Spatially restricted patterning cues provided by heparin-binding VEGF-A control blood vessel branching morphogenesis

Abstract

Branching morphogenesis in the mammalian lung and Drosophila trachea relies on the precise localization of secreted modulators of epithelial growth to select branch sites and direct branch elongation, but the intercellular signals that control blood vessel branching have not been previously identified. We found that VEGF(120/120) mouse embryos, engineered to express solely an isoform of VEGF-A that lacks heparin-binding, and therefore extracellular matrix interaction domains, exhibited a specific decrease in capillary branch formation. This defect was not caused by isoform-specific differences in stimulating endothelial cell proliferation or by impaired isoform-specific signaling through the Nrp1 receptor. Rather, changes in the extracellular localization of VEGF-A in heparin-binding mutant embryos resulted in an altered distribution of endothelial cells within the growing vasculature. Instead of being recruited into additional branches, nascent endothelial cells were preferentially integrated within existing vessels to increase lumen caliber. The disruption of the normal VEGF-A concentration gradient also impaired the directed extension of endothelial cell filopodia, suggesting that heparin-binding VEGF-A isoforms normally provide spatially restricted stimulatory cues that polarize and thereby guide sprouting endothelial cells to initiate vascular branch formation. Consistent with this idea, we found opposing defects in embryos harboring only a heparin-binding isoform of VEGF-A, including excess endothelial filopodia and abnormally thin vessel branches in ectopic sites. We conclude that differential VEGF-A isoform localization in the extracellular space provides a control point for regulating vascular branching pattern.

Figure 1

Loss of heparin-binding VEGF-A reduces vascular branching complexity during embryogenesis. (A) VEGF-A isoforms in the mouse; the isoforms differ by the absence or presence of heparin-binding domains encoded by exons 6 and 7 (highlighted in yellow); the VEGF120 isoform (asterisk) is the only isoform expressed when exons 6 and 7 are ablated. (B) RT-PCR of cDNA derived from 12.5-dpc hindbrains (wt/wt, wt/120, and 120/120) using oligonucleotides specific for VEGF-A or GAPDH; cloned cDNAs (pBS-120, pBS-164, and pBS-188) were used as controls. (C,D) Whole-mount immunohistochemistry of wt/wt (C) and 120/120 (D) littermate embryos at 11.25 dpc using an anti-PECAM mAb shows that loss of heparin-binding VEGF-A does not ablate vascularization during embryogenesis. (E–H) Vascular networks in the limb at 12.5 dpc (E,F) and 13.5 dpc (G,H). (I–L) Vascular networks in the hindbrain at 11.25 dpc (I,J) and in the posterior lateral hindbrain at 13.5 dpc (K,L). (M–P) Vascular networks in the stomach at 11.25 dpc (M,N) and 12.5 dpc (O,P). Blood vessels in stage matched wt/wt (C,E,G,I,K,M,O) and 120/120 (D,F,H,J,L,N,P) littermates were visualized by immunohistochemistry using an antibody to PECAM (C–F,I–P) or a Tie2LacZ reporter (G,H). Examples of vessel abnormalities are highlighted. Stretches devoid of branchpoints are labeled with black arrowheads; regions severely impaired in branching are indicated with Δ; vessel branches with coiling ends are labeled with black arrows; and a large vessel tuft is with a star. Bars: C,D, 1 mm; E–H, 500 μm; I,J, 500 μm; K,L, 100 μm; M,N, 100 μm; O,P, 200 μm.

