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.

  • Article
  • Published:

Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues

Nature Cell Biology volume 19, pages 653–665 (2017)Cite this article

Subjects

Abstract

The hierarchical organization of properly sized blood vessels ensures the correct distribution of blood to all organs of the body, and is controlled via haemodynamic cues. In current concepts, an endothelium-dependent shear stress set point causes blood vessel enlargement in response to higher flow rates, while lower flow would lead to blood vessel narrowing, thereby establishing homeostasis. We show that during zebrafish embryonic development increases in flow, after an initial expansion of blood vessel diameters, eventually lead to vessel contraction. This is mediated via endothelial cell shape changes. We identify the transforming growth factor beta co-receptor endoglin as an important player in this process. Endoglin mutant cells and blood vessels continue to enlarge in response to flow increases, thus exacerbating pre-existing embryonic arterial–venous shunts. Together, our data suggest that cell shape changes in response to biophysical cues act as an underlying principle allowing for the ordered patterning of tubular organs.

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: Zebrafish eng mutants develop AVMs.
Figure 2: Zebrafish 72 hpf trunk vasculature recapitulates an AVM-like phenotype.
Figure 3: Analysis of DA and PCV diameter during development.
Figure 4: Blood flow patterns adapt to the expansion of the embryonic vasculature.
Figure 5: Eng does not affect klf2a-mediated shear stress sensing.
Figure 6: Loss of eng function leads to blood vessel enlargement in an EC autonomous manner.
Figure 7: Endoglin function is necessary for blood-flow-induced cell shape changes.
Figure 8: Role of endoglin in controlling the morphology of mammalian ECs exposed to flow.

Similar content being viewed by others

References

  1. Ochoa-Espinosa, A. & Affolter, M. Branching morphogenesis: from cells to organs and back. Cold Spring Harb. Perspect. Biol. 4, a008243 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. Filosa, J. A., Morrison, H. W., Iddings, J. A., Du, W. & Kim, K. J. Beyond neurovascular coupling, role of astrocytes in the regulation of vascular tone. Neuroscience 323, 96–109 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Joyner, M. J. & Casey, D. P. Regulation of increased blood flow (hyperemia) to muscles during exercise: a hierarchy of competing physiological needs. Physiol. Rev. 95, 549–601 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Baeyens, N. & Schwartz, M. A. Biomechanics of vascular mechanosensation and remodeling. Mol. Biol. Cell 27, 7–11 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Kamiya, A. & Togawa, T. Adaptive regulation of wall shear stress to flow change in the canine carotid artery. Am. J. Physiol. 239, H14–H21 (1980).

    CAS  PubMed  Google Scholar 

  6. Langille, B. L. Arterial remodeling: relation to hemodynamics. Can. J. Physiol. Pharmacol. 74, 834–841 (1996).

    Article  CAS  PubMed  Google Scholar 

  7. Langille, B. L., Bendeck, M. P. & Keeley, F. W. Adaptations of carotid arteries of young and mature rabbits to reduced carotid blood flow. Am. J. Physiol. 256, H931–H939 (1989).

    CAS  PubMed  Google Scholar 

  8. Langille, B. L. & O’Donnell, F. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelium-dependent. Science 231, 405–407 (1986).

    Article  CAS  PubMed  Google Scholar 

  9. Tuttle, J. L. et al. Shear level influences resistance artery remodeling: wall dimensions, cell density, and eNOS expression. Am. J. Physiol. 281, H1380–H1389 (2001).

    CAS  Google Scholar 

  10. Baeyens, N. et al. Vascular remodeling is governed by a VEGFR3-dependent fluid shear stress set point. eLife 4, e04645 (2015).

    Article  PubMed Central  Google Scholar 

  11. Kamiya, A., Bukhari, R. & Togawa, T. Adaptive regulation of wall shear stress optimizing vascular tree function. Bull. Math. Biol. 46, 127–137 (1984).

