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Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis

Nature volume 445, pages 776–780 (2007)Cite this article

Abstract

In sprouting angiogenesis, specialized endothelial tip cells lead the outgrowth of blood-vessel sprouts towards gradients of vascular endothelial growth factor (VEGF)-A1,2. VEGF-A is also essential for the induction of endothelial tip cells2, but it is not known how single tip cells are selected to lead each vessel sprout, and how tip-cell numbers are determined. Here we present evidence that delta-like 4 (Dll4)–Notch1 signalling regulates the formation of appropriate numbers of tip cells to control vessel sprouting and branching in the mouse retina. We show that inhibition of Notch signalling using γ-secretase inhibitors, genetic inactivation of one allele of the endothelial Notch ligand Dll4, or endothelial-specific genetic deletion of Notch1, all promote increased numbers of tip cells. Conversely, activation of Notch by a soluble jagged1 peptide leads to fewer tip cells and vessel branches. Dll4 and reporters of Notch signalling are distributed in a mosaic pattern among endothelial cells of actively sprouting retinal vessels. At this location, Notch1-deleted endothelial cells preferentially assume tip-cell characteristics. Together, our results suggest that Dll4–Notch1 signalling between the endothelial cells within the angiogenic sprout serves to restrict tip-cell formation in response to VEGF, thereby establishing the adequate ratio between tip and stalk cells required for correct sprouting and branching patterns. This model offers an explanation for the dose-dependency and haploinsufficiency of the Dll4 gene3,4,5, and indicates that modulators of Dll4 or Notch signalling, such as γ-secretase inhibitors developed for Alzheimer’s disease, might find usage as pharmacological regulators of angiogenesis.

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Figure 1: γ-Secretase inhibitor experiments in vivo.
Figure 2: Dll4 and Notch1 loss-of-function analyses in vivo.
Figure 3: Endothelial Dll4–Notch signalling in vivo.
Figure 4: Notch gain-of-function analysis in vivo.

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Acknowledgements

We thank F. Radke for providing Notch1floxed/floxed mice. Support from the following foundations and granting agencies is acknowledged: Swedish Cancer Society, Association for International Cancer Research, European Union, the Novo Nordisk, Strategic Research, Söderberg, Hedlund, Wallenberg and Inga-Britt and Arne Lundberg Foundations (to C.B.); National Institutes of Health (US, NIH) and JH (USPHS National Research Service Award) (to L.I.-A.). H.G., L.-K.P. and P.L. are supported by Cancer Research UK. We acknowledge the Swegene Centre for Cellular Imaging at Gothenburg University for the use of imaging equipment, and the Light Microscopy Service and Peptide Synthesis Laboratory, London Research Institute (Cancer Research UK) for technical assistance.

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Author notes

  1. Per Lindblom, Ann-Katrin Nilsson & Linda Karlsson

    Present address: Present addresses: Molecular Toxicology, Safety Assessment, AstraZeneca R&D, SE-151 85 Södertälje, Sweden (P.L.); Stem Cell Center, BMC B10, Klinikg. 26, Lund University, SE-221 84 Lund, Sweden (A.-K.N.); Department of Physiology, Göteborg University, P.O. Box 434, SE-405 30 Göteborg, Sweden (L.K.).,

  2. Mattias Kalén, Holger Gerhardt and Christer Betsholtz: These authors contributed equally to this work.

Authors and Affiliations

  1. AngioGenetics Sweden AB, Scheeles väg 2, SE-171 77 Stockholm, Sweden,

    Mats Hellström, Elisabet Wallgard, Ann-Katrin Nilsson, Linda Karlsson & Mattias Kalén

  2. Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, and,

    Mats Hellström, Elisabet Wallgard, Mattias Kalén & Christer Betsholtz

  3. Department of Medicine, Karolinska Institutet, SE 171 77 Stockholm, Sweden,

    Christer Betsholtz

  4. Vascular Biology Laboratory, London Research Institute, Cancer Research UK, London WC2A 3PX, UK,

    Li-Kun Phng, Per Lindblom & Holger Gerhardt

  5. Department of Molecular, Cell and Developmental Biology and Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA,

    Jennifer J. Hofmann, Jackelyn Alva & M. Luisa Iruela-Arispe

  6. Program for Developmental Biology, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada,

    Leigh Coultas & Janet Rossant

  7. Neurology, Neuroscience and Oncology, Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland 21205, USA,

    Nicholas Gaiano & Keejung Yoon

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Correspondence to Mats Hellström or Holger Gerhardt.

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[Competing Interests Statement: M.H. and M.K. are employed by AngioGenetics Sweden AB. C.B. receives funding from and is a consultant of AngioGenetics Sweden AB.]

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This file contains Supplementary Methods, Supplementary Figures S1-S11 with Legends and additional references. (PDF 7910 kb)

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Hellström, M., Phng, LK., Hofmann, J. et al. Dll4 signalling through Notch1 regulates formation of tip cells during angiogenesis. Nature 445, 776–780 (2007). https://doi.org/10.1038/nature05571

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