Article Figures & Tables
Figures
- Fig. 1.
Multiple endothelial cells occupy the tip position in blood vessel sprouts. (A) Common inferred endothelial cell topology in sprouts (left), and the observed topology of overlapping cells (right). (B) Sprout consisting of HUVECs infected with either GFP-expressing lentivirus or tdTomato-expressing lentivirus and mixed prior to assay. (C) Schematic of percentage cell overlap measurements. (D) Frequency of topologies with indicated percentage of cell overlap in HUVEC sprouting assay. (E) Left, mosaic vessel sprout from P6 mouse retina of Ub-CreER×tdTomatoflox/+ cross, stained with isolectin (green, endothelial marker) and DAPI (blue, nuclear marker). Right, orthogonal views and diagrams show overlap of endothelial cells near the tip. (F) Frequency of topologies with indicated percentage of tip cell overlap in mouse retina.
- Fig. 2.
Endothelial cell dynamics lead to tip cell overlap. (A) Time-lapse imaging of HUVEC sprout. Red and green arrows, tips of tdTomato- and GFP-expressing cells, respectively. (B) Frequency of topologies with indicated percentage of tip cell overlap in live-imaged HUVEC sprouts. (C) Mosaic HUVEC sprout with individual cells expressing either LifeAct-GFP or LifeAct-RFP. (D) Percentage of mosaic sprouts with filopodia of one color (red) or two colors (blue), n=20 sprouts. (E,G) Vessel sprouts from P6 mouse retina of Ub-CreER×tdTomatoflox/+ cross, stained with isolectin (white, endothelial marker). Asterisks, tomato-positive filopodia; arrowheads, tomato-negative filopodia. (E) Sprout with labeled and unlabeled cells at tip. (F) Percentage of sprouts with only red filopodia (red) or red and negative filopodia (blue), n=20 sprouts. (G) Sprout with only labeled cells at tip. (H) Percentage of filopodia that are tomato-positive (red) or tomato-negative (blue) in sprouts of indicated compositions. Statistical comparisons using χ2. *P≤0.001.
- Fig. 3.
Polarity markers localize in sprout tips and define a longitudinal apical border. (A) Lumenized HUVEC sprout stained for collagen IV (blue, basal), podocalyxin (PODXL, red, apical), and DAPI (white, nuclear). Left, en face view of compressed z-stacks; right, orthogonal z-planes through horizontal line after deconvolution analysis. (B) Lumenized retinal vein behind vascular front stained for isolectin (green, endothelial), PODXL (red, apical), β1 integrin (blue, basal) and DAPI (white, nuclear). Left, en face view of compressed z-stacks; right, orthogonal z-planes through horizontal line after deconvolution analysis. (C) Line-scan of marker intensity from the luminal space of the vessel outwards; isolectin (green, endothelial), PODXL (red, apical), β1 integrin (blue, basal), dextran perfusion (D488, purple, patent lumen), and DAPI (black, nuclear). Black arrows, intensity peaks corresponding to apical and basal areas. (D) Average ratio of basal to apical pixel intensity for PODXL (n=26), collagen IV (n=12) and β1 integrin (n=200), normalized to isolectin. Statistical comparisons versus Isolectin by Student's t-test±s.e.m. *P≤0.05. (E) Unlumenized HUVEC sprout tip, stained with β1 integrin (blue, basal), PODXL (red, apical) and DAPI (white, nuclear). Note polarization near the sprout tip. Left, en face view of compressed z-stacks; right, orthogonal z-planes through horizontal line after deconvolution analysis. (F) Retinal sprout tip stained with PODXL (red, apical), isolectin (green, endothelial) and DAPI (white, nuclear). Left, en face view of compressed z-stacks; right, orthogonal z-planes through horizontal lines after deconvolution analysis. Note PODXL localization at the longitudinal cell-cell border. (G) Retinal sprout tip stained with PODXL (red, apical), Isolectin (green, endothelial) and β1 integrin (blue, basal). Arrow, lateral cell-cell border; arrowhead, longitudinal cell-cell border. (H) Line scan of marker intensity at the indicated borders. Black arrows, intensity peaks for isolectin. (I) Relative PODXL intensity in longitudinal versus lateral borders in retinal sprouts. Error bars±s.e.m., Student's t-test. *P≤0.05. (J) Retinal sprout perfused with FITC-D488 (green), then stained with isolectin (white, endothelial) and DAPI (blue, nuclear). Note perfusion along longitudinal border. (A,B,E-G) Diagrams show approximate marker localization. Scale bars: 10 µm in A,B,E,F,J; 5 µm in G.
- Fig. 4.
Loss of aPKCζ disrupts endothelial cell overlap and lumen formation. (A,C,E) Sprouts of indicated genotypes stained for isolectin (green, endothelial cells), DAPI (blue, nuclear), and PODXL (red, apical) or D488 (red, perfusion). (B) Relative amount of nuclear overlap in sprout tips of indicated genotypes. Statistical comparisons using χ2. **P≤0.01. (D,F) Distance from sprout tip to PODXL (D) or to D488 (F). Error bars±s.e.m., Student's t-test. *P≤0.05; **P≤0.01. (G) Model showing proposed sprout tip topology in different genetic backgrounds, and proposed relationship between topology, polarization and lumen formation.
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