    Article  CAS  PubMed  Google Scholar 

  12. Rodbard, S. Vascular caliber. Cardiology 60, 4–49 (1975).

    Article  CAS  PubMed  Google Scholar 

  13. Thoma, R. Untersuchungen über die Histogenese und Histomechanik des Gefässsystems (Verlag von Ferdinand Enke, 1893).

    Google Scholar 

  14. Pries, A. R., Hopfner, M., le Noble, F., Dewhirst, M. W. & Secomb, T. W. The shunt problem: control of functional shunting in normal and tumour vasculature. Nat. Rev. Cancer 10, 587–593 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tual-Chalot, S., Oh, S. P. & Arthur, H. M. Mouse models of hereditary hemorrhagic telangiectasia: recent advances and future challenges. Front. Genet. 6, 25 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  16. Roman, B. L. et al. Disruption of acvrl1 increases endothelial cell number in zebrafish cranial vessels. Development 129, 3009–3019 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Mahmoud, M. et al. Pathogenesis of arteriovenous malformations in the absence of endoglin. Circ. Res. 106, 1425–1433 (2010).

    Article  CAS  PubMed  Google Scholar 

  18. Rochon, E. R., Menon, P. G. & Roman, B. L. Alk1 controls arterial endothelial cell migration in lumenized vessels. Development 143, 2593–2602 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Tual-Chalot, S. et al. Endothelial depletion of Acvrl1 in mice leads to arteriovenous malformations associated with reduced endoglin expression. PLoS ONE 9, e98646 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. Kamaid, A. et al. Betaglycan knock-down causes embryonic angiogenesis defects in zebrafish. Genesis 53, 583–603 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Lee, N. Y. et al. Endoglin regulates PI3-kinase/Akt trafficking and signaling to alter endothelial capillary stability during angiogenesis. Mol. Biol. Cell 23, 2412–2423 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Li, D. Y. et al. Defective angiogenesis in mice lacking endoglin. Science 284, 1534–1537 (1999).

    Article  CAS  PubMed  Google Scholar 

  23. Gougos, A. & Letarte, M. Primary structure of endoglin, an RGD-containing glycoprotein of human endothelial cells. J. Biol. Chem. 265, 8361–8364 (1990).

    Article  CAS  PubMed  Google Scholar 

  24. Seghers, L. et al. Shear induced collateral artery growth modulated by endoglin but not by ALK1. J. Cell Mol. Med. 16, 2440–2450 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Xu, C. et al. Arteries are formed by vein-derived endothelial tip cells. Nat. Commun. 5, 5758 (2014).

    CAS  PubMed  Google Scholar 

  26. Childs, S., Chen, J. N., Garrity, D. M. & Fishman, M. C. Patterning of angiogenesis in the zebrafish embryo. Development 129, 973–982 (2002).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. 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  PubMed  PubMed Central  Google Scholar 

  29. Atri, D., Larrivee, B., Eichmann, A. & Simons, M. Endothelial signaling and the molecular basis of arteriovenous malformation. Cell Mol. Life Sci. 71, 867–883 (2014).

    Article  CAS  Google Scholar 

  30. Bagatto, B. & Burggren, W. A three-dimensional functional assessment of heart and vessel development in the larva of the zebrafish (Danio rerio). Physiol. Biochem. Zool. 79, 194–201 (2006).

    Article  PubMed  Google Scholar 

  31. Malone, M. H. et al. Laser-scanning velocimetry: a confocal microscopy method for quantitative measurement of cardiovascular performance in zebrafish embryos and larvae. BMC Biotechnol. 7, 40 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. Jamison, R. A., Samarage, C. R., Bryson-Richardson, R. J. & Fouras, A. In vivo wall shear measurements within the developing zebrafish heart. PLoS ONE 8, e75722 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Pappano, A. J. & Wier, W. G. Cardiovascular Physiology (Elsevier/Mosby, 2013).

