Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Blood flow drives lumen formation by inverse membrane blebbing during angiogenesis in vivo

Nature Cell Biology volume 18, pages 443–450 (2016)Cite this article

Subjects

Abstract

How vascular tubes build, maintain and adapt continuously perfused lumens to meet local metabolic needs remains poorly understood. Recent studies showed that blood flow itself plays a critical role in the remodelling of vascular networks1,2, and suggested it is also required for the lumenization of new vascular connections3,4. However, it is still unknown how haemodynamic forces contribute to the formation of new vascular lumens during blood vessel morphogenesis. Here we report that blood flow drives lumen expansion during sprouting angiogenesis in vivo by inducing spherical deformations of the apical membrane of endothelial cells, in a process that we have termed inverse blebbing. We show that endothelial cells react to these membrane intrusions by local and transient recruitment and contraction of actomyosin, and that this mechanism is required for single, unidirectional lumen expansion in angiogenic sprouts. Our work identifies inverse membrane blebbing as a cellular response to high external pressure. We show that in the case of blood vessels such membrane dynamics can drive local cell shape changes required for global tissue morphogenesis, shedding light on a pressure-driven mechanism of lumen formation in vertebrates.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Blood pressure drives unicellular and multicellular lumen expansion in angiogenic sprouts.
Figure 2: Apical membrane undergoes inverse blebbing during lumen expansion.
Figure 3: Blood pressure drives inverse blebbing at the apical membrane of angiogenic sprouts.
Figure 4: Endothelial cells retract inverse blebs by recruiting and contracting actomyosin at the apical membrane.
Figure 5: Apical membrane contractility regulates lumen formation during sprouting angiogenesis.

Similar content being viewed by others

References

  1. Chen, Q. et al. Haemodynamics-driven developmental pruning of brain vasculature in zebrafish. PLoS Biol. 10, e1001374 (2012).

    Article  CAS  Google Scholar 

  2. Kochhan, E. et al. Blood flow changes coincide with cellular rearrangements during blood vessel pruning in zebrafish embryos. PLoS ONE 8, e75060 (2013).

    Article  CAS  Google Scholar 

  3. Herwig, L. et al. Distinct cellular mechanisms of blood vessel fusion in the zebrafish embryo. Curr. Biol. 21, 1942–1948 (2011).

    Article  CAS  Google Scholar 

  4. Lenard, A. et al. In vivo analysis reveals a highly stereotypic morphogenetic pathway of vascular anastomosis. Dev. Cell 25, 492–506 (2013).

    Article  CAS  Google Scholar 

  5. Potente, M., Gerhardt, H. & Carmeliet, P. Basic and therapeutic aspects of angiogenesis. Cell 146, 873–887 (2011).

    Article  CAS  Google Scholar 

  6. Jakobsson, L. et al. Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat. Cell Biol. 12, 943–953 (2010).

    Article  CAS  Google Scholar 

  7. Pelton, J. C., Wright, C. E., Leitges, M. & Bautch, V. L. Multiple endothelial cells constitute the tip of developing blood vessels and polarize to promote lumen formation. Development 141, 4121–4126 (2014).

    Article  CAS  Google Scholar 

  8. Jin, S. W., Beis, D., Mitchell, T., Chen, J. N. & Stainier, D. Y. Cellular and molecular analyses of vascular tube and lumen formation in zebrafish. Development 132, 5199–5209 (2005).

    Article  CAS  Google Scholar 

  9. Strilic, B. et al. The molecular basis of vascular lumen formation in the developing mouse aorta. Dev. Cell 17, 505–515 (2009).

    Article  CAS  Google Scholar 

  10. Charpentier, M. S., Tandon, P., Trincot, C. E., Koutleva, E. K. & Conlon, F. L. A distinct mechanism of vascular lumen formation in Xenopus requires EGFL7. PLoS ONE 10, e0116086 (2015).

    Article  Google Scholar 

  11. Wang, Y. et al. Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis. Development 137, 3119–3128 (2010).

    Article  CAS  Google Scholar 

  12. Cunningham, C. C. Actin polymerization and intracellular solvent flow in cell surface blebbing. J. Cell Biol. 129, 1589–1599 (1995).

    Article  CAS  Google Scholar 

  13. Keller, H. & Eggli, P. Protrusive activity, cytoplasmic compartmentalization, and restriction rings in locomoting blebbing Walker carcinosarcoma cells are related to detachment of cortical actin from the plasma membrane. Cell Motil. Cytoskeleton 41, 181–193 (1998).

