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The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system

Abstract

Blood vessels and nerves are complex, branched structures that share a high degree of anatomical similarity. Guidance of vessels and nerves has to be exquisitely regulated to ensure proper wiring of both systems. Several regulators of axon guidance have been identified and some of these are also expressed in endothelial cells; however, the extent to which their guidance functions are conserved in the vascular system is still incompletely understood. We show here that the repulsive netrin receptor UNC5B is expressed by endothelial tip cells of the vascular system. Disruption of the Unc5b gene in mice, or of Unc5b or netrin-1a in zebrafish, leads to aberrant extension of endothelial tip cell filopodia, excessive vessel branching and abnormal navigation. Netrin-1 causes endothelial filopodial retraction, but only when UNC5B is present. Thus, UNC5B functions as a repulsive netrin receptor in endothelial cells controlling morphogenesis of the vascular system.

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Figure 1: Unc5b expression in the vascular system.
Figure 2: Increased capillary branching and tip-cell filipodia extension in Unc5b mouse and zebrafish mutant embryos.
Figure 3: Abnormal morphology of Unc5b mouse mutant arteries.
Figure 4: Netrin-1 reduces endothelial cell migration and filopodial extension in vitro.
Figure 5: Netrin-1 stimulates filopodial retraction of endothelial cells in vivo.
Figure 6: Netrin-1-induced filopodial retraction is lost in the absence of Unc5b.

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Acknowledgements

We thank members of U36 for critical discussions; E. Etienne, A. Réaux, C. Esguerra and S. Maity for their expertise; C. Betsholtz for the Pdgfrb probe; H. Gerhardt for sharing unpublished data; and H. Rayburn, C. Jolicoeur, J. Zhong, S. Terclavers, A. Claes, A. Vanhuffelen and S. Wyns for technical support. This work was supported by grants from Inserm (Avenir), Fondation Schlumberger pour l'Education et la Recherche (FSER), Ministère de l'Education et de la Recherche (ACI Biologie du Développement), Association pour la Recherche contre le Cancer (ARC) to A.E., a European Union grant to A.E. and P.C., and grants to M.T.-L. from the NIMH and the NHLBI. F.l.N. was supported by the Royal Dutch Academy of Science (KNAW) and Avenir; X.L. by the Damon Runyon Cancer Research Foundation; and M.T.-L. by the Howard Hughes Medical Institute.

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Correspondence to Marc Tessier-Lavigne or Anne Eichmann.

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Competing interests

M.T.-L. is an inventor of patents covering members of the mammalian UNC5 and netrin families. He is a member of the scientific advisory board and a shareholder of Renovis Inc., and is employed by and a shareholder of Genentech Inc. Both companies have a commercial interest in members of these families.

Supplementary information

Supplementary Figure 1

Unc5b expression in arterial endothelial cells in the mouse retina. Double-labelings of retina wholemounts with isolectinB4, lacZ, Unc5b and pdgfr-β in situ hybridizations. (PPT 6999 kb)

Supplementary Figure 2

Generation of Unc5b mutant mice. Representation of gene inactivation strategy and evidence for absence of Unc5b transcripts in homozygous mutant mouse embryos. (PPT 1596 kb)

Supplementary Figure 3

Specificity of zebrafish morpholino knockdowns (a) and Northern blot analysis of Unc5b expression in primary human endothelial cells (b). (PPT 955 kb)

Supplementary Figure 4

Aortic ring assays. Overview of aortic ring cultures in controls, in the presence of netrin-1/UNC5B-Fc and in the presence of netrin-1, as well as statistical analysis of endothelial cell movement. (PPT 171 kb)

Supplementary Movie 1

Control endothelial tip cell. Random filopodial movement. (MOV 3447 kb)

Supplementary Movie 2

Endothelial tip cells faced with a gradient of pre-clustered Netrin-1/UNC5B-Fc. Random filopodial movement. (MOV 1745 kb)

Supplementary Movie 3

Endothelial tip cells faced with a gradient of Netrin-1, note filopodial retraction and backward movement of the cells. (MOV 3369 kb)

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Lu, X., le Noble, F., Yuan, L. et al. The netrin receptor UNC5B mediates guidance events controlling morphogenesis of the vascular system. Nature 432, 179–186 (2004). https://doi.org/10.1038/nature03080

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