Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2007 Feb 27;104(9):3225-30.
doi: 10.1073/pnas.0611177104. Epub 2007 Feb 12.

The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching

Steven Suchting  1 , Catarina Freitas, Ferdinand le Noble, Rui Benedito, Christiane Bréant, Antonio Duarte, Anne Eichmann
Affiliations
Comparative Study

The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching

Steven Suchting et al. Proc Natl Acad Sci U S A. .

Abstract

Delta-like 4 (Dll4) is a transmembrane ligand for Notch receptors that is expressed in arterial blood vessels and sprouting endothelial cells. Here we show that Dll4 regulates vessel branching during development by inhibiting endothelial tip cell formation. Heterozygous deletion of dll4 or pharmacological inhibition of Notch signaling using gamma-secretase inhibitor revealed a striking vascular phenotype, with greatly increased numbers of filopodia-extending endothelial tip cells and increased expression of tip cell marker genes compared with controls. Filopodia extension in dll4(+/-) retinal vessels required the vascular growth factor VEGF and was inhibited when VEGF signaling was blocked. Although VEGF expression was not significantly altered in dll4(+/-) retinas, dll4(+/-) vessels showed increased expression of VEGF receptor 2 and decreased expression of VEGF receptor 1 compared with wild-type, suggesting they could be more responsive to VEGF stimulation. In addition, expression of dll4 in wild-type tip cells was itself decreased when VEGF signaling was blocked, indicating that dll4 may act downstream of VEGF as a "brake" on VEGF-mediated angiogenic sprouting. Taken together, these data reveal Dll4 as a negative regulator of vascular sprouting and vessel branching that is required for normal vascular network formation during development.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Vessel branching defect in dll4+/− embryos. (A–D) Staining for β-gal activity in E10.5 unc5b+/− and dll4+/− embryos and for PECAM-1 in E10.5 wild-type and dll4+/− embryos. dll4+/− embryos show increased vessel branching from the internal carotid artery (ica, white arrow; dots represent arterial branchpoints; e, eye; v, vein). (E and F) Aortic explants from dll4+/− branch more profusely than wild-type. (G) Quantification of aortic sprout area after 48 h (n = 7 per group, three litters). (H–N) Increased branching and filopodia extension in E11.5 dll4+/− hindbrain vessels compared with wild-type. (H and I) Confocal images of isolectin B4-stained hindbrain vessels (dots indicate branchpoints). (J) Quantification of hindbrain vessel branchpoints (n = 8 wild-type, 10 dll4+/−). (K–N) High-magnification confocal images showing excessive filopodial extensions in dll4+/− compared with wild-type. Error bars, SD; ∗∗, P < 0.001, Mann–Whitney U test. [Scale bars, 270 μm (A); 330 μm (B and D); 300 μm (C); 350 μm (E and F); 150 μm (H and I); 40 μm (K and L); 20 μm (M and N).]
Fig. 2.
Fig. 2.
Increased tip cell formation in dll4+/− retinal vessels. (A and B) Isolectin B4-stained P5 dll4+/− retinal vessels show a hyperfused plexus compared with wild-type (a, artery; v, vein). (C and D) dll4+/− vessels extend many more filopodia within the vascular plexus compared with wild-type (arterial zone shown; dots indicate filopodia extensions). (E–H) Quantification of filopodial bursts at the vascular front, branchpoints in the vascular plexus, percentage of retina covered by vessels, and BrdU-labeled retinal ECs in wild-type and dll4+/−. (I–P) Whole-mount ISH shows expanded expression of tip cell marker genes pdgfb and unc5b in dll4+/− compared with wild-type (arrowheads indicate tip cell expression at vascular front). (M–P) Corresponding isolectin B4 staining of retinal vessels. Error bars, SD; ∗∗, P < 0.001, Mann–Whitney U test. [Scale bars, 250 μm (A and B); 40 μm (C and D); and 100 μm (I–P).]
Fig. 3.
Fig. 3.
Regulation of dll4+/− tip cells by VEGF. (A–D) Loss of dll4 mRNA expression after intraocular injection of soluble VEGFR1 (sFlt1) in wild-type pups. (A and B) Isolectin B4 staining shows tip cell filopodia (A, white arrowheads), and whole-mount ISH shows dll4 expression (B, blue arrowheads) in uninjected eyes. (C and D) Five hours after sFlt1 injection, filopodia are not observed, and dll4 expression is decreased. (E–I) Intraocular injections in P5 dll4+/− pups show inhibition of filopodia extension by antibodies blocking VEGFR2 (E and F) but not VEGFR1 (G and H). Dots indicate tip cells. (I) Quantification of total filopodia length per section of retina for injected eyes (n = four to five pups per group, three independent experiments). (J–R) Altered VEGFR expression in dll4+/− vessels compared with wild type. (J) qPCR of isolated retinal ECs (pooled from 10 retinas per group) show lower expression of dll4 (control) and vegfr1 and higher expression of vegfr2 in dll4+/− compared with wild type. (K–R) Whole-mount ISH shows up-regulation of vegfr2 and down-regulation of vegfr1 expression in dll4+/− compared with wild-type vessels (arrowheads indicate tip cell expression at vascular front). (O–R) Corresponding isolectin B4 staining of retinal vessels (a, arteries; v, veins). Error bars, SD; ∗∗, P < 0.001, Mann–Whitney U test. [Scale bars, 45 μm (A–D); 40 μm (E–H); and 200 μm (K–R).]
Fig. 4.
Fig. 4.
Inhibition of Notch signaling in retinas. (A and B) Isolectin B4 staining of wild-type P5 retinal vessels after 2 days of treatment with γ-secretase inhibitor DAPT or DMSO control. (C and D) Quantification of vessel branchpoints and percentage of retina covered by vessels in DAPT-compared with DMSO-treated retinas. (E–L) Whole-mount ISH shows expanded expression of tip cell marker genes pdgfb and dll4 in DAPT-treated retinas (arrowheads indicate tip cell expression at vascular front). (I–L) Corresponding isolectin B4 staining of retinal vessels (a, arteries; v, veins). Error bars, SD; ∗∗, P < 0.001, Mann–Whitney U test. [Scale bars, 40 μm (A and B); 130 μm (E, F, I, and J); and 100 μm (G, H, K, and L).]
See this image and copyright information in PMC

References

    1. Artavanis-Tsakonas S, Rand MD, Lake RJ. Science. 1999;284:770–776. - PubMed
    1. Shawber CJ, Kitajewski J. BioEssays. 2004;26:225–234. - PubMed
    1. Lawson ND, Scheer N, Pham VN, Kim CH, Chitnis AB, Campos-Ortega JA, Weinstein BM. Development. Vol. 128. Cambridge, UK: 2001. pp. 3675–3683. - PubMed
    1. Lawson ND, Vogel AM, Weinstein BM. Dev Cell. 2002;3:127–136. - PubMed
    1. Villa N, Walker L, Lindsell CE, Gasson J, Iruela-Arispe ML, Weinmaster G. Mech Dev. 2001;108:161–164. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources