Figure 1
Figure 1. mDll4 overexpression causes major defects in the developing vascular system. (A) pZ/EG-mDll4 transgenesis vector and result of Cre recombination. (B) LacZ staining of a ZEG-Dll4 embryo at embryonic day 8.0 (E8.0). (C) EGFP expression in the double transgenic (DT) embryos at E8.5. (D) Haemorrhaging in the DT embryos at E9.0. Whole-mount PECAM1 immunostaining of E9.0 DT (E) and control (F) embryos. (F) DT embryo at E9.0 showing a hypertrophied dorsal aorta (red arrow), ramified ACV (green arrow), and an unbranched vascular plexus in the head region (blue arrow), relative to the control embryo (E). PECAM1 immunostaining of vitelline membranes from WT (G) and DT embryos (H) show a blockage in the angiogenic remodeling of the vitelline vasculature of mutant embryos. (I,J) Serial sections of an E9.5 WT embryo demonstrating the region where the ACV (green arrow) connects to the sinus venosus (blue arrow; K-M) Serial sections of a E9.5 DT embryo (anterior-posterior) showing a fusion between the aorta (red arrow) and the ACV (green arrow) just before its connection to the sinus venosus (blue arrow). In panel K the ACV consists of a plexus of small capillaries (green arrow) that join to form a single vessel with a large lumen just before its fusion with the dorsal aorta (M). Microangiography with India Ink injection confirmed the existence of functional connections between the DA and the ACV of DT embryos (O), with ink flowing directly from the aortae (red arrow) to the sinus venosus (blue arrow), in contrast to the regular flow of the control embryos (N). Endothelial-specific overexpression of mDll4 (Dll4e) causes the same vascular defects as observed in DT embryos. (P) Immunofluorescence with anti-PECAM1 and anti-Dll4 antibodies on WT embryo at E9.5. Immunofluorescence with anti-PECAM1 and anti-Dll4 antibodies on DT embryo (Q,R) and Dll4e embryos at E9.5 (S,T), confirming that Dll4 is ubiquitously expressed in DT embryos and that it is pan-endothelial in Dll4e embryos, while WT embryos show only Dll4 expression on the dorsal aortae (DA).

mDll4 overexpression causes major defects in the developing vascular system. (A) pZ/EG-mDll4 transgenesis vector and result of Cre recombination. (B) LacZ staining of a ZEG-Dll4 embryo at embryonic day 8.0 (E8.0). (C) EGFP expression in the double transgenic (DT) embryos at E8.5. (D) Haemorrhaging in the DT embryos at E9.0. Whole-mount PECAM1 immunostaining of E9.0 DT (E) and control (F) embryos. (F) DT embryo at E9.0 showing a hypertrophied dorsal aorta (red arrow), ramified ACV (green arrow), and an unbranched vascular plexus in the head region (blue arrow), relative to the control embryo (E). PECAM1 immunostaining of vitelline membranes from WT (G) and DT embryos (H) show a blockage in the angiogenic remodeling of the vitelline vasculature of mutant embryos. (I,J) Serial sections of an E9.5 WT embryo demonstrating the region where the ACV (green arrow) connects to the sinus venosus (blue arrow; K-M) Serial sections of a E9.5 DT embryo (anterior-posterior) showing a fusion between the aorta (red arrow) and the ACV (green arrow) just before its connection to the sinus venosus (blue arrow). In panel K the ACV consists of a plexus of small capillaries (green arrow) that join to form a single vessel with a large lumen just before its fusion with the dorsal aorta (M). Microangiography with India Ink injection confirmed the existence of functional connections between the DA and the ACV of DT embryos (O), with ink flowing directly from the aortae (red arrow) to the sinus venosus (blue arrow), in contrast to the regular flow of the control embryos (N). Endothelial-specific overexpression of mDll4 (Dll4e) causes the same vascular defects as observed in DT embryos. (P) Immunofluorescence with anti-PECAM1 and anti-Dll4 antibodies on WT embryo at E9.5. Immunofluorescence with anti-PECAM1 and anti-Dll4 antibodies on DT embryo (Q,R) and Dll4e embryos at E9.5 (S,T), confirming that Dll4 is ubiquitously expressed in DT embryos and that it is pan-endothelial in Dll4e embryos, while WT embryos show only Dll4 expression on the dorsal aortae (DA).

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