Endothelial Notch signaling enhances cardiac Bmp2/4 expression levels and induces pSmad 1/5 in the dorsal pericardium and proepicardium. A and B, Whole‐mount in situ hybridization for bmp4 or bmp2b in control, RO‐treated and animals overexpressing NICD in endothelial cells (fli1a:Gal4). A, 60 hpf. B, 80 hpf. White arrows, venous pole. Black arrows, atrioventricular canal of the heart tube. C, Quantification of pSmad 1/5+ DP cell numbers at 60 hpf. D, Quantification of pSmad1/5+ PE cell numbers at 60 hpf. E, epi:GFP embryos immunostained for GFP (green), myosin heavy chain (MHC, red), pSmad1/5 (white) and nuclei counterstained with DAPI (blue). Top panels, optical sections of a 60 hpf control zebrafish heart compared with hearts from zebrafish overexpressing NICD in pericardial and PE cells (wt1b:Gal4) or in endothelial cells (fli1a:Gal4). Zoomed views are shown below. Arrowheads, PE cluster. Yellow asterisks, PE pSmad1/5+ cells. F, Quantification of DP pSmad1/5+ cell number at 80 hpf. G, 3D projections, optical sections and zoomed images of 80 hpf control zebrafish heart compared to those overexpressing NICD in pericardial and proepicardial cells (wt1b:Gal4) or in endothelial cells (fli1a:Gal4). The DP was digitally isolated in 3D projections. Arrowheads, PE cluster. Arrows, epicardial cells. Yellow asterisks, pSmad1/5+ PE cells. H, Quantification of PE pSmad 1/5+ cell number at 80 hpf. at, atrium; DP, dorsal pericardium; hpf; hours post fertilization; PE, proepicardium; v, ventricle. Scale bar: 50 μm (25 μm in zoomed images E, G). Data in C, D, F, and H are means ± SD, one‐way ANOVA followed by Kruskal‐Wallis significant difference test. *P < .05, **P < .01, ***P < .001
|