Figure 2

The balanced expression of heparin-binding VEGF-A versus VEGF120 controls microvessel branching and vessel caliber. (A) Schematic representation of hindbrain vascularization between 10.0 (1) and 10.5 (4) dpc; between 9.5 and 10.0 dpc, the perineural vascular plexus in the pial membrane begins to extend sprouts into the neural tube (1), which grow perpendicularly toward the ventricular zone (2), where they branch out to form the subventricular vascular plexus (3,4). (B,C) Microvessel appearance on the pial and ventricular sides of a flat-mounted 12.5-dpc hindbrain; the midline region is indicated with an asterisk; the pial side of the hindbrain with P, the ventricular side with V. (D–F) Visualization of vascular networks in representative 500-μm² areas of the 13.5-dpc midbrain of wt/wt (D), wt/120 (E), and 120/120 (F) littermates; blood vessels were visualized with a Tie2LacZ reporter. (G) Number of microvessel branch points per 500-μm² area in the subventricular zone of the embryonic central nervous system in wt/wt (black), wt/120 (red), and 120/120 (green) littermates; n = 3 for each specimen; 10.75-dpc hindbrain (1); 11.25-dpc hindbrain (2); 13.5-dpc hindbrain (3); 11.25 spinal cord (4); and 13.5-dpc midbrain (5). (H,I) Ultrastructure of microvessels in wt/wt (H) and 120/120 (I) hindbrains of littermates at 10.75 dpc, visualized by transmission electron microscopy (TEM); endothelial nuclei are indicated with red stars. (J) Number of endothelial cell nuclei per capillary cross section in the hindbrain in wt/wt (n = 96) and 120/120 (n = 106) littermates at 10.75 dpc (P ≤ 0.0001, Mann-Whitney test). (K,L) Endothelial cell proliferation in hindbrain vessel cross sections of wt/wt (K) and 120/120 (L) littermates at 11.25 dpc; PECAM-positive vessels are shown in green, BrdU-positive endothelial nuclei (white stars) in red. (M) HUVEC proliferation assay; VEGF-A (VEGF121 or VEGF165) concentration: 0.5 ng/mL (1) , 2 ng/mL (2) , 10 ng/mL (3) , 20 ng/mL (4); and basic fibroblast growth factor concentration 3 ng/mL (5). Bars: B,C, 500 μm; D–F, 100 μm; K,L, 5 μm. Magnification: H,I, 5500×.

Figure 3

Altered distribution of secreted VEGF-A protein in the absence of heparin-binding isoforms. (A) VEGF-A gene expression near the hindbrain midline at 10.5 dpc, monitored with a VEGF-A lacZ reporter. (B) At 10.5 dpc, extracellular VEGF-A protein (red) concentrates near the midline, and the PECAM-positive microvessel network (green) is closing in on the VEGF-positive area. (C,D) Comparison of VEGF-A protein distribution in the midline region of wt/wt (C) and 120/120 (D) hindbrains at 10.5 dpc. (E,F) Pseudo-coloring of C,D to highlight regions of highest (red) and lowest (blue) staining intensity. The midline is labeled with an asterisk. Bar, 50 μm.

Figure 4

Impaired filopodia extension at the leading front of growing microvessel networks in the absence of heparin-binding VEGF-A. (A–H) PECAM-positive microvessels with laterally (arrowheads) and perpendicularly (arrows) extending filopodia in wt/wt (A,C,E,F) and 120/120 (B,D,G,H) hindbrains at 10.75 (A,B) and 11.25 (C–H) dpc; the midline is situated off the left side of the image in A and B and is indicated with a red line in C and D; examples of fusing microvessels are indicated with open arrowheads; examples of dilated or bulbous vessel ends in the 120/120 hindbrains are indicated with stars; examples of abnormally localized perpendicular filopodia in D are circled; and the corresponding region devoid of filopodia in C is indicated with a hash. (E,F) The lateral (E) and perpendicular (F) filopodial extensions in the areas boxed in C, magnified 2.5×. (G,H) The lateral (G) and perpendicular (H) filopodial extensions in the areas boxed in D, magnified 2.5×. Bars: A,B, 10 μm; C,D, 20 μm.