    Article  CAS  Google Scholar 

  14. Paluch, E., Piel, M., Prost, J., Bornens, M. & Sykes, C. Cortical actomyosin breakage triggers shape oscillations in cells and cell fragments. Biophys. J. 89, 724–733 (2005).

    Article  CAS  Google Scholar 

  15. Charras, G. T., Coughlin, M., Mitchison, T. J. & Mahadevan, L. Life and times of a cellular bleb. Biophys. J. 94, 1836–1853 (2008).

    Article  CAS  Google Scholar 

  16. Charras, G. & Paluch, E. Blebs lead the way: how to migrate without lamellipodia. Nat. Rev. Mol. Cell Biol. 9, 730–736 (2008).

    Article  CAS  Google Scholar 

  17. Martin-Belmonte, F. et al. PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42. Cell 128, 383–397 (2007).

    Article  CAS  Google Scholar 

  18. Sauteur, L. et al. Cdh5/VE-cadherin promotes endothelial cell interface elongation via cortical actin polymerization during angiogenic sprouting. Cell Rep. 9, 504–513 (2014).

    Article  CAS  Google Scholar 

  19. Phng, L. K. et al. Formin-mediated actin polymerization at endothelial junctions is required for vessel lumen formation and stabilization. Dev. Cell 32, 123–132 (2015).

    Article  CAS  Google Scholar 

  20. Kamei, M. et al. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442, 453–456 (2006).

    Article  CAS  Google Scholar 

  21. Yu, J. A., Castranova, D., Pham, V. N. & Weinstein, B. M. Single-cell analysis of endothelial morphogenesis in vivo. Development 142, 2951–2961 (2015).

    Article  CAS  Google Scholar 

  22. Iwasaki, T., Murata-Hori, M., Ishitobi, S. & Hosoya, H. Diphosphorylated MRLC is required for organization of stress fibers in interphase cells and the contractile ring in dividing cells. Cell Struct. Funct. 26, 677–683 (2001).

    Article  CAS  Google Scholar 

  23. Emelyanov, A. & Parinov, S. Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish. Dev. Biol. 320, 113–121 (2008).

    Article  CAS  Google Scholar 

  24. Tinevez, J. Y. et al. Role of cortical tension in bleb growth. Proc. Natl Acad. Sci. USA 106, 18581–18586 (2009).

    Article  CAS  Google Scholar 

  25. Davis, G. E. & Camarillo, C. W. An α2β1 integrin-dependent pinocytic mechanism involving intracellular vacuole formation and coalescence regulates capillary lumen and tube formation in three-dimensional collagen matrix. Exp. Cell Res. 224, 39–51 (1996).

    Article  CAS  Google Scholar 

  26. Xu, K. et al. Blood vessel tubulogenesis requires Rasip1 regulation of GTPase signaling. Dev. Cell 20, 526–539 (2011).

    Article  CAS  Google Scholar 

  27. Martin, M. et al. PP2A regulatory subunit Bα controls endothelial contractility and vessel lumen integrity via regulation of HDAC7. EMBO J. 32, 2491–2503 (2013).

    Article  CAS  Google Scholar 

  28. Riedl, J. et al. Lifeact mice for studying F-actin dynamics. Nat. Methods 7, 168–169 (2010).

    Article  CAS  Google Scholar 

  29. Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. & Schilling, T. F. Stages of embryonic development of the zebrafish. Dev. Dyn. 203, 253–310 (1995).

    Article  CAS  Google Scholar 

  30. Hogan, B. M. et al. Ccbe1 is required for embryonic lymphangiogenesis and venous sprouting. Nat. Genet. 41, 396–398 (2009).

    Article  CAS  Google Scholar 

  31. Lawson, N. D. & Weinstein, B. M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318 (2002).

    Article  CAS  Google Scholar 

  32. Phng, L. K., Stanchi, F. & Gerhardt, H. Filopodia are dispensable for endothelial tip cell guidance. Development 140, 4031–4040 (2013).

