Endothelial Cell Dynamics during Blood Vessel Morphogenesis

  • Li-Kun PhngEmail author


Blood vessels, together with the heart, have a fundamental role in supporting the metabolic demands of tissues not only during development but also in adults. New blood vessels are frequently generated through angiogenesis when new vessels emerge from pre-existing ones (Fig. 2.1a). Initially, endothelial cells (ECs) lining an existing vessel are selected to become tip cells to spearhead the formation of new vascular sprouts. New sprouts grow through EC proliferation and the polarized collective migration of both tip and trailing stalk cells into the avascular tissue. In order to generate a network of interconnecting vessel segments, tip cells anastomose with neighboring tip cells to establish new vascular loops. Importantly, vascular sprouts develop into tubes through which oxygen, metabolites, cells, and waste products can circulate around the body. Finally, the tubular network of blood vessels are either maintained or, depending on the tissue requirements in which the vessels pervade, remodeled through pruning into a more refined vascular network that carries blood flow optimally to tissues (Fig. 2.1b).

Over the past few decades, many key signaling pathways that regulate blood vessel development have been identified using primarily the mouse as the model organism. These include the Neuropilin (NRP)/Vascular Endothelial Growth Factor (VEGF)/Vascular Endothelial Growth Factor Receptor (VEGFR), Jagged/Delta-like/Notch, Transforming Growth Factor β (TGFβ)/Bone Morphogenic Protein (BMP) and EphrinB/EphB signaling cascades (Adams RH, Alitalo K. Nat Rev Mol Cell Biol 8:464–478, 2007; Potente M, Makinen T. Nat Rev Mol Cell Biol 18:477, 2017). Although these studies have uncovered the fundamental principles of angiogenesis, temporal information on the cellular dynamics of angiogenesis has been lacking due to difficulties in performing live imaging in mouse embryos and tissues. These challenges are alleviated by the use of zebrafish, whose embryos develop ex utero, are optically transparent and are therefore highly suited for live imaging. Combined with recent advances in imaging techniques and the development of fluorescent biosensors or reporters, it is now possible to observe the dynamics of ECs at cellular and subcellular resolution as blood vessel morphogenesis takes place. Imaging vascular morphogenesis in the zebrafish embryo has been indispensable in the identification of morphogenetic events such as apical membrane invagination and the elucidation of the cellular mechanisms of anastomosis and vessel pruning, which are dynamic processes that are difficult to visualize and investigate in mouse models.

In this chapter, I will summarize recent findings from zebrafish studies that highlight the dynamic nature of ECs during angiogenesis and vessel remodeling and focus on how the actin cytoskeleton regulates EC morphogenesis and behavior.


Angiogenesis vascular morphogenesis endothelial cells actin cytoskeleton membrane junction zebrafish live imaging 



I would like to thank Henry Belting for critical reading of the manuscript. I apologize to authors whose work in this research area was not cited due to space restrictions.


