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First published online 14 May 2008
doi: 10.1242/dev.016378

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Cell-autonomous requirement for β1 integrin in endothelial cell adhesion, migration and survival during angiogenesis in mice

Timothy R. Carlson^*, Huiqing Hu, Rickmer Braren, Yung Hae Kim and Rong A. Wang

Pacific Vascular Research Laboratory, Division of Vascular Surgery, Department of Surgeryand Department of Anatomy, University of California, San Francisco, CA 94143, USA.

View larger version (112K): [in this window] [in a new window]   Fig. 1. Tie2- and Tie1-Cre mediate efficient deletion of β1 integrins in mouse embryos. Gene deletion analyses of Tie2-Cre (A-D) and Tie1-Cre (E,F) mutants as compared with controls. (A) Genomic PCR analysis of e8.5 embryos demonstrates recombination (rec.) of β1 integrin in embryos carrying Tie2-Cre and two floxed alleles of β1. (B) e8.5 cryosections were stained with anti-CD31 (red), anti-β1 integrin (Ha2/5, green) and DAPI (blue). (C) Collagenase-dissociated e8.5 embryonic cells were plated onto fibronectin and stained with anti-β1 (HMβ1-1, green), anti-β3 integrin (red) and DAPI (blue). Endothelial cell (EC) identity in C was determined by co-staining with anti-CD31 (not shown). (D) Focal contacts in isolated ECs. Bars are means + s.e.m. of two (β1) or three (β3) experiments. **P<0.01 by one sample t-test and *P<0.05 by Student's t-test. (E) Genomic PCR analysis of e10.5 embryos from Tie1-Cre matings. (F) e9.5 cryosections were stained with anti-CD31 (red), anti-β1 integrin (green) and DAPI (blue). Arrows, primary head veins; arrowheads, β1 integrin-negative endothelium; ys, yolk sac blood islands; a-da, anterior dorsal aortae; p-da, posterior dorsal aortae; nt, neural tube; g, gut; ve, visceral endoderm. Also see Fig. 3F for diagram of embryonic structures. Scale bars: 20 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 2. Endothelial deletion of β1 integrins causes EC disorganization leading to cardiovascular defects and lethality at midgestation. Whole-mount anti-CD31 immunostained mouse yolk sacs at the indicated embryonic stages. The left panels of each e9.0 are capillary regions and the right panels are regions apparently undergoing arteriovenous remodeling. Note the disconnected capillaries and overall vascular disorganization in all mutants. Images are representative of at least five control/mutant embryo pairs at each stage. Scale bars: 100 µm for e9.5 and 50 µm for e9.0 and e10.5 Tie2-Cre; 25 µm for Tie1-Cre.

View larger version (73K): [in this window] [in a new window]   Fig. 3. Endocardial cell patterning defects and abnormal cardiac morphogenesis in the absence of β1 integrins. Endothelial staining of Tie2-Cre (B,D,H) and Tie1-Cre (J) mutants as compared with controls (A,C,G,I). (A-D) Whole-mount anti-CD31 immunostained control (A,C) and Tie2-Cre mutant (B,D) embryos at e9.0 (A,B) and e9.5 (C,D). (E) Diagram of an e9.5 mouse embryo. The dashed line indicates the approximate plane of the cross-section shown in F, and of the e9.5 control (G) and Tie2-Cre mutant (H) cryosections stained with anti-CD31 antibodies. The dashed box in F indicates the approximate location of the e10.5 control (I) and Tie1-Cre mutant (J) paraffin sections stained with anti-VE-cadherin antibodies and DAPI. In F, the endocardium (green) is separated from the myocardium (red) by the cardiac jelly (cj). Arrows, endocardium; arrowheads, intersomitic vessels; da, dorsal aorta; h, heart; a, atrium; v, ventricle; m, myocardium; fg, foregut; hg, hindgut. All images are representative of at least four control/mutant embryo pairs. Scale bars: 100 µm in A-D,G,H; 20 µm in I,J.