Figure 5

Filopodia extension from retinal cells. (A) Endothelial cells at the front of the growing vascular network (green) extend numerous filopodia into their surrounds and toward each other (arrowheads). (B,C) Endothelial filopodia (green, A) are enriched in actin (red, B); regions of overlapping staining appear yellow (C). (D–H) Endothelial cells at the front of the growing vascular network (green, D) track GFAP-positive astrocytes (red, E); some regions in which endothelial filopodia follow astrocyte processes are indicated with arrowheads (D–F). (G,H) An area shown in D and F, in which endothelial filopodia (G) follow astrocyte processes (H), magnified 2×; an undulating filopodium that has not lined up with astrocyte processes is indicated with an arrow (G,H). (A–H) Isolectin B4-positive blood vessels are shown in green, GFAP-positive astrocytes in red; isolectin B4-positive microglial cells in D, F, G, and H are labeled with stars. (I) Endothelial cells at the front of the growing vascular network extend filopodia (arrowheads) toward VEGF-A-expressing astrocytes (asterisks); isolectin B4-positive blood vessels are shown in brown; VEGF-A mRNA expression is shown in purple. Bars: A–C, 8.5 μm; D–F, 20 μm; G,H, 10 μm; I, 25 μm.

Figure 6

Filopodia extension toward VEGF-A deposits is impaired in the absence of heparin-binding isoforms. (A–D) Distribution of extracellular VEGF-A protein near the midline region of an 11.25-dpc wild type (A,C) and 120/120 (B,D) hindbrain. (A,B) Highest VEGF-A staining intensities are shown in red, lowest in blue; the shape of the VEGF-A gradient is shown at the bottom of the two panels. (C,D) PECAM-positive microvessels (green) extend many new branches (junctions labeled by white stars) toward the midline region in wt/wt (C), but not in 120/120 (D) hindbrains, in which, instead, the vessel network often terminates in closed loops. The microvessels closest to the midline extend multiple filopodia toward extracellular VEGF-A patches (red) in wt/wt hindbrains (C), but make only few filopodia in 120/120 hindbrains (D). (E) The area boxed in C, magnified 5×. A microvessel sprout extends filopodia (green) to contact VEGF-A patches (red); regions of overlap are indicated in yellow. (F,G) PECAM-positive vessel tips (green) near the midline region in wt/wt (F) and 120/120 (G) hindbrains; BrdU-positive (red) proliferating neuronal nuclei (in F,G) or double-positive endothelial nuclei (in G; open arrows). Some filopodia extending from the wild-type tip cell are indicated with arrowheads (F). The midline position in all panels is labeled with an asterisk. Bars: A–D, 20 μm; F,G, 100 μm.

Figure 7

VEGF-A isoform and Nrp-1 mutants exhibit distinct embryonic vessel defects. VEGF188 promotes ectopic filopodia extension (A,B) and excess branching (C,D) during brain vascularization. Blood vessels in the left wt/wt (A,C) or right 188/188 (B,D) hindbrain are shown at 10.75 (A,B) and 13.5 (C,D) dpc. The direction of filopodia extension is indicated with arrows in A,B. Clusters of perpendicularly extending filopodia near the midline are labeled with stars in B. Vessels near the midline (indicated with an asterisk) are labeled with black arrowheads in C and D; examples of ectopic branching with single arrows in D; and long thin vessel stretches with a double arrow in D. (E,F) Ectopic expression of the VEGF188 isoform (E) rescues vascular defects in hindbrains expressing VEGF120, but lacking VEGF164 (F). (G,H) Vascular networks covering the somites of littermate embryos producing (G) or lacking (H) heparin-binding VEGF-A at 10.5 dpc; an exemplary vessel stretch devoid of branch points is labeled with black arrowheads. (I,J) Vascular networks covering the somites of littermate embryos producing (I) or lacking (J) Nrp-1 at 10.5 dpc. (K) Working model for the role of VEGF-A isoforms in vascular patterning. The balanced production of soluble VEGF120 versus heparin-binding VEGF-A (HB-VEGF) determines the shape of the VEGF-A gradient around producing cells and thereby controls the decision of nearby vessels to invest growth into new branches (right) or luminal caliber (left). Accordingly, vessel size and branching pattern are dramatically different in the 120/120 and 188/188 midbrain at 13.5 dpc. Blood vessels were visualized by immunohistochemistry using an antibody to PECAM. Bars: A,B, 10 μm; C,D, 200 μm; E,F, 100 μm; G,H, 200 μm; I,J, 200 μm; K, 25 μm.