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First published online 11 July 2007
doi: 10.1242/dev.002576

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EGFL7 regulates the collective migration of endothelial cells by restricting their spatial distribution

¹ Tumor Biology and Angiogenesis Department, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
² Lexicon Genetics Inc., 8800 Technology Forest Place, The Woodlands, TX 77381-1160, USA.
³ Cell Microscopy Center, Department of Cell Biology, University Medical Center Utrecht and Institute for Biomembranes, 3584CX, Utrecht, Netherlands.
⁴ Translational Oncology Department, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
⁵ Pathology Department, Genentech Inc., 1 DNA Way, South San Francisco, CA 94080, USA.

View larger version (116K): [in this window] [in a new window]   Fig. 1. General phenotypes of the Egfl7-knockout mouse. (A,B) Egfl7+/- (A) and Egfl7-/- (B) E14.5 littermates. Arrowhead indicates systemic oedema in the Egfl7-/- mouse. (C,D) CD31 [also known as PECAM1; brown, endothelial cell (EC) marker] and Hematoxylin (blue, H, nuclear marker) staining on transverse sections of the neck dermis from Egfl7+/+ (C) and Egfl7-/- (D) E14.5 littermates. Red bars indicate the thickness of the epidermis. Increased thickness in the Egfl7-/- tissue is a hallmark of oedema. (E) Body weights of multiple pairs of Egfl7+/+ and Egfl7-/- littermates at the indicated ages. Adults are all between 4-7 months. P values are calculated with unpaired, two-tailed t-test with 95% confidence interval (the same method is used for all P values unless specified otherwise). (F-M) Hypoxyprobe (brown) and Hematoxylin (blue) staining on sections of adult organs from Egfl7+/+ and Egfl7-/- littermates. Genotype and organ names are indicated. Arrowheads indicate example hypoxic cells. The actual size represented by the width of the panel: 8.5 mm (A,B); 0.87 mm (C,D); 430 µm (F-M).

View larger version (77K): [in this window] [in a new window]   Fig. 2. Loss of Egfl7 results in delay of vascularization. (A-D) CD31 wholemount staining on embryonic mouse hearts from Egfl7+/+ (A,B) and Egfl7-/- (C,D) littermates at E12.5 (A,C, dorsal view of the left ventricles) and E14.5 (B,D, lateral view of the left ventricles, dorsal is to the right). Asterisks, areas without coronary vasculature; weak signal is from the underlying endocardium. (E,F) CD31 wholemount staining on embryonic heads from Egfl7+/+ (E) and Egfl7-/- (F) littermates at E10.5. Open arrowheads, major cranial vessels. (G) Ventral coronary vascular coverage of multiple pairs of Egfl7+/+ and Egfl7-/- littermates at E12.5 and E14.5. Horizontal bars represent means. Because of large inter-litter variation at E12.5, littermate relationship is specified with numbers in the graph. Significant difference is marked with an asterisk next to each P value. (H) Major cranial vessel counts of multiple pairs of Egfl7+/+ and Egfl7-/- E10.5 littermates from two knockout lines. ins, insertional knockout line; hom, homologous recombination knockout line. (I-N) Isolectin B4 (EC marker) staining on flatmount neonatal retinas from Egfl7+/+ (I-K) and Egfl7-/- (L-N) littermates at P2 (I,L), P5 (J,M) and P8 (K,N). (O,P) Retinal vascular coverage (O) and size (P) of multiple pairs of Egfl7+/+ and Egfl7-/- newborn littermates at the indicated ages. Solid red arrowheads indicate example aberrant EC clusters; arrows indicate sprouts at the retinal vascular migration front. The actual size represented by the width of the panel: 0.9 mm (A,C); 1.4 mm (B,D); 3.6 mm (E,F); 3 mm (I,J,L,M); 4.7 mm (K,N); K and N are assembled from overlapping 4x images.

View larger version (118K): [in this window] [in a new window]   Fig. 3. Vascular phenotypes in the adult Egfl7-knockout mouse. (A-H) Fluorescent angiography (A-D) and CD31 fluorescent staining (E-H) in the retinas of multiple pairs of Egfl7+/+ (A,B,E,F) and Egfl7-/- (C,D,G,H) littermates. (I-L) High-magnification views of aberrant structures in the Egfl7-/- retinas. L is a z-section showing two juxtaposed vessels. Blue, DAPI (I-L, nuclear marker); green, CD31 (I-L); red, collagen IV (K,L only, vascular basement membrane marker). (M-P) CD31 staining on 50 µm-thick sections of peritonea (M,N) and kidney (O,P) from Egfl7+/+ (M,O) and Egfl7-/- (N,P) littermates. Arrowhead, tortuous vessels; red arrows, knots formed by multiple vessels; yellow arrows, vessels of similar diameter running close to each other; asterisks, vascular lumen. The actual size represented by the width of the panel: 1.2 mm (A-D); 330 µm (E-H); 88 µm (I-K); 25 µm (L); 275 µm (M-P).