  1. Adams RH, Alitalo K (2007) Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8:464–478CrossRefPubMedGoogle Scholar
  2. Anderson MJ, Pham VN, Vogel AM, Weinstein BM, Roman BL (2008) Loss of unc45a precipitates arteriovenous shunting in the aortic arches. Dev Biol 318:258CrossRefPubMedPubMedCentralGoogle Scholar
  3. Aydogan V, Lenard A, Denes AS, Sauteur L, Belting H-G, Affolter M (2015) Endothelial cell division in angiogenic sprouts of differing cellular architecture. Biol Open 4:1259CrossRefPubMedPubMedCentralGoogle Scholar
  4. Baeyens N, Mulligan-Kehoe MJ, Corti F, Simon DD, Ross TD, Rhodes JM, Wang TZ, Mejean CO, Simons M, Humphrey J, Schwartz MA (2014) Syndecan 4 is required for endothelial alignment in flow and atheroprotective signaling. Proc Natl Acad Sci 111:17308CrossRefPubMedGoogle Scholar
  5. Baeyens N, Nicoli S, Coon BG, Ross TD, Van den Dries K, Han J, Lauridsen HM, Mejean CO, Eichmann A, Thomas J-L, Humphrey JD, Schwartz MA (2015) Vascular remodeling is governed by a VEGFR3-dependent fluid shear stress set point. elife 4:1CrossRefGoogle Scholar
  6. Baeyens N, Bandyopadhyay C, Coon BG, Yun S, Schwartz MA (2016) Endothelial fluid shear stress sensing in vascular health and disease. J Clin Invest 126:821CrossRefPubMedPubMedCentralGoogle Scholar
  7. Blaser H, Reichman-Fried M, Castanon I, Dumstrei K, Marlow FL, Kawakami K, Solnica-Krezel L, Heisenberg C-P, Raz E (2006) Migration of zebrafish primordial germ cells: a role for myosin contraction and cytoplasmic flow. Dev Cell 11:613CrossRefPubMedGoogle Scholar
  8. Blum Y, Belting H-G, Ellertsdottir E, Herwig L, Lüders F, Affolter M (2008) Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo. Dev Biol 316:312CrossRefPubMedGoogle Scholar
  9. Bussmann J, Wolfe SA, Siekmann AF (2011) Arterial-venous network formation during brain vascularization involves hemodynamic regulation of chemokine signaling. Development 138:1717CrossRefPubMedPubMedCentralGoogle Scholar
  10. Charras GT (2008) A short history of blebbing. J Microsc 231:466CrossRefPubMedGoogle Scholar
  11. Chen Q, Jiang L, Li C, Hu D, J-w B, Cai D, J-l D (2012) Haemodynamics-driven developmental pruning of brain vasculature in zebrafish. PLoS Biol 10:e1001374CrossRefPubMedPubMedCentralGoogle Scholar
  12. Collins C, Osborne LD, Guilluy C, Chen Z, O’Brien ET, Reader JS, Burridge K, Superfine R, Tzima E (2014) Haemodynamic and extracellular matrix cues regulate the mechanical phenotype and stiffness of aortic endothelial cells. Nat Commun 5:3984CrossRefPubMedPubMedCentralGoogle Scholar
  13. Conway DE, Breckenridge MT, Hinde E, Gratton E, Chen CS, Schwartz MA (2013) Fluid shear stress on endothelial cells modulates mechanical tension across VE-cadherin and PECAM-1. Curr Biol 23(11):1024–1030CrossRefPubMedPubMedCentralGoogle Scholar
  14. Coon BG, Baeyens N, Han J, Budatha M, Ross TD, Fang JS, Yun S, Thomas J-L, Schwartz MA (2015) Intramembrane binding of VE-cadherin to VEGFR2 and VEGFR3 assembles the endothelial mechanosensory complex. J Cell Biol 208:975CrossRefPubMedPubMedCentralGoogle Scholar
  15. Costa G, Harrington KI, Lovegrove HE, Page DJ, Chakravartula S, Bentley K, Herbert SP (2016) Asymmetric division coordinates collective cell migration in angiogenesis. Nat Cell Biol 18:1292CrossRefPubMedPubMedCentralGoogle Scholar
  16. Fraccaroli A, Franco CA, Rognoni E, Neto F, Rehberg M, Aszodi A, Wedlich-Söldner R, Pohl U, Gerhardt H, Montanez E (2012) Visualization of endothelial actin cytoskeleton in the mouse retina. PLoS One 7:e47488CrossRefPubMedPubMedCentralGoogle Scholar
  17. Franco CA, Jones ML, Bernabeu MO, Geudens I, Mathivet T, Rosa A, Lopes FM, Lima AP, Ragab A, Collins RT, Phng L-K, Coveney PV, Gerhardt H (2015) Dynamic endothelial cell rearrangements drive developmental vessel regression. PLoS Biol 13:e1002125CrossRefPubMedPubMedCentralGoogle Scholar
  18. Gebala V, Collins R, Geudens I, Phng L-K, Gerhardt H (2016) Blood flow drives lumen formation by inverse membrane blebbing during angiogenesis in vivo. Nat Cell Biol 18:443CrossRefPubMedGoogle Scholar
