Fig. 2.
Phenotypic analysis of the GNAQ R183Q Tie2-Cre endothelial mutants. (A) RT-PCR showing the expression of GNAQ R183Q in the E14.5 skin after doxycycline treatment in the tet-GNAQ R183Q; Rosa26-LSL-rtTA; Tie2-Cre triple transgenic mouse embryos. Gapdh was used as a control. (B) qRT-PCR showing the relative transgene expression level induced by doxycycline treatment. (C) Expression of GNAQ R183Q in endothelial cells with Tie2-Cre causes several blood vessel malformations in mouse embryos. Doxycycline treatment started at E12.5 and embryos were harvested at E15.5. (D) H&E staining on sections of E15.5 embryos showing blood-filled regions in multiple anatomical areas, including the craniofacial region, body trunk, liver and skin of mutant embryos (arrows). Scale bars: 1 mm (left); 0.5 mm (magnifications, right). (E) Whole-mount immunostaining of the E15.5 back skin with PECAM1 antibody. Although back skin from control embryos showed clear vascular networks (a), GNAQ R183Q mutant skin displayed many dilated vessels filled with blood cells (b). a’ and b’ show converted black and white images of a and b, respectively. Scale bar: 100 μm. Bar graphs showing the quantification of vein and capillary diameters in the control and mutant skin (three embryos per genotype). **P<0.01; ns, not significant. (F) Sagittal sections of E14.5 back skin stained with ZO-1 and collagen IV (green). PECAM1 (magenta) was used to highlight the endothelial cells. DAPI was used to highlight nuclei. Scale bar: 50 μm. Bar graphs showing the quantification of staining intensity of ZO-1 and collagen IV in endothelial cells (n=20 cells per genotype): no significant differences in the expression levels of these markers were found. Data are mean±s.e.m.