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.

Formation of the yolk sac vascular system and its connection to the embryonic circulation is crucial for embryo survival in both mammals and birds. Most mice with mutations in genes involved in vascular development die because of a failure to establish this circulatory loop. Surprisingly,formation of yolk sac arteries and veins has not been well described in the recent literature. Using time-lapse video-microscopy, we have studied arterial-venous differentiation in the yolk sac of chick embryos. Immediately after the onset of perfusion, the yolk sac exhibits a posterior arterial and an anterior venous pole, which are connected to each other by cis-cis endothelial interactions. To form the paired and interlaced arterial-venous pattern characteristic of mature yolk sac vessels, small caliber vessels of the arterial domain are selectively disconnected from the growing arterial tree and subsequently reconnected to the venous system, implying that endothelial plasticity is needed to fashion normal growth of veins. Arterial-venous differentiation and patterning are controlled by hemodynamic forces, as shown by flow manipulation and in situ hybridization with arterial markers ephrinB2 and neuropilin 1, which show that expression of both mRNAs is not genetically determined but plastic and regulated by flow. In vivo application of ephrinB2 or EphB4 in the developing yolk sac failed to produce any morphological effects. By contrast, ephrinB2 and EphB4 application in the allantois of older embryos resulted in the rapid formation of arterial-venous shunts. In conclusion, we show that flow shapes the global patterning of the arterial tree and regulates the activation of the arterial markers ephrinB2 and neuropilin 1.

Netrins are secreted molecules with roles in axonal growth and angiogenesis. The Netrin receptor UNC5B is required during embryonic development for vascular patterning, suggesting that it may also contribute to postnatal and pathological angiogenesis. Here we show that unc5b is down-regulated in quiescent adult vasculature, but re-expressed during sprouting angiogenesis in matrigel and tumor implants. Stimulation of UNC5B-expressing neovessels with an agonist (Netrin-1) inhibits sprouting angiogenesis. Genetic loss of function of unc5b reduces Netrin-1-mediated angiogenesis inhibition. Expression of UNC5B full-length receptor also triggers endothelial cell repulsion in response to Netrin-1 in vitro, whereas a truncated UNC5B lacking the intracellular signaling domain fails to induce repulsion. These data show that UNC5B activation inhibits sprouting angiogenesis, thus identifying UNC5B as a potential anti-angiogenic target.[Keywords: Vessel guidance; axon guidance molecules; tip cell; neovascularization; tumor angiogenesis] Supplemental material is available at http://www.genesdev.org. Three members of the netrin gene family, netrin-1, netrin-3, and ␤-netrin/netrin-4, have been identified in mammals (Serafini et al. 1996;Van Raay et al. 1997;Wang et al. 1999;Koch et al. 2000;Yin et al. 2000). Netrins are bifunctional guidance cues, attracting some axons while repelling others (Dickson 2002). Netrin-1 is secreted from cells at the ventral midline of the central nervous system and attracts commissural axons toward the midline. Netrins can, however, also repel certain axons, including the trochlear motor axons in vertebrates (Colamarino and Tessier-Lavigne 1995). Attraction and repulsion are mediated via activation of receptors of the deleted in colorectal cancer (DCC) and uncoordinated 5 (UNC5) families, respectively. The DCC family consists of DCC and Neogenin Keino-Masu et al. 1996), while the UNC5 family comprises four members, UNC5A to UNC5D (Leung-Hagesteijn et al. 1992;Leonardo et al. 1997). Axon attraction is mediated by the DCC receptors (Fazeli et al. 1997), while repulsion requires signaling through UNC5-DCC receptor heterodimers or UNC5 receptor homodimers (Hedgecock et al. 1990;Hong et al. 1999;Keleman and Dickson 2001).In addition to their role in axon guidance, Netrins and their receptors have been implicated in other develop- Article is online at http://www.genesdev.org/cgi