The Notch ligand Delta-like 4 negatively regulates endothelial tip cell formation and vessel branching
Author contributions: S.S., C.F., F.l.N., and A.E. designed research; S.S., C.F., F.l.N., R.B., and C.B. performed research; R.B. and A.D. contributed new reagents/analytic tools; S.S., C.F., F.l.N., and A.E. analyzed data; and S.S., C.F., and A.E. wrote the paper.
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 γ-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.
Notch signaling controls cell fate specification in a variety of cell contexts during embryonic and postnatal development (1). Genetic deletion of multiple components of the Notch pathway has revealed a critical role for Notch in vascular development (2). In mice, the absence of Notch signaling results in defective yolk sac vascular remodeling and aberrant formation of arterial-venous circuits in the embryo, often leading to embryonic death (2). Studies in zebrafish and in vitro cultured cells have implicated Notch signaling in arterial-venous specification through regulation of arterial genes such as EphrinB2 (efnb2) (2, 3) and have identified Notch as a downstream target of the vascular growth factor VEGF (4). Delta-like 4 (Dll4) is a transmembrane ligand for Notch receptors that shows restricted expression to endothelial cells (ECs), in particular to arteries and capillaries (5–8). Deletion of a single dll4 allele in mice results in early embryonic death [from embryonic day (E)9.5] associated with major defects in vascular remodeling in the yolk sac and embryo (9–11). Haploinsufficiency within the vascular system has previously been observed only for VEGF (12, 13), suggesting that an appropriate dosage of both of these genes is critical for correct vascular development. The degree of lethality associated with heterozygous dll4 deletion depends on the mouse strain, and a small percentage of outbred CD1 dll4 embryos can survive, allowing for more detailed analysis of roles for dll4 at later stages of vascular development. Here we describe a previously uncharacterized function for dll4 during sprouting angiogenesis, with dll4 suppressing endothelial tip cell formation and vessel branching by inhibiting the response of sprouting ECs to VEGF.
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We thank G. Breier (Technical University, Dresden, Germany) for the vegf probe and R. del Toro and B. deLafarge for help with quantifications. This work was supported by grants from Institut National de la Santé et de la Recherche Médicale (Avenir), Ministère de la Recherche (Agence Nationale de la Recherche (ANR) Programme Cardiovasculaire, Obésité, Diabète, No. 05-022-03), the Association pour la Recherche sur le Cancer (No. 3124), the Institut de France (Cino Del Duca), and the European Community (Grant LSHG-CT-2004-503573). S.S. is supported by INSERM (poste vert). C.F. is supported by Fundação para a Ciência e a Tecnologia (Fellowship No. 20225/2004).
Abbreviations
Dll4 | delta-like 4 |
EC | endothelial cell |
ISH | in situ hybridization |
En | embryonic day n |
PECAM-1 | platelet-EC adhesion molecule 1 |
Pn | postnatal day n |
qPCR | quantitative PCR |
DAPT | N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester |
VEGFR1/2 | VEGF receptor 1/2. |
Footnotes
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0611177104/DC1.
References
- 1. Artavanis-Tsakonas S, Rand MD, Lake RJ. Science. 1999;284:770–776.[PubMed]
- 2. Shawber CJ, Kitajewski J. BioEssays. 2004;26:225–234.[PubMed]
- 3. 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][Google Scholar]
- 4. Lawson ND, Vogel AM, Weinstein BM. Dev Cell. 2002;3:127–136.[PubMed]
- 5. Villa N, Walker L, Lindsell CE, Gasson J, Iruela-Arispe ML, Weinmaster G. Mech Dev. 2001;108:161–164.[PubMed]
- 6. Shutter JR, Scully S, Fan W, Richards WG, Kitajewski J, Deblandre GA, Kintner CR, Stark KL. Genes Dev. 2000;14:1313–1318.
- 7. Claxton S, Fruttiger M. Gene Expr Patterns. 2004;5:123–127.[PubMed]
- 8. Benedito R, Duarte A. Gene Expr Patterns. 2005;5:750–755.[PubMed]
- 9. Gale NW, Dominguez MG, Noguera I, Pan L, Hughes V, Valenzuela DM, Murphy AJ, Adams NC, Lin HC, Holash J, et al Proc Natl Acad Sci USA. 2004;101:15949–15954.[Google Scholar]
- 10. Krebs LT, Shutter JR, Tanigaki K, Honjo T, Stark KL, Gridley T. Genes Dev. 2004;18:2469–2473.