  19. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C (2003) VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161:1163CrossRefPubMedPubMedCentralGoogle Scholar
  20. Goetz JG, Steed E, Ferreira RR, Roth S, Ramspacher C, Boselli F, Charvin G, Liebling M, Wyart C, Schwab Y, Vermot J (2014) Endothelial cilia mediate low flow sensing during zebrafish vascular development. Cell Rep 6:799CrossRefPubMedGoogle Scholar
  21. Grashoff C, Hoffman BD, Brenner MD, Zhou R, Parsons M, Yang MT, McLean MA, Sligar SG, Chen CS, Ha T, Schwartz MA (2010) Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466:263. Nature Publishing GroupCrossRefPubMedPubMedCentralGoogle Scholar
  22. Herwig L, Blum Y, Krudewig A, Ellertsdottir E, Lenard A, Belting H-G, Affolter M (2011) Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo. Current Biology: CB 21:1942CrossRefPubMedGoogle Scholar
  23. Hultin S, Zheng Y, Mojallal M, Vertuani S, Gentili C, Balland M, Milloud R, Belting H-G, Affolter M, Helker CSM, Adams RH, Herzog W, Uhlén P, Majumdar A, Holmgren L (2014) AmotL2 links VE-cadherin to contractile actin fibres necessary for aortic lumen expansion. Nat Commun 5:3743CrossRefPubMedGoogle Scholar
  24. Iomini C, Tejada K, Mo W, Vaananen H, Piperno G (2004) Primary cilia of human endothelial cells disassemble under laminar shear stress. J Cell Biol 164:811CrossRefPubMedPubMedCentralGoogle Scholar
  25. Jakobsson L, Franco CA, Bentley K, Collins RT, Ponsioen B, Aspalter IM, Rosewell I, Busse M, Thurston G, Medvinsky A, Schulte-Merker S, Gerhardt H (2010) Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat Cell Biol 12(10):943–953. Nature Publishing GroupCrossRefPubMedGoogle Scholar
  26. Kamei M, Brian Saunders W, Bayless KJ, Dye L, Davis GE, Weinstein BM (2006) Endothelial tubes assemble from intracellular vacuoles in vivo. Nat Cell Biol 442:453Google Scholar
  27. Kochhan E, Lenard A, Ellertsdottir E, Herwig L, Affolter M, Belting H-G, Siekmann AF (2013) Blood flow changes coincide with cellular rearrangements during blood vessel pruning in zebrafish embryos. PLoS One 8:e75060CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kurz H, Gärtner T, Eggli PS, Christ B (1996) First blood vessels in the avian neural tube are formed by a combination of dorsal angioblast immigration and ventral sprouting of endothelial cells. Dev Biol 173:133CrossRefPubMedGoogle Scholar
  29. Kwon H-B, Wang S, Helker CSM, Rasouli SJ, Maischein H-M, Offermanns S, Herzog W, Stainier DYR (2016) In vivo modulation of endothelial polarization by Apelin receptor signalling. Nat Commun 7:11805CrossRefPubMedPubMedCentralGoogle Scholar
  30. Lawson ND, Weinstein BM (2002) In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev Biol 248:307CrossRefGoogle Scholar
  31. Lenard A, Ellertsdottir E, Herwig L, Krudewig A, Sauteur L, Belting H-G, Affolter M (2013) In vivo analysis reveals a highly stereotypic morphogenetic pathway of vascular anastomosis. Dev Cell 25:492CrossRefPubMedGoogle Scholar
  32. Lenard A, Daetwyler S, Betz C, Ellertsdottir E, Belting H-G, Huisken J, Affolter M (2015) Endothelial cell self-fusion during vascular pruning. PLoS Biol 13:e1002126CrossRefPubMedPubMedCentralGoogle Scholar
  33. Li J, Hou B, Tumova S, Muraki K, Bruns A, Ludlow MJ, Sedo A, Hyman AJ, McKeown L, Young RS, Yuldasheva NY, Majeed Y, Wilson LA, Rode B, Bailey MA, Kim HR, Fu Z, Carter DAL, Bilton J, Imrie H, Ajuh P, Dear TN, Cubbon RM, Kearney MT, Prasad RK, Evans PC, Ainscough JFX, Beech DJ (2014) Piezo1 integration of vascular architecture with physiological force. Nature 515(7526):279–282. Nature Publishing GroupCrossRefPubMedPubMedCentralGoogle Scholar
  34. Nakajima H, Yamamoto K, Agarwala S, Terai K, Fukui H, Fukuhara S, Ando K, Miyazaki T, Yokota Y, Schmelzer E, Belting H-G, Affolter M, Lecaudey V, Mochizuki N (2017) Flow-dependent endothelial YAP regulation contributes to vessel maintenance. Dev Cell 40:523. Elsevier IncCrossRefPubMedGoogle Scholar