    Google Scholar 

  34. Zhong, M. C., Wei, X. B., Zhou, J. H., Wang, Z. Q. & Li, Y. M. Trapping red blood cells in living animals using optical tweezers. Nat. Commun. 4, 1768 (2013).

    PubMed  Google Scholar 

  35. Levesque, M. J. & Nerem, R. M. The elongation and orientation of cultured endothelial cells in response to shear stress. J. Biomech. Eng. 107, 341–347 (1985).

    Article  CAS  PubMed  Google Scholar 

  36. Boon, R. A. et al. KLF2-induced actin shear fibers control both alignment to flow and JNK signaling in vascular endothelium. Blood 115, 2533–2542 (2010).

    Article  CAS  PubMed  Google Scholar 

  37. Nicoli, S. et al. MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Nature 464, 1196–1200 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Melchionna, R. et al. Laminar shear stress inhibits CXCR4 expression on endothelial cells: functional consequences for atherogenesis. FASEB J. 19, 629–631 (2005).

    Article  CAS  PubMed  Google Scholar 

  39. Hultin, S. et al. AmotL2 links VE-cadherin to contractile actin fibres necessary for aortic lumen expansion. Nat. Commun. 5, 3743 (2014).

    CAS  PubMed  Google Scholar 

  40. Corti, P. et al. Interaction between alk1 and blood flow in the development of arteriovenous malformations. Development 138, 1573–1582 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ong, A. C., Devuyst, O., Knebelmann, B. & Walz, G. Autosomal dominant polycystic kidney disease: the changing face of clinical management. Lancet 385, 1993–2002 (2015).

    Article  PubMed  Google Scholar 

  42. McDonald, J., Bayrak-Toydemir, P. & Pyeritz, R. E. Hereditary hemorrhagic telangiectasia: an overview of diagnosis, management, and pathogenesis. Genet. Med. 13, 607–616 (2011).

    Article  PubMed  Google Scholar 

  43. Sigurbjornsdottir, S., Mathew, R. & Leptin, M. Molecular mechanisms of de novo lumen formation. Nat. Rev. Mol. Cell Biol. 15, 665–676 (2014).

    Article  PubMed  CAS  Google Scholar 

  44. Costantini, F. & Kopan, R. Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development. Dev. Cell 18, 698–712 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Forster, D. & Luschnig, S. Src42A-dependent polarized cell shape changes mediate epithelial tube elongation in Drosophila. Nat. Cell Biol. 14, 526–534 (2012).

    Article  PubMed  CAS  Google Scholar 

  46. Lubarsky, B. & Krasnow, M. A. Tube morphogenesis: making and shaping biological tubes. Cell 112, 19–28 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Zuo, L., Iordanou, E., Chandran, R. R. & Jiang, L. Novel mechanisms of tube-size regulation revealed by the Drosophila trachea. Cell Tissue Res. 354, 343–354 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. Dietrich, A. C., Lombardo, V. A., Veerkamp, J., Priller, F. & Abdelilah-Seyfried, S. Blood flow and Bmp signaling control endocardial chamber morphogenesis. Dev. Cell 30, 367–377 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Kim, C., Ye, F. & Ginsberg, M. H. Regulation of integrin activation. Annu. Rev. Cell Dev. Biol. 27, 321–345 (2011).

    Article  CAS  PubMed  Google Scholar 

  50. Munjal, A. & Lecuit, T. Actomyosin networks and tissue morphogenesis. Development 141, 1789–1793 (2014).

    Article  CAS  PubMed  Google Scholar 

  51. Tzima, E., del Pozo, M. A., Shattil, S. J., Chien, S. & Schwartz, M. A. Activation of integrins in endothelial cells by fluid shear stress mediates Rho-dependent cytoskeletal alignment. EMBO J. 20, 4639–4647 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Muenzner, P., Bachmann, V., Zimmermann, W., Hentschel, J. & Hauck, C. R. Human-restricted bacterial pathogens block shedding of epithelial cells by stimulating integrin activation. Science 329, 1197–1201 (2010).