- 11. Duarte A, Hirashima M, Benedito R, Trindade A, Diniz P, Bekman E, Costa L, Henrique D, Rossant J. Genes Dev. 2004;18:2474–2478.
- 12. Ferrara N, Carver-Moore K, Chen H, Dowd M, Lu L, O'Shea KS, Powell-Braxton L, Hillan KJ, Moore MW. Nature. 1996;380:439–442.[PubMed]
- 13. Carmeliet P, Ferreira V, Breier G, Pollefeyt S, Kieckens L, Gertsenstein M, Fahrig M, Vandenhoeck A, Harpal K, Eberhardt C, et al Nature. 1996;380:435–439.[PubMed][Google Scholar]
- 14. Lu X, Le Noble F, Yuan L, Jiang Q, De Lafarge B, Sugiyama D, Breant C, Claes F, De Smet F, Thomas JL, et al Nature. 2004;432:179–186.[PubMed][Google Scholar]
- 15. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, et al J Cell Biol. 2003;161:1163–1177.[Google Scholar]
- 16. Isogai S, Lawson ND, Torrealday S, Horiguchi M, Weinstein BM Development. Vol. 130. Cambridge, UK: 2003. pp. 5281–5290. [[PubMed][Google Scholar]
- 17. Gariano RF, Gardner TW. Nature. 2005;438:960–966.[PubMed]
- 18. West H, Richardson WD, Fruttiger M Development. Vol. 132. Cambridge, UK: 2005. pp. 1855–1862. [[PubMed][Google Scholar]
- 19. Stone J, Itin A, Alon T, Pe'er J, Gnessin H, Chan-Ling T, Keshet E. J Neurosci. 1995;15:4738–4747.
- 20. Liu ZJ, Shirakawa T, Li Y, Soma A, Oka M, Dotto GP, Fairman RM, Velazquez OC, Herlyn M. Mol Cell Biol. 2003;23:14–25.
- 21. Olsson AK, Dimberg A, Kreuger J, Claesson-Welsh L. Nat Rev Mol Cell Biol. 2006;7:359–371.[PubMed]
- 22. Williams CK, Li JL, Murga M, Harris AL, Tosato G. Blood. 2005
- 23. MacKenzie F, Duriez P, Larrivee B, Chang L, Pollet I, Wong F, Yip C, Karsan A. Blood. 2004;104:1760–1768.[PubMed]
- 24. Taylor KL, Henderson AM, Hughes CC. Microvasc Res. 2002;64:372–383.[PubMed]
- 25. Yang K, Cepko CL. J Neurosci. 1996;16:6089–6099.
- 26. Gariano RF, Hu D, Helms J. Gene Expr Patterns. 2006;6:187–192.[PubMed]
- 27. Fischer A, Schumacher N, Maier M, Sendtner M, Gessler M. Genes Dev. 2004;18:901–911.
- 28. Krebs LT, Xue Y, Norton CR, Shutter JR, Maguire M, Sundberg JP, Gallahan D, Closson V, Kitajewski J, Callahan R, et al Genes Dev. 2000;14:1343–1352.[Google Scholar]
- 29. Limbourg FP, Takeshita K, Radtke F, Bronson RT, Chin MT, Liao JK. Circulation. 2005;111:1826–1832.
- 30. Xue Y, Gao X, Lindsell CE, Norton CR, Chang B, Hicks C, Gendron-Maguire M, Rand EB, Weinmaster G, Gridley T. Hum Mol Genet. 1999;8:723–730.[PubMed]
- 31. Nakajima M, Yuasa S, Ueno M, Takakura N, Koseki H, Shirasawa T. Mech Dev. 2003;120:657–667.[PubMed]
- 32. Moyon D, Pardanaud L, Yuan L, Breant C, Eichmann A Development. Vol. 128. Cambridge, UK: 2001. pp. 3359–3370. [[PubMed][Google Scholar]
- 33. Dovey HF, John V, Anderson JP, Chen LZ, de Saint Andrieu P, Fang LY, Freedman SB, Folmer B, Goldbach E, Holsztynska EJ, et al J Neurochem. 2001;76:173–181.[PubMed][Google Scholar]