    Article  CAS  Google Scholar 

  33. Kwan, K. M. et al. The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, 3088–3099 (2007).

    Article  CAS  Google Scholar 

  34. Kawakami, K. et al. A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev. Cell 7, 133–144 (2004).

    Article  CAS  Google Scholar 

  35. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank members of the Vascular Biology and Vascular Patterning Laboratories for helpful discussions. We thank the Cancer Research UK London Research Institute Animal and Fish Facilities, and the Aquatic Facilities at the Max Delbrück Center for Molecular Medicine and Vesalius Research Center for animal care. We thank T. Surrey and N. Cade for access to the inverted 3i spinning-disc confocal, and P. Vanden Berghe for access to the Andor spinning-disc confocal of the Cell Imaging Core facility at KU Leuven. We thank G. Kelly from the Francis Crick Institute (London, UK) for help with statistics. V.G. is financially supported by Cancer Research UK. L.-K.P. is financially supported by a HFSP Long-Term Fellowship. H.G. is financially supported by Cancer Research UK, the Lister Institute for Preventive Medicine, a European Research Council starting grant REshape (311719) and the Berlin Institute of Health (BIH). This work was supported by the DZHK (German Center for Cardiovascular Research) and the BMBF (German Ministry of Education and Research).

Author information

Author notes

  1. Li-Kun Phng and Holger Gerhardt: These authors contributed equally to this work.

Authors and Affiliations

  1. The Francis Crick Institute, Lincoln’s Inn Fields Laboratory, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK

    Véronique Gebala

  2. Integrative Vascular Biology Laboratory, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Robert-Rössle-Strasse 10, 13125 Berlin, Germany

    Véronique Gebala, Russell Collins & Holger Gerhardt

  3. Department of Oncology, Vascular Patterning Laboratory, Vesalius Research Center, VIB, KU Leuven, Herestraat 49, 3000 Leuven, Belgium

    Ilse Geudens, Li-Kun Phng & Holger Gerhardt

  4. Department of Cell Biology, National Cerebral and Cardiovascular Center Research Institute, Osaka 565-8565, Japan

    Li-Kun Phng

  5. DZHK (German Center for Cardiovascular Research), partner site Berlin, Germany

    Holger Gerhardt

  6. Berlin Institute of Health (BIH), Berlin, Germany

    Holger Gerhardt

Authors
  1. Véronique Gebala
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Russell Collins
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ilse Geudens
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Li-Kun Phng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Holger Gerhardt
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

V.G., L.-K.P. and H.G. designed the experiments. V.G. and L.-K.P. performed the experiments and analysed the data. R.C. generated the Tg(fli1ep:PLC∂-PH–RFP) zebrafish line. I.G. generated the Tg(fli1ep:EGFP–CAAX) zebrafish line. V.G. and H.G. wrote the manuscript.

Corresponding author

Correspondence to Holger Gerhardt.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 (Related to Figure 2.)

(a–e) Tg(kdr-l:ras-Cherry)s916 embryos with mosaic expression of Lifeact-EGFP were used to measure expansion time (a), retraction time (b), expansion speed (c) and retraction speed (d) in relation to bleb size (n = 31 blebs from 3 cells). Dots in (a–d) correspond to single blebs. (e) shows mean and standard deviation for each property. Values for classical blebs come from1. (f) Tg(fli1ep:EGFP;fli1ep:PLCδ-PH-RFP) embryos were imaged from 35 hpf. Arrowhead, inverse bleb. C, cytoplasm. E, extracellular space. L, lumen. Time is in hours:minutes:seconds. Scale bar is 5 μm. Images are representative of 3 embryos analysed.

Supplementary Figure 2 (Related to Figure 4.)

(a) Embryos with mosaic expression of Myl9b-EGFP and Lifeact-mCherry were imaged at 2 dpf. Arrowheads show co-localisation of F-actin and Myosin-II at cell junctions (A), at the apical membrane (B), and at the base of filopodia (C). Scale bars are 10 μm. Images are representative of 6 embryos analysed. (b) Tg(kdr-l:ras-Cherry)s916 embryos with mosaic expression of Myl9b-EGFP were imaged from 34 hpf. Dotted line, apical membrane. Arrow, expanding apical membrane. Arrowhead, onset of Myosin-II recruitment. C, cytoplasm. E, extracellular space. L, lumen. Time is in hours:minutes:seconds. Scale bar is 5 μm. Images are representative of 3 embryos analysed. (c) Kymograph generated along the magenta line in b. X axis, time (t) in seconds. Y axis, distance (d) in μm.

Supplementary information

Supplementary Information

Supplementary Information (PDF 536 kb)

(related to Fig. 2a). Apical membrane undergoes inverse blebbing during lumen expansion in sprouting ISVs.

Time-lapse series of an endothelial sprout with mosaic expression of EGFP-CAAX imaged from 36 hpf. The apical membrane shows inverse blebs as the lumen expands into the sprout (black arrow). The red arrow shows a disconnected lumen fragment originating from the collapse of the lumen. Time is in hours:minutes:seconds. (AVI 5674 kb)

(related to Supplementary Fig. 1f). Early apical determinants localise at the apical membrane during inverse blebbing.

