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. 2016 Aug 24;11(8):e0161205. doi: 10.1371/journal.pone.0161205

Fig 5. Ethanol exposure led to the failure of suppression of Bmp activity from the chamber cells during valvulogenesis.

Fig 5

(A-C) In situ hybridization detecting bmp4 expression showed restricted expression at the AV boundary and outflow tract in control embryo (A) and widely distributed throughout the ventricle in ethanol treated embryos (B, C). (D-F) 3D reconstruction of confocal sections of phospho-Smad-1/5/9 immunostained embryos showed Bmp responsive phospho-Smad-1/5/9 positive cardiomyocytes at the base of the atrium, near AVC and inner curvature of the ventricle (white arrow) in the control embryo (D); phospho-Smad-1/5/9 positive cardiomyocytes were seen throughout the venricle in ethanol exposed embryos. Note: Please see S1 Fig for high magnification images. (G-L) Single Z-sections of phospho Smad-1/5/9 and MF20 (cardiac myosin) double immunostained embryos at the AV boundary showed phospho-Smad-1/5/9 positive endocardial cells at AVC in the control embryos (G, J) and throughout the entire ventricular endocardium in ethanol exposed embryos (H, I, K, L). Arrowheads: phospho-Smad-1/5/9 positive endocardial cells, Arrows: AVC myocardium. Note phospho-Smad-1/5/9 positive myocardial cells at the AVC towards the inner curvature of the ventricle in control embryo (G; arrow) and all ventricular myocardial cells in ethanol-exposed embryos (H, I). (M-O”) Time lapse images of Tg(bre:EGFP) embryos from 48–52 hpf showed progressive loss of GFP signal from chamber cardiomyocytes, and strong GFP signal at the AV canal towards the inner curvature (arrow) and at the base of the atrium in control embryos (M-M’”); GFP signal was less suppressed from the chamber cardiomyocytes in E100 ethanol exposed embryos (N-N’”) and not suppressed in E150 ethanol exposed embryos (O-O’”). (P-R”) BODIPY-ceramide labeled embryos showed expansion of cardiac jelly (blue line) region between myocardium and endocardium in the atrium (P-R), AVC (P’-R’) and in the ventricle (P”-R”) in ethanol-exposed embryos (Q-R”) compared to control (P-P”). A: atrium, V: ventricle. White dotted line demarcates the heart.