    Article  CAS  PubMed  Google Scholar 

  53. Tulis, D. A., Unthank, J. L. & Prewitt, R. L. Flow-induced arterial remodeling in rat mesenteric vasculature. Am. J. Physiol. 274, H874–H882 (1998).

    CAS  PubMed  Google Scholar 

  54. Unthank, J. L., Nixon, J. C., Burkhart, H. M., Fath, S. W. & Dalsing, M. C. Early collateral and microvascular adaptations to intestinal artery occlusion in rat. Am. J. Physiol. 271, H914–H923 (1996).

    CAS  PubMed  Google Scholar 

  55. Jin, Y. et al. Endoglin prevents vascular malformation by regulating flow-induced cell migration and specification through VEGFR2 signalling. Nat. Cell Biol. http://dx.doi.org/10.1038/ncb3534 (2017).

  56. Whitesell, T. R. et al. An α-smooth muscle actin (acta2/αsma) zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells. PLoS ONE 9, e90590 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Mancini, M. L. et al. Endoglin plays distinct roles in vascular smooth muscle cell recruitment and regulation of arteriovenous identity during angiogenesis. Dev. Dyn. 238, 2479–2493 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Westerfield, M. The Zebrafish Book (University of Oregon Press, 1993).

    Google Scholar 

  59. Allinson, K. R., Carvalho, R. L., van den Brink, S., Mummery, C. L. & Arthur, H. M. Generation of a floxed allele of the mouse Endoglin gene. Genesis 45, 391–395 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Wang, Y. et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483–486 (2010).

    Article  CAS  PubMed  Google Scholar 

  61. Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39, e82 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Villefranc, J. A., Amigo, J. & Lawson, N. D. Gateway compatible vectors for analysis of gene function in the zebrafish. Dev. Dyn. 236, 3077–3087 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Bussmann, J. & Schulte-Merker, S. Rapid BAC selection for tol2-mediated transgenesis in zebrafish. Development 138, 4327–4332 (2011).

    Article  CAS  PubMed  Google Scholar 

  64. Siekmann, A. F. & Lawson, N. D. Notch signalling limits angiogenic cell behaviour in developing zebrafish arteries. Nature 445, 781–784 (2007).

    Article  CAS  PubMed  Google Scholar 

  65. Mori, N. & Chang, K. PIV toolbox in MATLAB-Getting Started with mPIV (2004).

    Google Scholar 

  66. Hove, J. R. et al. Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis. Nature 421, 172–177 (2003).

    Article  CAS  PubMed  Google Scholar 

  67. Anton, H. et al. Pulse propagation by a capacitive mechanism drives embryonic blood flow. Development 140, 4426–4434 (2013).

    Article  CAS  PubMed  Google Scholar 

  68. Fåhræus, R. & Lindqvist, T. The viscosity of the blood in narrow capillary tubes. Am. J. Physiol. 96, 562–568 (1931).

    Google Scholar 

  69. Goetz, J. G. et al. Endothelial cilia mediate low flow sensing during zebrafish vascular development. Cell Rep. 6, 799–808 (2014).

    Article  CAS  PubMed  Google Scholar 

  70. Taubin, G. Estimation of planar curves, surfaces, and nonplanar space-curves defined by implicit equations with applications to edge and range image segmentation. IEEE Trans. Pattern Anal. 13, 1115–1138 (1991).

    Article  Google Scholar 

  71. Eberly, D. H. 3D Game Engine Design: A Practical Approach to Real-time Computer Graphics 2nd edn (Morgan Kaufmann Publishers, 2007).