  35. Nicoli S, Standley C, Walker P, Hurlstone A, Fogarty KE, Lawson ND (2010) MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Nature 464:1196CrossRefPubMedPubMedCentralGoogle Scholar
  36. Paluch EK, Raz E (2013) The role and regulation of blebs in cell migration. Curr Opin Cell Biol 25:582CrossRefPubMedPubMedCentralGoogle Scholar
  37. Panciera T, Azzolin L, Cordenonsi M, Piccolo S (2017) Mechanobiology of YAP and TAZ in physiology and disease. Nat Rev Mol Cell Biol 18:758CrossRefPubMedGoogle Scholar
  38. Pelton JC, Wright CE, Leitges M, Bautch VL (2014) Multiple endothelial cells constitute the tip of developing blood vessels and polarize to promote lumen formation. Development 141:4121CrossRefPubMedPubMedCentralGoogle Scholar
  39. Phng LK, Stanchi F, Gerhardt H (2013) Filopodia are dispensable for endothelial tip cell guidance. Development 140:4031CrossRefPubMedGoogle Scholar
  40. Phng L-K, Gebala V, Bentley K, Philippides A, Wacker A, Mathivet T, Sauteur L, Stanchi F, Belting H-G, Affolter M, Gerhardt H (2015) Formin-mediated actin polymerization at endothelial junctions is required for vessel lumen formation and stabilization. Dev Cell 32:123CrossRefPubMedGoogle Scholar
  41. Pollard TD, Cooper JA (2009) Actin, a central player in cell shape and movement. Science 326:1208CrossRefPubMedPubMedCentralGoogle Scholar
  42. Potente M, Makinen T (2017) Vascular heterogeneity and specialization in development and disease. Nat Rev Mol Cell Biol 18:477. Nature Publishing GroupCrossRefPubMedGoogle Scholar
  43. Ridley AJ (2011) Life at the leading edge. Cell 145:1012. Elsevier IncCrossRefPubMedGoogle Scholar
  44. Sauteur L, Krudewig A, Herwig L, Ehrenfeuchter N, Lenard A, Affolter M, Belting H-G (2014) Cdh5/VE-cadherin promotes endothelial cell Interface elongation via cortical actin polymerization during Angiogenic sprouting. Cell Rep 9:504. The AuthorsCrossRefPubMedGoogle Scholar
  45. Sauteur L, Affolter M, Belting H-G (2017) Distinct and redundant functions of Esama and VE-cadherin during vascular morphogenesis. Development 144:1554CrossRefGoogle Scholar
  46. Siekmann AF, Lawson ND (2007) Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature 445:781CrossRefPubMedGoogle Scholar
  47. Sugden WW, Meissner R, Aegerter-Wilmsen T, Tsaryk R, Leonard EV, Bussmann J, Hamm MJ, Herzog W, Jin Y, Jakobsson L, Denz C, Siekmann AF (2017) Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues. Nat Cell Biol 19:653CrossRefPubMedPubMedCentralGoogle Scholar
  48. Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, Cao G, DeLisser H, Schwartz MA (2005) A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 437:426CrossRefGoogle Scholar
  49. Wakayama Y, Fukuhara S, Ando K, Matsuda M, Mochizuki N (2015) Cdc42 mediates bmp-induced sprouting angiogenesis through Fmnl3-driven assembly of endothelial Filopodia in zebrafish. Dev Cell 32:109. Elsevier IncCrossRefPubMedGoogle Scholar
  50. Wang Y, Kaiser MS, Larson JD, Nasevicius A, Clark KJ, Wadman SA, Roberg-Perez SE, Ekker SC, Hackett PB, McGrail M, Essner JJ (2010) Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis. Development 137:3119CrossRefPubMedPubMedCentralGoogle Scholar
  51. Wiley DM, Kim J-D, Hao J, Hong CC, Bautch VL, Jin S-W (2011) Distinct signalling pathways regulate sprouting angiogenesis from the dorsal aorta and the axial vein. Nat Cell Biol 13:686CrossRefPubMedPubMedCentralGoogle Scholar
  52. Xu C, Hasan SS, Schmidt I, Rocha SF, Pitulescu ME, Bussmann J, Meyen D, Raz E, Adams RH, Siekmann AF (2014) Arteries are formed by vein-derived endothelial tip cells. Nat Commun 5:5758CrossRefPubMedPubMedCentralGoogle Scholar
  53. Yu JA, Castranova D, Pham VN, Weinstein BM (2015) Single-cell analysis of endothelial morphogenesis in vivo. Development 142:2951CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Laboratory for Vascular MorphogenesisRIKEN Center for Biosystems Dynamics ResearchKobeJapan

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