DEV02294 Supplementary Material

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• Supplemental Figure 1 -

  Fig. S1. Histological analysis of Par3^ΔE3/ΔE3 embryos at E11.5. (A,B) Hematoxylin and Eosin staining of sections obtained from wild-type (A) and Par3^ΔE3/ΔE3 (B) littermates at E11.5. Both embryos show the developing endocardial cushions (ec) and trabeculations (arrowheads) in the ventricles (v). Scale bars: 100 mm. (A¢, B¢) Enlarged views of the ventricular walls indicated in A and B, respectively. Although the wild-type heart chambers are covered with a single layer of epicardial cells (arrowheads), the Par3^ΔE3/ΔE3 myocardium is still naked (arrows). Scale bars: 25 mm. (C) The Par3^ΔE3/ΔE3 embryo has a hypoplastic heart with an extremely thin atrial wall (arrowheads). a, atrium; v, ventricle. Scale bar: 25 mm.

• Supplemental Figure 2 -

  Fig. S2. No significant difference in the myocardial and endocardial proliferations in Par3^ΔE3/ΔE3 and control littermate embryos. (A,B) Representative immunofluorescence images for BrdU (cyan) and DAPI (red) in wild-type (A) and Par3^ΔE3/ΔE3 (B) hearts pulse-labeled for 1 hour with BrdU at E9.5. The BrdU-positive (A¢,B¢, cyan) or -negative (red) myocardial and endocardial cells were counted in three pairs of Par3^ΔE3/ΔE3 and control littermates at E9.5 and E10.5. Scale bars: 50 mm. (C) No significant difference in the proliferation of myocardial and endocardial cells between Par3^ΔE3/ΔE3 and control littermates at E9.5 and E10.5. Values shown are mean±s.d of three independent pairs of littermates.

• Supplemental Figure 3 -

  Fig. S3. Mesonephric development is disturbed in Par3^ΔE3/ΔE3 embryos. (A,B) Hematoxylin and Eosin staining of sections obtained from wild-type (A) and Par3^ΔE3/ΔE3 (B) embryos at E11.5. The wild-type mesonephric vesicles (mv) are composed of a simple columnar epithelium and each nucleus is positioned on the basal side of the epithelium (arrowhead). By contrast, the Par3^ΔE3/ΔE3 mesonephric vesicles consist of multilayered epithelium with randomly positioned nuclei (arrowhead). wd, Wolffian duct. Scale bars: 25 mm. (C,D) Immunohistochemistry revealed WT1 in the glomerular structures in the mesonephric vesicles of wild-type (C) and Par3^ΔE3/ΔE3 (D) embryos at E10.5. Whereas the wild-type mesonephric vesicles are organized to form epithelial cysts and the glomerular structures have nuclei positive for WT1, the Par3^ΔE3/ΔE3 mesonephric vesicles and the glomerular structures are disorganized. Scale bars: 25 mm. (E,F) Immunofluorescence for PAR6b (red) and ZO1 (green) in the mesonephric vesicles of wild-type (C) and Par3^ΔE3/ΔE3 (D) embryos at E10.5. In the wild-type mesonephric vesicles, PAR6b exclusively localizes at the apical membrane domains and ZO-1 concentrates at the apical cell-cell junctions. By contrast, in the Par3^ΔE3/ΔE3 mesonephric vesicles, PAR6b generally mislocalizes to the cytoplasm and the apical domains are disorganized.