    Google Scholar 

Download references

Acknowledgements

This work was funded by the Max Planck Society (A.F.S.), the Deutsche Forschungsgemeinschaft (DFG SI-1374/3-2; DFG SI-1374/4-1; DFG SI-1374/5-1; A.F.S.), and a European Research Council (ERC) starting grant (260794-ZebrafishAngio; A.F.S.). This work was supported by the Deutsche Forschungsgemeinschaft (DFG) Cells-in-Motion Cluster of Excellence (EXC 1003-CIM), University of Münster, Germany. J.B. was supported by a long-term EMBO post-doctoral fellowship. L.J. was supported by The Swedish Research Council, The Swedish Cancer Society and Karolinska Institutet. We thank H. Arthur for providing the Engflox/flox mice and R. Adams for the Cdh5(PAC)-CreERT2 mice.

Author information

Author notes

  1. Jeroen Bussmann

    Present address: Department of Supramolecular & Biomaterials Chemistry, Leiden Institute of Chemistry (LIC), Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA, Leiden, The Netherlands

Authors and Affiliations

  1. Max Planck Institute for Molecular Biomedicine, Roentgenstrasse 20, D-48149, Muenster, Germany

    Wade W. Sugden, Roman Tsaryk, Elvin V. Leonard, Jeroen Bussmann, Mailin J. Hamm, Wiebke Herzog & Arndt F. Siekmann

  2. Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), University of Muenster, D-48149, Muenster, Germany

    Wade W. Sugden, Robert Meissner, Roman Tsaryk, Elvin V. Leonard, Mailin J. Hamm, Wiebke Herzog, Cornelia Denz & Arndt F. Siekmann

  3. Institute of Applied Physics and Center for Nonlinear Science (CeNoS), University of Muenster, D-48149, Muenster, Germany

    Robert Meissner & Cornelia Denz

  4. Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, 8057, Zurich, Switzerland

    Tinri Aegerter-Wilmsen

  5. University of Muenster, D-48149, Muenster, Germany

    Mailin J. Hamm & Wiebke Herzog

  6. Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE 171 77, Stockholm, Sweden

    Yi Jin & Lars Jakobsson

Authors
  1. Wade W. Sugden
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert Meissner
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tinri Aegerter-Wilmsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roman Tsaryk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Elvin V. Leonard
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jeroen Bussmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Mailin J. Hamm
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Wiebke Herzog
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yi Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Lars Jakobsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Cornelia Denz
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Arndt F. Siekmann
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

W.W.S. and A.F.S. designed and interpreted the experiments and wrote the manuscript. J.B. identified the zebrafish eng homologue and generated the Tg(klf2a:YFP)mu107 zebrafish. R.T. performed cell culture experiments. E.V.L. performed FACS sorting of zebrafish embryos and performed qPCR experiments. R.M. and C.D. performed particle velocimetry, the optical rail experiments and determined flow parameters. T.A.-W. performed zebrafish cell shape analysis. M.J.H. and W.H. generated Tg(fli1a:lifeactEGFP)mu240 zebrafish. Y.J. and L.J. determined cell shape changes in eng mutant mice.

Corresponding author

Correspondence to Arndt F. Siekmann.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of a zebrafish endoglin orthologue.

