Fig. 5. The epicardial progenitor cells in Par3^E3/E3 embryos do not form cell cysts but retain migration and proliferation activities. (A,B) Immunohistochemistry revealed WT1 in the nuclei of epicardial progenitor cells and epicardial cells in wild-type (A) and Par3^E3/E3 (B) littermates at E10.5. A wild-type embryo forms typical WT1-positive EPP cell cysts (arrow), and the ventricle (v) and atrium (a) are covered with a single layer of epicardial cells (arrowheads). Although a Par3^E3/E3 heart has an abnormal proepicardial serosa (ps) lacking typical budding cysts, the atrium, not the ventricle, is partially covered with a single layer of epicardial cells (arrowheads). Scale bars: 20 µm. (C) Representative immunofluorescence images for WT1 and BrdU in wild-type and Par3^E3/E3 proepicardial serosae (ps) pulse-labeled for 1 hour with BrdU at E10.5. The BrdU-positive (blue spots) and -negative (white spots) epicardial progenitor cells expressing WT1 were counted in three pairs of Par3^E3/E3 and control littermates at E9.5 and E10.5. Scale bars: 20 µm. (D) The proportion of BrdU-positive epicardial progenitor cells shows no significant difference between Par3^E3/E3 and control littermates at E9.5 and E10.5. The proportion of the BrdU-positive cells with respect to the corresponding control littermates is considered as 1.0 and values shown are mean±s.d of three independent pairs of littermates. (E,F) Modified Boyden chamber assay on epicardial explant cells from wild-type and Par3^E3/E3 embryos at E9.5. After a 24-hour incubation, cells that remained on the upper surface were removed and the epicardial progenitor cells migrated to the bottom surface were identified by staining for WT1 (cyan) and DNA (red). (E',F') High-magnification views of the regions shown in E,F, respectively. Scale bars: 100 µm for E,F; 20 µm for E',F'. (G) The quantification of the number of WT1-positive epicardial progenitor cells in modified Boyden chamber assay shows no significant difference in the migration between wild-type and Par3^E3/E3 epicardial progenitor cells. The explants from three wild-type and four Par3^E3/E3 embryos were analyzed. Error bars represent s.e.m.