View larger version (100K): [in this window] [in a new window]   Fig. 4. EC detachment and absence of neural tube invasion upon Tie1-Cre-mediated deletion of β1 integrins at e10.5. (A-B') Control (A) and Tie1-Cre mutant (B) mouse embryos were whole-mount stained with anti-VE-cadherin (red), embedded in paraffin, sectioned and counterstained with DAPI (blue). The dashed white line indicates the boundary between the neural tube (nt) and the mesenchyme. The arrow indicates an area of discontinuous endothelium in the cardinal vein (cv) and the arrowhead indicates an EC apparently undergoing detachment. Asterisks indicate dilated blood vessels. da, dorsal aortae. The cardinal veins in the boxed regions (green dashed lines) in A and B are shown at high magnification in A' and B', respectively. (C,D,F,G) Control (C,F) and Tie1-Cre mutant (D,G) mouse embryo cryosections stained with anti-CD31 (red), DAPI (blue), and anti-fibronectin (green, C,D) or anti-laminin (green, F,G). Arrowheads in D indicate discontinuous endothelium along the vascular basement membrane, and the dashed line encircles an EC within the lumen that has apparently detached. In F and G, the laminin-rich basement membrane (green) indicated by the dashed lines separates the neural tube from the mesenchyme. Note the absence of ECs in the neural tubes in mutants, and that significant laminin surrounds ECs within the control neural tubes. (E,H) Quantification of EC discontinuity in major blood vessels (E) and of EC failure to invade the neural tubes (H). Bars are the mean values + s.e.m. of four control/mutant pairs at e10.5. *P<0.001 by Student's t-test. Scale bars: 20 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 5. EC-autonomous role for β1 integrins in angiogenic remodeling. (A-D) Still images of control (A,C) and Tie2-Cre mutant (B,D) mouse embryonic P-Sp explant cultures at the indicated timepoints. GFP-positive ECs were visualized by a Tie1-GFP transgene. Aberrant EC clusters are evident in Tie2-Cre mutant explants, but not in control explants. The OP9 feeder cells, not visible in the fluorescence images, were a confluent monolayer on top of which the ECs grew out from the P-Sp. The dashed lines indicate the approximate location of the P-Sp explants. See also Movies 1, 2 in the supplementary material. (E-H) Capillary morphogenesis of embryonic Itgb1flox/flox ECs immortalized with polyoma middle T antigen and subsequently infected with adenovirus (Ad), in the absence of any antibodies (E,F) or in the presence of anti-v integrin plus anti-β3 (G) or anti-β1 (H) integrin function-blocking antibodies. Phase-contrast images were captured after 4 hours in culture. Scale bars: 100 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 6. β1 integrins are required for EC adhesion and migration in a matrix-specific manner. Analysis of immortalized embryonic Itgb1flox/flox ECs (A,B) or primary embryonic ECs (C-J). (A) Adhesion of adenovirus (Ad)-infected embryonic Itgb1flox/flox ECs in serum-free medium after 30 minutes. Microplate coating concentrations were: fibronectin (FN), 20 nM; laminin (LM), 25 nM; collagen I (Col I), 55 nM; collagen IV (Col IV), 40 nM; Matrigel (MG), 125 µg/ml; vitronectin (VN), 20 nM. (B) Haptotactic migration of embryonic Itgb1flox/flox ECs in serum-free medium after 4 hours. The ECM that served as the stimulus for migration was coated to the underside of a filter at the following concentrations: FN, 40 nM; LM, 50 nM; MG, 500 µg/ml; VN, 20 nM. (C) Cell spreading after 20 hours of culture. (D) Migration speed measured over 24 hours (fibronectin) or 14 hours (laminin) by timelapse videomicroscopy. (E-H) Representative migration tracks along with a phase-contrast image overlaid with DiI-Ac-LDL uptake fluorescence. Units on migration tracks are pixels. Coating concentrations were: fibronectin, 40 nM; laminin, 15 nM. Focal adhesion formation in control (I) and mutant (J) embryonic ECs on fibronectin as assessed by anti-FAKpY397 (green), anti-CD31 (red) and DAPI (blue) staining. Values in A and B are means + s.d., and in C and D are means + s.e.m. **P<0.01 and *P<0.05 by Student's t-test. All experiments were performed at least twice and representative results are shown. Scale bars: 20 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 7. β1 integrins regulate EC growth through effects on survival rather than proliferation. In vivo (A,B) and in vitro (C-E) effects of β1 deletion on EC growth. The ratios of BrdU/CD31 (A) and TUNEL/CD31 (B) double-positive ECs relative to total ECs were calculated from multiple immunostained cryosections prepared from three (A) or four (B) pairs of control and Tie2-Cre mutant mouse embryos at e9.0, prior to the onset of overt morbidity. Bars in A and B are means + s.e.m., and differences are not statistically significant by Student's t-test (A, n=1200 control and n=965 Tie2-Cre mutant ECs, P=0.625; B, n=1299 control and n=1116 mutant ECs, P=0.36). (C) Growth rate of embryonic Itgb1flox/flox ECs cultured on gelatin (Gel) or vitronectin (VN). (D) Incorporation of BrdU into DNA after 6 hours of culture. (E) Entry of the apoptotic-cell-permeant dye, YO-PRO-1, into proliferating EC cultures. In C-E, data are means + s.d. of replicate measurements, and each experiment was repeated with similar results. **P<0.01 and *P<0.05 by Student's t-test.

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