(a) Schematic representation of the endoglin locus showing conservation of gene order (synteny) between human chromosome 9q33-34 and two paralogous loci on zebrafish chromosome 6 containing the Pregnancy-associated plasma protein A (pappa), Astrotactin 2 (astn2), a retrotransposed Atrial myosin light chain (myl4-RT), Deleted in bladder cancer 1 (dbc1), endoglin (eng), adenylate kinase 1 (ak1) and G-protein signaling modulator 1 (gpsm1) genes. Paralogous genes that were likely duplicated during the teleost whole genome duplication (WGD) event are indicated in blue. Subsequent gene loss on the paralogous chromosomes (lost genes in grey) most likely resulted in the current gene order. Pink shading indicates a small synteny block containing the Endoglin and AK1 genes. Dashed lines indicate synteny breaks between human and zebrafish. (b) Alignment of the conserved C-terminal region of zebrafish and human Endoglin. Asterisks indicate conserved residues. (c) Phylogenetic tree based on the alignment of the C-terminal sequences (transmembrane region and C-terminal domain) of vertebrate Tgfbr3 (green shading) and Endoglin (red shading). Numbers at branchpoints represent bootstrap values (percentage out of 1000 replicates). (d) Whole-mount in situ hybridization of eng shows vascular-restricted expression in 52 hpf zebrafish embryos. Expression is detected in the basal communicating artery (BCA), the posterior communicating segments (PCS), basilar artery (BA), primordial hindbrain channels (PHBC) and central arteries (CtAs) of the hindbrain as well as the dorsal aorta (DA, arrows), posterior cardinal vein (PCV) and dorsal longitudinal anastomotic vessel (DLAV, arrowheads) of the trunk vasculature. Scale bar in overview picture is 500 μm, in high magnification images scale bar is 100 μm. Images are representative of 48 out of 49 embryos. (e) Whole-mount in situ hybridization of eng expression in WT embryos treated with 15 uM nifedipine from 48–52 hpf. Note that blood flow reduction does not affect hindbrain expression, but decreases eng mRNA levels in the DLAV (arrowheads). Scale bar in overview picture is 500 μm, in high magnification images scale bar is 100 um. Images are representative of 45 out of 46 embryos.

Supplementary Figure 2 eng mRNA is subject to nonsense-mediated decay in engmu130 mutants.

(a) qPCR to detect zebrafish eng in FACS-sorted arterial and venous ECs from pooled 72 hpf embryos. Data is shown as relative expression normalized to WT arterial EC levels. In WTs, eng transcript can be detected at similar levels in the arterial and venous populations, and is almost non-detectable in engmu130 mutant ECs. Experiments were performed n = 3 times independently and were analysed by paired Student’s t-test. (b) Whole-mount in situ hybridization for endoglin expression at 32 hpf in embryos from WT, engmu130 and engmu132 heterozygous incrosses. All embryos carrying engmu132 alleles (which contain a 7nt substitution that does not introduce a frameshift) show the same staining intensity as WT, with particularly strong expression in the PCV (arrowhead). engmu130 +/− embryos already show decreased staining, which disappears almost entirely from the head and trunk vasculature in engmu130 mutants (arrows). Scale bar is 500 um. Images are representative of the number of embryos as indicated in the respective panel. NS, not significant, ∗P < 0.05,∗∗P < 0.01, ∗∗∗P < 0.001, error bars indicate s.e.m.

Supplementary Figure 3 ISV remodelling in WT and engmu130 17 dpf juveniles.

(a,b) Maximum intensity projections of confocal z-stacks of 17 dpf WT (a) and engmu130 mutants (b). Yellow arrowheads point to remodelled ISVs that no longer maintain connection to the DLAV. Scale bar is 100 μm. (c–f) Quantification of aISV identity (c), vISV identity (d), ISVs containing DsRed + erythrocytes (e) and remodelled ISVs (f). WTs and engmu130 mutants have no difference in ISV distribution or presence of RBCs, but engmu130 mutants show increased remodelling (n = 15 siblings (158 ISVs analysed) and n = 12 muts (122 ISVs analysed)). Analysed by unpaired Student’s t-test. NS, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, error bars indicate s.e.m.

Supplementary Figure 4 Arterial-venous specification is unaffected in endoglin mutants.

