Figure 1.
CAR deficiency in mouse embryos after heart development causes subcutaneous edema, hemorrhage and lethality.
(A) Western blot analysis of CAR protein levels in E14.5 and E15.5 embryos from F/F;Cre (cKO, n = 3) and littermate F/F control (ctrl, n = 3) animals from two litters after administration of tamoxifen at E12.5. Primary CAR antibody was RP 291. An antibody against calnexin was used as a loading control. (B) Macroscopic images of E16.5 embryos given tamoxifen at E12.5 showing subcutaneous edema and hemorrhage in cKO (n = 12) embryos but not in littermate F/F (n = 18) control embryos (ctrl), which appeared normal. Embryos were from 5 litters. Images were taken with a 4× objective. (C) Histological analysis of transverse sections of E16.5 embryos given tamoxifen at E12.5 showing subcutaneous edema in tamoxifen-treated cKO (n = 3) embryos while the littermate F/F controls (ctrl, n = 3) were normal. Embryos were from 3 litters. Images were taken with a 20× objective. Scale bar = 50 µm.
Table 1.
The proportion of embryos with macroscopic phenotypic changes following tamoxifen administration at E12.5.
Figure 2.
CAR is expressed in lymphatic endothelial cells during mouse development and localizes to cell-cell junctions.
Confocal images of whole-mount skin preparations from wildtype (A, B) (n = 3) and cKO (C) (n = 3) E16.5 embryos stained by immunofluorescence with the antibodies rabbit anti-CAR IG1 (green), hamster anti CD31 (red) and rat anti LYVE-1 (purple) to visualize CAR expression in blood vessels (CD31highLYVE-1neg) and lymphatic vessels (CD31lowLYVE-1pos). Animals were from 3 different litters (A) Low magnification images showing CAR expression in lymphatic vessels but not in blood vessels. Scale bar = 50 µm (B). High-magnification images showing distribution and partial co-localization of CAR with CD31, and LYVE-1 at lymphatic endothelial cell-cell junctions (arrows). Scale bar = 50 µm. (C) Low magnification images showing efficient downregulation of CAR in lymphatic vessels of cKO embryos. Scale bar = 50 µm.
Figure 3.
Lymphatic vessels of CAR deficient mouse embryos are structurally abnormal.
(A, C) Confocal immunofluorescence images of whole-mount skin preparations from cKO (n = 5) and littermate F/F controls (ctrl, n = 4) at E16.5 co-stained with the antibodies rabbit anti-LYVE-1 (green) and hamster anti-CD31 (red) to visualize lymphatic vessels (A) and blood vessels (C), respectively. Embryos were from 2 litters. Scale bar = 50 µm. (B, D) Measure of vessel diameter. Data is presented as means ± SEM with at least four to six mice per group. (A) Immunofluorescence images showing dilated lymphatic vessels in cKO compared to ctrl embryos. (B) Bar graph showing significantly increased average diameter of subcutaneous lymphatic vessels in cKO versus F/F control (ctrl) embryos at E16.5. P<0.001. (C) Immunofluorescence images showing no differences in blood vessels in cKO compared to ctrl embryos. (D) Bar graph showing no significant differences in average diameter of subcutaneous blood vessels in cKO versus F/F control (ctrl) embryos at E16.5. P = 0.76.
Figure 4.
Lymphatic vessels of CAR deficient mouse embryos display endothelial gaps.
(A, B) High-resolution confocal images of whole-mount skin preparations from E16.5 cKO (n = 3) and littermate F/F control (ctrl, n = 3) embryos stained in three independent experiments by immunofluorescence with antibodies to visualize lymphatic vessels (LYVE-1, green) and junctions between lymphatic endothelial cells (CD31/VE-cadherin, red). DAPI was used to visualize nuclei (blue). (A) Immunofluorescence image showing a dilated LYVE-I positive lymphatic vessel in an E16.5 cKO embryo (left). Gaps between endothelial cells are present and even more visible at higher magnification (arrows in right image, which is a magnification of the white box outlined in the left image). Primary antibody was rat anti-LYVE-1. Scale bar = 50 µm. (B) Top panel: High-magnification images of a dilated LYVE-I positive lymphatic vessel from an E16.5 cKO embryo showing the presence of lymphatic endothelial gaps (arrows) at, or close to, cell-cell junctions visualized by co-staining with antibodies against CD31 (red, middle image) or VE-cadherin (red, right image). Bottom panel: no gaps were present in similarly stained F/F littermate controls (ctrl). Primary antibodies were rabbit anti-LYVE-1, hamster anti CD31 and rat anti VE-cadherin. Scale bar = 150 µm. (C) Transmission electron microscopy (TEM) images of subcutaneous lymphatic vessels (L) in E16.5 cKO (n = 4) and littermate F/F control (ctrl, n = 4) embryos. Embryos were from two litters. In ctrl embryos, lymphatic endothelial cells with clearly visible nuclei (N) formed an intact lining around the entire lumen of the lymphatic vessels. In cKO embryos, lymphatic endothelial cell nuclei were smaller and the cells appeared locally damaged (arrow) and did not form an intact lining of the vessel lumen. Regions of the luminal surface were devoid of lymphatic endothelial cells (arrowheads). Scale bars = 10 µm (left and middle images), 5 µm (right image).
Figure 5.
CAR deficiency during mouse development causes incomplete separation of the blood and lymphatic systems.
(A–C) Confocal images of whole-mount skin preparations from E16.5 cKO (n = 3) and littermate F/F control (ctrl, n = 3) embryos stained in three independent experiments by immunofluorescence with rat anti-TER 119 (TER, green) to visualize erythrocytes, and the vascular markers hamster anti-CD31 (red) and rabbit anti-LYVE-1 (purple) to visualize blood vessels (CD31highLYVE-1neg) and lymphatic vessels (CD31lowLYVE-1pos). Scale bar = 50 µm. (A, B) Images showing staining for TER in blood but not in lymphatic vessels in ctrl embryos (A), and staining for TER in dilated lymphatic vessels in cKO embryos (B). Lower panels show z projections of the upper images at levels indicated by the white lines. (C) Images showing leakage of TER-positive erythrocytes from dilated lymphatic vessels in cKO but not in ctrl embryos.