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Additional Supporting Information may be found in the online version of this article.

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DVDY_22762_sm_suppinfofig1.tif12312KSupporting Information Supp. Fig. S1. Expression patterns in the myocardium of Id2 wildtype embryos. a–d: Stage E9.5. a: Overview section of MLC-2a expression. b: Level of the common atrium (A). c: Level of the right side of the common ventricle (V). d: Level of the left side of the common ventricle. b–d are enlargements of the boxed areas in a. At this early stage, expression of Id2 (b–d) is observed in a mosaic pattern in the myocardium, i.e., Id2-expressing cells (arrows) are interposed between Id2-non-expressing cells (open arrows). Prominent Id2 expression can also be observed in endocardial cells (open arrowheads in b and d). e–h: Stage E10.5. e: Overview section of MLC-2a expression. f–j: Enlargements of the boxed areas in e, indicating the level of the primary fold (PF), situated in between the primitive right (RV) and left ventricle (LV). f: Expression of MLC-2a. The arrowhead indicates the epicardium. g: Expression of Id2 in the primary fold. Although the majority of the myocardial cells of the primitive RV and LV now do not express Id2 anymore ( h, i), in the area of the primary fold numerous Id2-expressing cells are observed (g). Id2-positive endocardial cells are still observed (open arrowheads in h and i). Epicardial cells also express Id2 (arrowhead in g) at this stage. j: WT-1 expression in epicardial cells. The arrowhead indicates the epicardium. A subepicardial layer has not been formed yet, and epicardial-derived cells are not observed in the myocardium yet. k–n: Stage E12.5. k: Overview section of MLC-2a expression. l–n are enlargements of the boxed area in k, indicating the level of the ventricular septum (IVS). Arrowheads in l–n indicate epicardial cells, which show Id2 and WT-1 expression. l: Expression of MLC-2a. m: Expression of Id2: Numerous Id2-expressing cells are still observed in the IVS. n: In contrast to the previous stage, WT-1-positive epicardial-derived cells are now also observed in the subepicardium and in the area of the IVS. LV, left ventricle; RV, right ventricle. Scale bars: 100 μm, except h and I = 50 μm.
DVDY_22762_sm_suppinfofig2.tif9072KSupporting Information Supp. Fig. S2. E9.5: Pro-epicardial organ in Id2 wildtype (+/+) and knockout (−/−) embryos. a,b: 3D-reconstructions, dorso-caudal views of an Id2 wildtype (a) and Id2 knockout (b) embryo. Color coding: light blue/grey, myocardium; blue, lumen of the cardinal veins; lime green, MLC-2a-positive and Nkx2.5-negative sinus venosus myocardium; shaded red, lumen ventricles; pink, pulmonary pit. Cells of the pro-epicardial organ (PEO) are depicted in purple. c, e: Sections of a wildtype (c) and a knockout (e) embryo at the level of the PEO, showing WT-1 expression. d: Enlargement of the boxed area in c. f: Enlargement of the boxed area in e. In the knockout embryo, the volume of the PEO appears smaller than in the wildtype. Also note the reduced amount of sinus venosus myocardium in the knockout embryo (green area in b), as compared to the wildtype (green area in a). A, common atrium; LCV, left cardinal vein; RCV, right cardinal vein; V, ventricle. Scale bars = 100 μm.
DVDY_22762_sm_suppinfofig3.tif3383KSupporting Information Supp. Fig. S3. Expression of Id2 and WT-1 at stage E10.5. a, b: Overview sections stained with Id2 (a) and WT-1 (b) at the level of the outflow tract (OFT). c, d: Enlargements of the boxed areas in a and b that depict the part of the OFT bordering the dorsal mesenchyme. Cells in this area profoundly express Id2 and WT-1 (arrowheads in c and d), and are cuboid in shape, indicating active epithelial- to-mesenchymal transformation (EMT) at this site. e,f: Overview sections stained with Id2 (e) and WT-1 (f) at the level of the common atrium. g, h: Enlargements of the boxed areas in e and f that depict the right cardinal vein (RCV) and surrounding mesenchyme. Marked expression of Id2 and WT-1 is observed in this mesenchyme, as well as in de bordering coelomic epithelial cells that are cuboid in shape (arrowheads in g, h), indicating active EMT at this site where the sinus venosus myocardium will develop. LV, left ventricle. Scale bars = 100 μm.
DVDY_22762_sm_suppinfofig4.tif4749KSupporting Information Supp. Fig. S4. Development of the myocardium in Id2 wildtype (+/+) and knockout (−/−) embryos. a, d: Stage E11.5. In the wildtype embryo (a), a significant compact myocardial layer has not developed yet at this stage, and the myocardium has a relatively thin and trabeculated appearance. In the knockout embryo of the same stage (d), the thin appearance of the myocardium is more profound. Also, the atrial myocardium is thinner, and the atria more dilated as compared to the wildtype embryo. b,e, c,f: In the wildtype embryos (b, c), a compact myocardial layer has started to develop (starting in the left ventricle [LV]), and is increasing in volume from stage E12.5 (b), to stage E13.5 (c). In contrast, in the knockout embryos of the same stage no compact layer has been developed, and the myocardium still has a thin appearance at stage E12.5 (e) and E13.5 (f) as compared to wildtypes. LA, left atrium; RA, right atrium. Scale bars = 100 μm.

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