(a,b) Arterial-venous identity of ISVs as determined by flow direction and/or morphology (connection to DA or PCV) in WTs and engmu130 mutants. WTs and mutants have similar distribution of arteries and veins, with a slight increase in the number of unresolved ISV connections in engmu130 mutants. Indicated numbers of ISVs from 9 WT and 12 mut embryos were analysed. (c) Maximum intensity projection of confocal z-stack of engmu130 mutants at 72 hpf. Tg(fli1a:eng-GFP)mu156 embryos exhibit vascular-specific GFP expression, evidenced by overlap with Tg(kdrl:Hsa.HRAS-mCherry)s916. Scale bar is 100 μm. (d,e) Quantification of DA (d) and PCV (e) diameters in engmu130 mutants with or without the Tg(fli1a:eng-GFP)mu156 rescue construct. Tagged endoglin-GFP modestly rescues DA, but not PCV diameters (n = 12 GFP-negative, n = 13 GFP-positive engmu130 mutants). Analysed by unpaired Student’s t-test. (f) Quantification of the number of ISVs actively carrying RBCs shows rescue in engmu130 embryos carrying the Tg(fli1a:eng-GFP)mu156 rescue construct. 683 ISVs from 13 GFP-positive engmu130 mut embryos were analysed. NS, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, error bars indicate s.e.m.

Supplementary Figure 5 Generation of the Tg(klf2a:YFP)mu107 reporter line.

(a) Schematic of the cloning strategy. A Citrine cassette was inserted behind the endogenous ATG of the klf2a gene contained in the CH73-180A21 BAC by homologous recombination. (b,c) Tg(klf2a:YFP)mu107 embryos display vascular-specific YFP expression, evidenced by overlap with Tg(kdrl:Hsa.HRAS-mCherry)s916. Embryos treated with tnnt2a MO lose YFP expression in the DA and PCV. Scale bar is 100 μm. (d–f) Quantification of YFP fluorescence intensity in the DA, PCV and ISVs in ctr MO and tnnt2a MO injected embryos (DA/PCV: n = 15 control MO, n = 15 tnnt2a MO; ISVs: n = 126 ISVs from 15 control MO, n = 133 ISVs from 15 tnnt2a MO). Analysed by Mann-Whitney U test. NS, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, error bars indicate s.e.m.

Supplementary Figure 6 Transplantation analysis of mosaic ISVs.

(a–c) Maximum intensity projections of confocal z-stack of mosaic ISVs from (a) WT- > WT, (b) Mut- > WT and (c) WT- > Mut transplantations. Optical sections reveal portions of lumen comprised only of donor cells, only of host cells, or a mosaic combination of host and donor cells. Scale bar on overview is 50 um, scale bar on lumen cross-section is 10 μm. (d-f) Quantification of mosaic ISV diameters depending on cellular composition from (d) WT- > WT, (e) Mut- > WT and (f) WT- > Mut transplantations. All ISVs from WT- > WT transplantations had normal diameters (d), while mutant cells caused dilation when they formed part or all of the lumen in Mut- > WT situations (e). WT cells were able to reduce ISV diameter when they formed all of the lumen in WT- > Mut scenarios (f). (WT- > WT n = 216 vessel segments from 54 ISVs; Mut- > WT n = 44 vessel segments from 11 ISVs; WT- > Mutn = 204 vessel segments from 51 ISVs). Analysed by One-Way ANOVA. (g) Schematic summarizing the requirement for eng function in every cell of a vascular tube to maintain proper dimensions. NS, not significant, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, error bars indicate s.e.m.

Supplementary Figure 7 Model for endoglin’s role in the biphasic response of ECs to blood flow.

(a) By 24 hpf, the first artery and vein have assembled by vasculogenesis. Blood flow starts. (b) As development proceeds, cardiac output rises. The axial vessels increase in diameter and carry an increasing volume of blood. At 48 hpf, the capillaries connecting the artery and vein have just begun to lumenize and carry flow. (c) Between 48 and 72 hpf, blood flow triggers shape changes in WT ECs. They elongate and align in the direction of flow, without changing their surface areas. This lengthens the vessel and reduces its diameter. The arterial constriction redirects blood flow through the capillary network. (d) In eng-deficient blood vessels, ECs are unable to limit their surface areas in response to blood flow. Although elongation still occurs to some extent, the increase in size impedes the ability of ECs to align to the flow. Ultimately, this results in vessel dilation and the formation of an arterio-venous shunt. The high velocities achieved in the shunt prevent RBCs from passing into capillaries, and the pulsatile quality of arterial flow is transmitted to the venous return.

Supplementary Figure 8

Original scanned blots used in Fig. 5e.

Supplementary information

Supplementary Information

Supplementary Information (PDF 2443 kb)

Distal blood flow in WT fin regenerate at 5 dpa.

Brightfield movie of blood flow in the distal regenerating fin of WT fish at 5 dpa. Numbers label individual bony rays. Arrows indicate flow direction, while dashed lines denote arteries (red) and veins (blue). Flow from a centrally located artery distributes through a plexus to two veins at the end of each ray. Note weak flow reversal in lateral vein of the first ray, arcing over an inactive artery. (MP4 19382 kb)

AVMs and disturbed flow in engmu130 fin regenerate at 5 dpa.

Brightfield movie of blood flow in the distal regenerating fin of engmu130 mutant at 5 dpa. Numbers label individual bony rays. Arrows indicate flow direction, while dashed lines denote arteries (red) and veins (blue). Note prominent AVMs in second ray from the left. In ray #3, more proximally located AVMs either stop or reverse blood flow in the veins, and the central artery is not active. Ray #4 has strong flow reversal in the left vein, arcing over an inactive central artery. Note bleedings at distal end of all rays. (MP4 25340 kb)

ISV flow in 72 hpf sibling embryo.

Brightfield movie of RBC flow in the trunk of sibling embryo at 72 hpf. Nearly all ISVs on both sides of the embryo carry RBC flow. (MP4 23761 kb)

ISV flow in 72 hpf engmu130 embryo.

Brightfield movie of RBC flow in the trunk of engmu130 embryo at 72 hpf. Note that only 1 or 2 ISVs have persistent RBC flow, while most have no or only very intermittent RBC flow. (MP4 28677 kb)

Optical rail restores RBC flow in capillaries of engmu130 mutants.

Brightfield movie of RBC flow in the trunk of engmu130 embryo at 72 hpf. Holographic optical tweezers (HOT) are focused at entrance to an aISV, but laser is turned off. About halfway through movie the laser is turned on (red circle indicates HOT focal point), slows down RBCs that pass through the laser, and diverts them from the main arterial blood flow into the capillary. (MP4 7546 kb)

Time-lapse of cell shape changes in DA between 48 and 72 hpf in sibling embryo.

Maximum intensity projection of confocal z-stacks of Tg(fli1a:lifeactEGFP)mu240; Tg(fli1a:nEGFP)y7, and Tg(-0.8flt1:RFP)hu5333 in sibling embryo between 48 and 72 hpf. Acquisitions were made at 40 min intervals, shown at 5 frames per second. Note decrease in DA diameter between 48 and 72 hpf, and elongation of ECs. (MP4 1378 kb)

Time-lapse of cell shape changes in DA between 48 and 72 hpf in engmu130 embryo.

Maximum intensity projection of confocal z-stacks of Tg(fli1a:lifeactEGFP)mu240; Tg(fli1a:nEGFP)y7z, and Tg(-0.8flt1:RFP)hu5333 in engmu130 embryo between 48 and 72 hpf. Acquisitions were made at 40 min intervals, shown at 5 frames per second. Note a similar DA diameter at 48 hpf as sibling in Supplementary Video 4 that fails to decrease appreciably by 72 hpf. Note, additionally, the dramatic increase in EC sizes compared to siblings during the movie. (MP4 1437 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Sugden, W., Meissner, R., Aegerter-Wilmsen, T. et al. Endoglin controls blood vessel diameter through endothelial cell shape changes in response to haemodynamic cues. Nat Cell Biol 19, 653–665 (2017). https://doi.org/10.1038/ncb3528

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3528

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