a-E2:EGFP line 2 (green) in uninjured (Ctrl) and 3 dpa samples. Magnified views of the outlined region are shown in I. (I) Single optical section of confocal images of the outlined regions in H. Single-channel images are shown in grayscale. Arrows and arrowheads (presumed perivascular) indicate representative double-positive cells. Scale bar: 100 µm. (J) HCR staining result of pdgfrb (magenta) on a whole-mounted heart carrying the ncam1a-E2:EGFP reporter (line 2, green, anti-EGFP antibody staining). Single-channel image shows staining signals of pdgfrb. Arrows indicate EGFP+pdgfrb+ cells. Scale bar: 50 µm. (K) Section images demonstrating expression of ncam1a-E2:EGFP (green) in tcf21:H2A-mCherry+ (magenta) cells. Magnified views of the outlined regions are shown on the right. White dashed lines indicate the injury sites. Scale bar: 100 µm. Arrows indicate representative double-positive cells. (L) HCR staining of ncam1a (magenta dots) on ventricular sections (ctrl, 3 dpa) showing ncam1a-E1:EGFP expression (line 1, green) in ncam1a+ cells. The outlined regions are enlarged on the right. Arrows indicate EGFP+ncam1a+ cells. No definitive epicardial expression of ncam1a was observed in the Ctrl samples. Some nonspecific background staining is evident in the muscle. Scale bar: 50 µm.