Figure 1.
Schematic diagram of murine PPM development.
(A–D) Scheme of PPM development based on transverse sections through the cardiac venous pole of wildtype embryos from E11.5 to E14.5. The lumen of the caval veins and the heart is marked in pale pink, the lung and trachea in pale blue, the (parietal) pleura in blue, the (parietal) pericardium in red, and the degenerating subcoelomic mesenchyme in green. Blue arrows demonstrate the direction of the detachment of the PPMs from the subcoelomic mesenchyme. Note the PPMs are double-layered consisting of the parietal layer of the pleura and the pericardium. Dorsal is up, ventral is down. h, heart; lu, lung; pc, pericardial cavity; pl, pleural cavity; ppc, pericardioperitoneal canal; ppm, pleuropericardial membranes.
Figure 2.
PPM defects in Tbx18-mutant embryos.
(A–H) Histological stainings with haematoxylin and eosin were performed on transverse (A–D) and sagittal (E–H) sections of the venous pole region of the heart at E18.5. They reveal that PPMs are only partly released from the lateral body wall and that they are not attached to the mediastinum resulting in bilateral communication between the pleural and pericardial cavities in the mutant. Arrowheads mark the attachment point of the PPMs to the lateral body wall; arrows highlight the persisting PPC in Tbx18-deficient embryos. lu, lung; lsh, left sinus horn; lv, left ventricle; pc, pericardial cavity; pl, pleural cavity; ppm, pleuropericardial membrane; ra, right atrium; rsh, right sinus horn; th, thymus.
Figure 3.
PPM development in control and Tbx18-deficient embryos.
(A–P), Histological analysis by haematoxylin and eosin stainings of PPM development was performed on transverse sections of a posterior and a mid-transverse section plane of control and Tbx18-deficient hearts from E11.5 to E14.5. Magnifications (C, D, G, H) highlight the sinuatrial mesenchymal ridges that protrude into the PPCs in control embryos at E11.5 and E12.5, and their absence in Tbx18-deficient embryos, respectively. Genotypes and stages are as indicated. Asterisks highlight the subcoelomic lateral body wall mesenchyme; black arrowheads mark the attachment point of the PPMs to the lateral body wall; black arrows point to the remaining PPCs; and green arrowheads mark the mesenchymal ridge that is necessary for the closure of the PPCs. icv, inferior cardinal vein; la, left atrium; lcv, left cardinal vein; li, liver; lu, lung; lsh, left sinus horn; lv, left ventricle; pc, pericardial cavity; ppc, pericardioperitoneal canal; pl, pleural cavity; ppm, pleuropericardial membrane; ra, right atrium; rcv, right cardinal vein; rsh, right sinus horn; rv, right ventricle.
Figure 4.
The PPCs in Tbx18-deficient embryos remain open due to the loss of GFP-positive sinuatrial mesenchymal ridges.
(A–J) In situ hybridization analysis of GFP expression on sagittal sections of control and mutant hearts from E10.5 to E14.5. In Tbx18-deficient embryos the mesenchymal ridges are not established (F–J). Stages are as indicated on top and the genotypes on the left side. Black arrows point to the open right PPC. Green arrowheads mark the GFP-positive mesenchymal ridges in control embryos at E11.5 and E12.5. icv, inferior cardinal vein; lu, lung; ppm, pleuropericardial membrane; ra, right atrium; rscv; right superior caval vein; rsh, right sinus horn; rv, right ventricle.
Figure 5.
PPC closure is disturbed in Wt1-deficient embryos.
(A–H) Histological analysis by haematoxylin and eosin stainings on sagittal sections through the PPCs of control (genotype: Wt1+/−) and Wt1-mutant embryos from E11.5 to E14.5. Black arrows highlight the pericardial defect in Wt1-deficient embryos at E13.5 and E14.5. Green arrowheads mark the mesenchymal ridges in control and Wt1-deficient embryos at E11.5 and E12.5. Stages and genotypes are as indicated. icv, inferior cardinal vein; lu, lung; ppm, pleuropericardial membrane; ra, right atrium; rsh, right sinus horn; rv; right ventricle.
Figure 6.
Sinuatrial mesenchymal ridges are highly proliferative.
(A–D) Immunohistochemical analysis for GFP protein (green) and BrdU (red) (A, B) and the TUNEL assay (C, D) on sagittal sections through the heart of Tbx18GFP/+ embryos at E11.5 and E12.5 show that the mesenchymal ridges (green arrowheads) are highly proliferative and that apoptosis is absent. (E) Quantitative analysis of proliferation by the BrdU assay reveals non-significant changes of proliferation at E11.5 and significantly increased proliferation in Wt1-deficient sinuatrial ridges at E12.5. (F, G) The TUNEL assay on sagittal sections through the heart of Wt1−/− at E11.5 and E12.5 shows that apoptosis is absent from mesenchymal ridges (green arrowheads) at these stages. lu, lung; ra, right atrium.
Figure 7.
Gene expression in and around the sinuatrial ridges in Wt1- and Tbx18-mutant hearts.
(A–D) In situ hybridization analysis of Wt1 (A), Aldh1a2 (B), Tbx18 (C) and Gata4 (D) on sagittal sections trough the venous pole region of control, Wt1- and Tbx18-deficient hearts at E11.5 and E12.5. Genotypes and stages are as indicated. Green arrowheads point to the mesenchymal ridges that are important for the closure of the PPCs in control embryos; black arrows mark the remaining PPCs. icv, inferior cardinal vein; lu, lung; ra, right atrium.
Figure 8.
Schematic diagram of PPC closure in mouse development.
(A–C) Scheme of PPC closure by sinuatrial mesenchymal ridges based on sagittal sections through the cardiac venous pole of wildtype embryos from E11.5 to E13.5. Sinuatrial mesenchymal ridges protrude into the PPCs at E11.5 and E12.5 and fuse with the overlying wall of the lung hilus at E13.5 to separate pleural and pericardial cavities. The lumen of the caval veins and the heart is marked in pale pink, the lung and trachea in pale blue, the mesenchymal sinuatrial ridges in green (additionally marked by green arrowheads), the (parietal) pericardium in red, the (parietal) pleura in dark blue and the double-walled PPMs in red and blue. icv; inferior caval vein; lu, lung; pc, pericardial cavity; pl, pleural cavity; ppc, pericardioperitoneal canal; ppm, pleuropericardial membranes; ra, right atrium; rcv, right superior caval vein; rv, right ventricle.