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Fig. 4. Epicardial progenitor and epicardial cells are selectively affected in Par3^E3/E3 embryos at E9.5. (A-E) Immunohistochemical identification of epicardium, myocardium and endocardium using antibodies against 4 integrin (A), VCAM1 (C) or smooth muscle actin (D; SMA), and PECAM1 (E), respectively. All of these tissues were also labeled with anti-GATA4 mAb. The ventricle (v) and atrium (a) of a wild-type heart, but not those of a Par3^E3/E3 heart, are enveloped with a single layer of cells positive for 4 integrin. Although the proepicardial serosae (ps) of wild-type and Par3^E3/E3 hearts are positive for 4 integrin and GATA-4, the cardiac morphology is different: the Par3^E3/E3 heart lacks cell cysts (A',B', arrowheads), which are formed in the wild-type heart (A,B). The staining patterns of GATA4, VCAM1 and SMA in the myocardium, and those for GATA4 and PECAM1 in the endocardium, are indistinguishable between wild-type and Par3^E3/E3 littermates. Scale bars: 50 µm. (F,G) Epicardial progenitor and epicardial cells express the PAR3 protein in the cell-cell junctions. Immunofluorescent microscopy images of 4 integrin (green), PAR3 (red), DAPI (cyan), PECAM1 (blue) and ZO1 (green) in wild-type and Par3^E3/E3 hearts of E9.5 littermates. The signals for PAR3 are detected in wild-type (F), but not in Par3^E3/E3 (G), proepicardial serosa (arrows) and epicardial cells (arrowheads). Although ZO1 colocalizes with PAR3 in the proepicardial serosa and epicardial cells (arrowheads, merged view in G), only ZO1 is detected in the endocardial cells (arrows). Par3^E3/E3 proepicardial serosa (F', ps) shows an aberrant accumulation of 4 integrin-positive cells. Scale bars: 50 µm.

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