View larger version (68K): [in this window] [in a new window]   Fig. 4. EGFL7 regulates the collective migration of ECs, but not the movement of individual ECs. (A,B) Live imaging at days 4-7 of cultured mouse aortic rings isolated from Egfl7+/+ (A) and Egfl7-/- (B) littermates. Colored arrowheads track individual sprouts over time. (C,D) Quantification of sprout length (C) and sprout extension velocity (D) on cultured aortic rings isolated from the Egfl7+/+ (blue bars) and Egfl7-/- (red bars) littermates. (E) Transwell migration assay performed on fibronectin- or EGFL7-coated inserts using ECs isolated from four pairs Egfl7+/+ and Egfl7-/- littermates. Migration efficiency was compared between Egfl7-/- (ko) and Egfl7+/+ (wt) samples and the ratios plotted. (F) Migration velocity of individual HUVECs on fibronectin (FN), EGFL7, or combined substrate. The actual size represented by the width of the panel: 1.09 mm (A); 0.73 mm (B).

View larger version (72K): [in this window] [in a new window]   Fig. 5. EGFL7 restricts the spatial distribution of migrating ECs. (A-F) Confocal images of vascular sprouts in P5 retinas from Egfl7+/+ (A-C) and Egfl7-/- (D-F) littermates stained with Isolectin B4 (white in A,D; green in B,C,E,F) and propidium iodide (PI) to indicate nuclei (red in B,C,E-F). C and F are z-sections of the retinal vascular migration front. These images were taken from areas similar to those indicated by the arrows and arrowheads in Fig. 2J,M. Crosses mark stalk cell nuclei; asterisks mark tip cell nuclei. (G,H) Tip (G) and stalk (H) cell counts in multiple 40 µm-long segments of sprouts from both genotypes. (I-L) Aortic rings isolated from Egfl7+/+ (I,J) and Egfl7-/- (K,L) littermates stained for CD31 (green) and with DAPI (red, pseudo color). White dashed lines outline the edges of the aortic rings. The CD31- DAPI+ signals in J and L are nuclei of fibroblasts and smooth muscle cells that have migrated away from the aortic rings. (M) EC counts in multiple 50 µm-long segments of sprouts from both genotypes. The actual size represented by the width of the panel: 230 µm (A,D); 76 µm (B,E); 298 µm (C,F); 1.3 mm (I,K); 294 µm (J-L).

View larger version (83K): [in this window] [in a new window]   Fig. 6. Aberrant localization of basement membrane marker in the Egfl7-/- vessels. Neonatal mouse retinas (A-F) or aortic rings (G-L) isolated from the Egfl7+/+ (A-C,G-I) and Egfl7-/- (D-F,J-L) littermates stained for collagen IV (green), CD31 (red) and with DAPI (blue). Representative single-frame confocal images (A,B,D,E,G,H,J,K) and optical z-sections (C,F,I,L) are shown. Arrowheads, vascular sprouts similar to those shown at higher magnification in the panels beneath; arrows, mislocalized collagen IV between ECs within a single sprout; asterisks, individual ECs. In C and F, two separate z-sections are shown in each panel. The actual size represented by the width of the panel: 732 µm (A,D); 108 µm (B,E); 36 µm (C,F); 366 µm (G,J); 72 µm (H,I,K,L).

View larger version (64K): [in this window] [in a new window]   Fig. 7. EGFL7 is associated with the interstitial ECM. (A) Anti-HA western blot on different fractions of chicken embryonic fibroblasts expressing GFP or HA-tagged human EGFL7 (top panel), and Coomassie Blue staining of the same cell fractions prepared from an equal number of cells (bottom panel). (B-G) The following cells were cultured for 3 days and then double stained for EGFL7 (green) and with DAPI (blue) in the absence of detergent: HUVECs (dark blue ovals in diagram) and human dermal fibroblasts (light blue ovals) in mixed culture (B); HUVECs alone (C); fibroblasts alone (E); HUVECs in the bottom well with fibroblasts in an insert (D); or HUVEC alone on fibronectin (F) or laminin (G). (H-M) Confocal images of aortic ring sprouts from Egfl7+/+ (H-K) or Egfl7-/- (L,M) littermates stained for fibronectin (blue), EGFL7 (red), CD31 (green) and with DAPI (gray). H-J show a segment of sprout with stalk cells; K shows tip cells; L,M show a segment of sprouts with both tip and stalk cells. The actual size represented by the width of the panel: 650 µm (B-G); 80 µm (H-J); 54 µm (K); 100 µm (L,M).

View larger version (41K): [in this window] [in a new window]   Fig. 8. A model describing how EGFL7 regulates sprout morphology and motility.

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