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Fig. 3. Histopathological analysis reveals that Par3^E3/E3 embryos lack epicardial cells. (A,B) Diagrams representing epicardial development by two cellular mechanisms. (1) Red arrow: epicardial progenitor cells form cell cysts with a central cavity. The cysts bud out from the pericardial surface of the septum transversum (proepicardial serosa: ps) and float to reach the myocardial surface of the heart (v, ventricle; a, atrium), and attached epicardial cells spread to cover the heart chambers (enlarged schema in B). (2) Blue arrow: epicardial progenitor cells migrate directly to and spread over the atrial chamber as a continuous epithelial sheet. Epicardial cells secrete soluble tropic factor(s) required for cardiomyocyte proliferation (B, arrows). In all the figures, anterior is towards the left and cranial is towards the top. (C-F) Hematoxylin and Eosin staining of sagittal sections obtained from wild-type (C,E) and Par3^E3/E3 littermates (D,F) at E9.5 (C,D) or E10.5 (E,F). C'-F' are higher magnifications of the regions shown in C-F, respectively. The specifications of the heart chambers (v, ventricle; a, atrium) and outflow tract (oft) are not affected in Par3^E3/E3 embryos. In wild-type embryos at E9.5 and E10.5, a substantial number of epicardial progenitor cell cysts (C',E', arrows) are formed and bud out from the proepicardial serosa (ps). However, no cell cysts are observed in the corresponding regions of Par3^E3/E3 embryos (D',F') at either stage. At E10.5, the myocardial surface (myo) in the wild-type ventricle, but not in the Par3^E3/E3 ventricle, is covered with a single layer of flat epicardial cells (E', arrowheads). Scale bars: 100 µm for C-F; 25 µm for C'-F'.

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