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Fig. 1 Partial VE-Cadherin Blockade in Organotypic Culture and VE-Cadherin Knockdown in Zebrafish Stimulate Sprouting
(A) VE-cadherin accumulation at junctions in established tubules. HUVEC-EGFP cocultured with HDF were fixed 7 days (migratory) or 12 days (established) after coculture and stained for VE-cadherin. Middle panels show magnifications of insets in left panels. Note the continuous distribution of VE-cadherin in established tubules. Scale bar represents 50 μm.
(B) Partial VE-cadherin blockade induces sprouting. Established tubules were treated with Cad5 (5 μg/ml, 10 μg/ml, or 50 μg) or control IgG and followed by time lapse microscopy. Video stills are at 12 hr intervals over 48 hr. Closed arrows show sprouting points; open arrows show areas of disassembly. Scale bar represents 50 μm.
(C) Partial VE-cadherin blockade increases tube formation. Established tubules treated with Cad5 (5 μg/ml) or control IgG for 30 hr were visualized by CD31 staining 3 days after the end of treatment. Number of tubules, total length, and number of branch points are represented as mean ± SEM (n = 9 microscopic fields from triplicate wells). Scale bar represents 100 μm.
(D) Increased sprouting of intersegmental vessels in zebrafish embryos with VE-cadherin knockdown. Shown is a lateral view of the trunk region of a representative control morpholino-injected transgenic fli1:EGFP zebrafish embryo (Cont-MO) or embryo injected with VE-cadherin morpholino oligonucleotide (VE-cad MO2) at 30 hpf. Upper panels show bright-field images of whole embryos showing normal development of VE-cad MO embryos. Middle panels show magnifications of top panel insets showing the notochord (nc) and somites (s). Lower panels show fluorescent images of trunk vasculature with three intersegmental vessels. Note the persistent sprouting (arrows) of intersegmental vessels (ISV) in VE-cadherin morphants. DA, dorsal aorta; PCV, posterior cardinal vein. Scale bar represents 50 μm. Histogram shows quantification of branching of ISVs.