Figure 1.
Embryonic lethality in Prkcd and Prkce double deficient mouse embryos at approximately E9.5.
A and B, embryonic stage E9.5. Double null embryos (B) display growth retardation, and swollen pericardium as main phenotypes. C and D, embryonic stage E10.5, when such phenotypes became more remarkable. Scale bars = 800 µm.
Figure 2.
Impaired vessel formation in Prkcd and Prkce double deficient mouse embryos.
Unlike in wild type counterparts (A), obvious absence of defined vascular network in double deficient embryos (B) could be observed at E9.5 by performing whole mount immunostaining of the endothelial marker CD31 (PECAM). C and D, zoom-in of PECAM immunostained wild type and double deficient embryonic heads, respectively. E–H, immunodetection via CD31 of blood vessels in head (E and F) and trunk (G and H) in E9.5 mouse embryonic transversal sections. Wild type endothelial tubes are readily detected (E and G), unlike double deficient counterparts (F and H), which are less abundant and show weaker positive staining (arrows). Scale bars = 1000 µm (A and B), 500 µm (C and D), and 50 µm (E–H).
Figure 3.
PRKCD and PRKCE are both expressed in mouse embryonic endothelium at E9.5.
A, whole mount LacZ staining suggests weak expression of PRKCD in few endothelial cells. B, PRKCE appears broadly expressed in endothelium in whole mount LacZ stained embryos. C and D, LacZ and eosin stained sections confirmed the endothelial expression pattern observed for PRKCD and PRKCE in whole embryos, respectively. E, western blotting shows that wild type mouse embryonic endothelial cells (MEECs) express PRKCD. Prkcd−/− MEECs were used as a negative control. F, CD31 positive signal in virtually all MEECs confirms the purity of WT and Prkcd−/− cell populations. Mouse embryonic fibroblasts (MEFs) were used as a negative control. Expression of PRKCE in MEECs was previously demonstrated in a similar manner [20]. Scale bars = 500 µm (A and B), 25 µm (C and D) and 200 µm (F).
Figure 4.
Impaired cell organization in cultured Prkcd and Prkce double deficient murine allantois.
A, in vitro culture of wild type allantois led to the formation of a vascular plexus. B, Prkcd and Prkce double deficient allantois in culture displayed impaired formation of a vascular network. C, blood vessel formation in cultured Prkcd and Prkce double heterozygous allantois does not significantly differ from wild type counterparts. D, quantifications with the software angiotool and student t-test analyses of 3 allantoises per genotype show significantly decreased average value for total number of junctions in Prkcd and Prkce double deficient allantoises versus their wild type counterparts. Scale bars = 500 µm. * and n.s. = Difference between average values is (p≤0.01) and is not statistically significant, respectively.
Figure 5.
Impaired vessel structure in Prkcd and Prkce double deficient embryos.
A–D, via transmission electron microscopy, wild type (A and B) sagittal sections of the E9.5 embryonic head showed endothelial cells with endothelial adherens junctions (arrows) as well as contact with surrounding mesenchymal cells (single arrowhead). However, Prkcd and Prkce double deficient sections (C and D) showed dilated vessels, and endothelial cells with decreased endothelial specific adherent junctions and decreased contact with surrounding cells. Hemidesmosomes (double arrowheads) were equally detectable in both wild type and Prkcd and Prkce double deficient embryo sections. E and F, comparison between hematoxylin & eosin stained wild type (E) versus Prkcd and Prkce double deficient (F) transversal embryo sections at E9.5 showed reduced size of dorsal aorta (arrows) in the absence of Prkcd and Prkce. G and H, immunofluorescent analyses showed that VE-cadherin is readily expressed in wild type dorsal aorta whereas its expression levels appeared reduced in double deficient sections on these embryos. E, endothelial cell; M, mesenchymal cell. Scale bars = 1 µm (A and C), 0.1 µm (B and D), 100 µm (E and F), and 50 µm (G and H).
Figure 6.
Undetectable vascular smooth muscle cell differentiation in Prkcd and Prkce double deficient dorsal aorta.
A and B, immunodetection of α-SMA in whole embryos resulted in positive staining of dorsal aorta in wild type (A) but not Prkcd and Prkce double deficient (B) embryos at E9.5. C and D, wild type sections showed positive staining for α-SMA at the dorsal aorta (C and D) and heart (C) at E9.5. E and F, Prkcd and Prkce double deficient sections showed no immunosignal over the background at dorsal aorta at E9.5. Arrows indicate dorsal aorta. DA, dorsal aorta; U, umbilical cord. Scale bars = 250 µm (A and B), 100 µm (C and E), and 50 µm (D and F).
Figure 7.
Decreased mRNA levels of vasculo and angiogenic genes in the absence of PRKCD and PRKCE.
Significant downregulation of several vasculo- angiogenic genes in double Prkcd and Prkce deficient embryos suggests an important role of PRKCD and PRKCE in the formation of the vasculature. * = p<0.05.