Time-lapse series of an endothelial sprout expressing cytoplasmic EGFP (left panel, green) and a PLCδ-PH-RFP reporter for PIP2 (right panel, magenta) imaged from 35 hpf. The apical membrane retains apical markers (PIP2) as it expands (white arrow). Time is in hours:minutes:seconds. (AVI 1106 kb)

(related to Fig. 2c). Inverse membrane blebbing drives multicellular lumen expansion in sprouting ISVs.

Time-lapse series of an endothelial sprout with mosaic expression of EGFP-CAAX (left panel, green) and expression of mCherry-CAAX (right panel, magenta) imaged from 32 hpf. Inverse blebbing occurs simultaneously in both cells forming the ISV as the lumen expands (white arrows). Time is in hours:minutes:seconds. (AVI 3628 kb)

(related to Fig. 3a). Interruption of blood flow by laser ablation inhibits inverse blebbing at the apical membrane of sprouting ISVs.

Time-lapse series of an endothelial sprout expressing EGFP-CAAX imaged from 33 hpf. Laser ablation was performed along a line spanning the entire thickness of the vessel at the place indicated by the red arrow, and at the time indicated. Ablation led to an immediate loss of the inverse blebs at the apical membrane and to gradual regression of the lumen (black arrow). Time is in hours:minutes:seconds. (AVI 3341 kb)

(related to Fig. 3b). Interruption of blood flow by tricaine treatment inhibits inverse blebbing at the apical membrane of sprouting ISVs.

Time-lapse series of an endothelial sprout expressing mCherry-CAAX imaged from 34 hpf, before, during and after treatment with 4× tricaine. Blood flow stops about 15–20 min after addition of 4× tricaine, leading to a loss of the inverse blebs at the apical membrane. Black arrows show expansion of the apical membrane by inverse blebbing before treatment with 4× tricaine and after washout. Time is in hours:minutes:seconds. (AVI 18761 kb)

(related to Fig. 4a, b). F-actin polymerises around inverse blebs as they retract

Time-lapse series of an endothelial sprout with mosaic expression of Lifeact-EGFP (left panel, green) and mCherry-CAAX (right panel, magenta) imaged from 35 hpf. F-actin polymerises around inverse blebs as they retract. Time is in hours:minutes:seconds. (AVI 4797 kb)

(related to Fig. 4f). Laser ablation of the cell cortex at the apical membrane of growing lumens leads to the expansion of inverse blebs.

Time-lapse series of an endothelial sprout expressing Lifeact-EGFP (left panel, green) and mCherry-CAAX (right panel, magenta) imaged from 33 hpf. Laser ablation of the cell cortex was performed along the indicated black/white line and led to the expansion of a bleb that later retracted (white arrow). Time is in hours:minutes:seconds. (AVI 1333 kb)

(related to Supplementary Fig. 5c). Apical contractility is required for lumen expansion in sprouting ISVs.

Time-lapse series of an endothelial sprout with mosaic expression of Myl9bAA-EGFP (left panel, green) and mCherry-CAAX (right panel, magenta) imaged from 35 hpf. The cell expressing Myl9bAA fails to lumenise from the ventral part of the ISV. Lumen pushes into the cell from the dorsal longitudinal anastomotic vessel (DLAV) but fails to expand (white arrows). Time is in hours:minutes:seconds. (AVI 8695 kb)

(related to Fig. 5b). Endothelial cells with decreased apical contractility show uncontrolled blebbing.

Time-lapse series of an endothelial sprout with mosaic expression of Myl9bAA-EGFP (left panel, green) and mCherry-CAAX (right panel, magenta) imaged from 48 hpf. The apical membrane undergoes excessive and uncoordinated blebbing and fails to expand. Time is in hours:minutes:seconds. (AVI 2466 kb)

(related to Fig. 5b). Partially lumenised endothelial cells with decreased apical contractility show side lumen branches.

Time-lapse series of an endothelial sprout with mosaic expression of Myl9bAA-EGFP (left panel, green) and mCherry-CAAX (right panel, magenta) imaged from 52 hpf. The ISV is dilated and shows side lumen branches that fail to retract (white arrows). Time is in hours:minutes:seconds. (AVI 9607 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gebala, V., Collins, R., Geudens, I. et al. Blood flow drives lumen formation by inverse membrane blebbing during angiogenesis in vivo. Nat Cell Biol 18, 443–450 (2016). https://doi.org/10.1038/ncb3320

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3320

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing