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

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.

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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.

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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).

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Authors and Affiliations

Authors

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.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 (Related to Figure 2.)

(ae) 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 (ad) 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)

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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

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