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First published online 1 March 2006
doi: 10.1242/dev.02294

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PAR3 is essential for cyst-mediated epicardial development by establishing apical cortical domains

¹ Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, Yokohama 236-0004, Japan.
² Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences, Yokohama 244-0813, Japan.
³ Department of Cell Biology, JFCR-Cancer Institute, Tokyo 135-8550, Japan.
⁴ Center for Translational and Advanced Animal Research (CTAAR), Tohoku University School of Medicine, Sendai 980-8575, Japan.
⁵ CREST, Japan Science and Technology Corporation (JST), Saitama 332-0012, Japan.

View larger version (67K): [in a new window]   Fig. 1. Targeted disruption of mouse Par3 gene. (A) Schematic structures of the full-length PAR3 protein (180 kDa) and a major splice variant (100 kDa) expressed in mouse embryos. The third exon (E3) encodes amino acids 75-135. Anti-PAR3 antibodies were raised against amino acids 1-115 (N2-3E1) or 712-936 (C2-3AP). CR1 and PDZ indicate the conserved oligomerization and PDZ domains, respectively. (B) Restriction maps of wild-type mouse Par3 genomic locus, targeting vector, targeted allele (Par3Neo) and Par3 deleted allele (Par3E3) obtained after cre-mediated excision of loxP-flanked region, including the third exon (E3, 181 bp, boxes), which encodes amino acids 75-135. The targeting vector has one loxP-flanked MC1-neo cassette in intron 2, one loxP site in intron 3 and one MC1-DT-A-negative selection marker. The loxP sequences are shown in triangles. The position of the 5' external probe for Southern blot analysis is indicated. RV, EcoRV; Ap, ApaI; Nc, NcoI; Ns, NsiI; Nd, NdeI. (C) Southern blotting of EcoRV- or ApaI-digested genomic DNA from wild-type (+/+) and heterozygous (Neo/+) littermates. The size of each band yielded by the 5' external probe is indicated. (D) Southern blotting of EcoRV-digested genomic DNA from wild-type (+/+), heterozygous (+/E3) and homozygous (E3/E3) littermates. The 5' external probe yields an 8.2 kb band from the wild-type allele, and a 7.2 kb band from the Par3E3 allele. (E) The loss of the PAR3 protein in embryos at E11.5 was examined by immunoblotting. Protein extracts from the heads of the embryos shown in D were probed with affinity-purified anti-PAR3 antibodies (C2-3AP, N2-3E1). An asterisk indicates nonspecific bands. The CBB staining of blotted membrane is shown as a loading control.

View larger version (80K): [in a new window]   Fig. 2. Par3E3/E3 embryos show growth retardation at E9.5 and cardiac defect at E10.5. (A-G) Gross appearance of wild-type (A,D,F) and Par3E3/E3 (B,C,E,G) embryos at E9.5 (A-C) and E10.5 (D-G; enlarged, D'-G'). Mild (B) to severe (C) growth retardation is observed in Par3E3/E3 embryos at E9.5. Although the common ventricular chamber (asterisk) and the common atrial chamber (arrowhead) are formed in Par3E3/E3 embryo at E10.5 (E'), the heart is hypoplastic. A severely affected Par3E3/E3 embryo at E10.5 (G,G') shows an enlarged atrium (arrow) and peripheral edema (arrowheads). Scale bars: 0.5 mm. (H,I) Histological analysis of the placentas from wild-type (H) and Par3E3/E3 (I) littermates at E10.5 shows no significant difference in the development of the giant cells (gc), spongiotrophoblasts (sp), labyrinth (la) and cholionic plates (cp). Both placentas show abundant embryonic blood vessels (arrows) and their close proximity to maternal blood sinuses (arrowheads). The indicated regions are enlarged in H' and I'. Scale bars: 200 µm.

View larger version (93K): [in a new window]   Fig. 3. Histopathological analysis reveals that Par3E3/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 Par3E3/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 Par3E3/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 Par3E3/E3 embryos (D',F') at either stage. At E10.5, the myocardial surface (myo) in the wild-type ventricle, but not in the Par3E3/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'.

View larger version (114K): [in a new window]   Fig. 4. Epicardial progenitor and epicardial cells are selectively affected in Par3E3/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 Par3E3/E3 heart, are enveloped with a single layer of cells positive for 4 integrin. Although the proepicardial serosae (ps) of wild-type and Par3E3/E3 hearts are positive for 4 integrin and GATA-4, the cardiac morphology is different: the Par3E3/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 Par3E3/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 Par3E3/E3 hearts of E9.5 littermates. The signals for PAR3 are detected in wild-type (F), but not in Par3E3/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). Par3E3/E3 proepicardial serosa (F', ps) shows an aberrant accumulation of 4 integrin-positive cells. Scale bars: 50 µm.

View larger version (90K): [in a new window]   Fig. 5. The epicardial progenitor cells in Par3E3/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 Par3E3/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 Par3E3/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 Par3E3/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 Par3E3/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 Par3E3/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 Par3E3/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 Par3E3/E3 epicardial progenitor cells. The explants from three wild-type and four Par3E3/E3 embryos were analyzed. Error bars represent s.e.m.

View larger version (113K): [in a new window]   Fig. 6. PAR6ß, aPKC and ezrin lose apical cortical association in Par3E3/E3 epicardial progenitor cells at E10.5. (A-D) Although PAR6ß and aPKC closely associate with the apical cortical domains in wild-type EPP cells, they show diffuse distribution in the EPP cells of Par3E3/E3 littermates (arrowheads). DNA was stained with DAPI (red in A'-D'). Scale bars: 10 µm. (E-J) Localization of PAR6ß, 4 integrin, ZO1, aPKC/, ß1 integrin, ezrin and PAR3 in cryosections of the EPP cells in wild-type and Par3E3/E3 littermates at E10.5. Merged images are shown in the fourth column: PAR-6ß, aPKC/ or ezrin, red; 4 or ß1 integrin, green; ZO1 or PAR3, blue. The apical cortical association of PAR6ß and aPKC/ in wild-type EPP cells is disturbed with the proteins diffusely distributed in the cytoplasm in Par3E3/E3 EPP cells (E-H, white arrowheads). However, both the wild-type and Par3E3/E3 EPP cells show the accumulation of 4 and ß1 integrins in the basal subdomains (E-J, arrows), and ZO1 concentrates immediately apical to 4 or ß1 integrins (E-H, black arrowheads). The apical cortical association of ezrin in wild-type EPP cells is mostly lost in Par3E3/E3 EPP cells (I,J, arrowheads). The asterisk in J indicates an aberrant accumulation of Par3E3/E3 EPP cells in the proepicardial serosa. Scale bars: 10 µm.

View larger version (30K): [in a new window]   Fig. 7. Model for EPP cell cyst formation. Mesenchymal-to-epithelial transition specifies EPP cells expressing WT1 and 4 integrin. Cell-cell contacts and integrin-mediated contacts to the extracellular matrix provide spatial cues for epithelial cell polarity. PAR3 interprets the spatial cell polarity cues to localize PAR6ß and aPKC, leading to the establishment of apical cortical domains of EPP cells. Completely polarized EPP cells can then form EPP cell cysts. By contrast, Par3E3/E3 EPP cells fail to localize PAR6ß and aPKC, and cannot establish the apical domains.

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