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

Robo4 is an endothelial cell-specific member of the Roundabout axon guidance receptor family. To identify Robo4 binding partners, we performed a protein-protein interaction screen with the Robo4 extracellular domain. We find that Robo4 specifically binds to UNC5B, a vascular Netrin receptor, revealing unexpected interactions between two endothelial guidance receptors. We show that Robo4 maintains vessel integrity by activating UNC5B, which inhibits signaling downstream of vascular endothelial growth factor (VEGF). Function-blocking monoclonal antibodies against Robo4 and UNC5B increase angiogenesis and disrupt vessel integrity. Soluble Robo4 protein inhibits VEGF-induced vessel permeability and rescues barrier defects in Robo4(-/-) mice, but not in mice treated with anti-UNC5B. Thus, Robo4-UNC5B signaling maintains vascular integrity by counteracting VEGF signaling in endothelial cells, identifying a novel function of guidance receptor interactions in the vasculature.

Basal cell carcinoma, the most frequent human skin cancer, arises from activating hedgehog (HH) pathway mutations; however, little is known about the temporal changes that occur in tumour-initiating cells from the first oncogenic hit to the development of invasive cancer. Using an inducible mouse model enabling the expression of a constitutively active Smoothened mutant (SmoM2) in the adult epidermis, we carried out transcriptional profiling of SmoM2-expressing cells at different times during cancer initiation. We found that tumour-initiating cells are massively reprogrammed into a fate resembling that of embryonic hair follicle progenitors (EHFPs). Wnt/ β-catenin signalling was very rapidly activated following SmoM2 expression in adult epidermis and coincided with the expression of EHFP markers. Deletion of β-catenin in adult SmoM2-expressing cells prevents EHFP reprogramming and tumour initiation. Finally, human basal cell carcinomas also express genes of the Wnt signalling and EHFP signatures.

Rationale The lymphatic vasculature plays a major role in fluid homeostasis, absorption of dietary lipids, and immune surveillance. Fluid transport depends on the presence of intraluminal valves within lymphatic collectors. Defective formation of lymphatic valves leads to lymphedema, a progressive and debilitating condition for which curative treatments are currently unavailable. How lymphatic valve formation is regulated remains largely unknown. Objective We investigated if the repulsive axon guidance molecule Semaphorin3A (Sema3A) plays a role in lymphatic valve formation. Methods and Results We show that Sema3A mRNA is expressed in lymphatic vessels and that Sema3A protein binds to lymphatic valves expressing the Neuropilin-1 (Nrp1) and PlexinA1 receptors. Using mouse knockout models, we show that Sema3A is selectively required for lymphatic valve formation, via interaction with Nrp1 and PlexinA1. Sema3a−/− mice exhibit defects in lymphatic valve formation, which are not due to abnormal lymphatic patterning or sprouting, and mice carrying a mutation in the Sema3A binding site of Nrp1, or deficient for Plxna1, develop lymphatic valve defects similar to those seen in Sema3a−/− mice. Conclusions Our data demonstrate an essential direct function of Sema3A-Nrp1-PlexinA1 signaling in lymphatic valve formation.

Autonomic sympathetic nerves innervate peripheral resistance arteries, thereby regulating vascular tone and controlling blood supply to organs. Despite the fundamental importance of blood flow control, how sympathetic arterial innervation develops remains largely unknown. Here, we identified the axon guidance cue netrin-1 as an essential factor required for development of arterial innervation in mice. Netrin-1 was produced by arterial smooth muscle cells (SMCs) at the onset of innervation, and arterial innervation required the interaction of netrin-1 with its receptor, deleted in colorectal cancer (DCC), on sympathetic growth cones. Function-blocking approaches, including cell type-specific deletion of the genes encoding Ntn1 in SMCs and Dcc in sympathetic neurons, led to severe and selective reduction of sympathetic innervation and to defective vasoconstriction in resistance arteries. These findings indicate that netrin-1 and DCC are critical for the control of arterial innervation and blood flow regulation in peripheral organs.

Focal adhesion kinase (FAK) regulates numerous cellular functions and is critical for processes ranging from embryo development to cancer progression. Although autophosphorylation on Tyr-397 appears required for FAK functions in vitro, its role in vivo has not been established. We addressed this question using a mutant mouse (fak⌬) deleted of exon 15, which encodes Tyr-397. The resulting mutant protein FAK⌬ is an active kinase expressed at normal levels. Our results demonstrate that the requirement for FAK autophosphorylation varies during development. FAK ⌬/⌬ embryos developed normally up to embryonic day (E) 12.5, contrasting with the lethality at E8.5 of FAK-null embryos. Thus, autophosphorylation on Tyr-397 is not required for FAK to achieve its functions until late mid-gestation. However, FAK ⌬/⌬ embryos displayed hemorrhages, edema, delayed artery formation, vascular remodeling defects, multiple organ abnormalities, and overall developmental retardation at E13.5-14.5, and died thereafter demonstrating that FAK autophosphorylation is also necessary for normal development. Fibroblasts derived from mutant embryos had a normal stellate morphology and expression of focal adhesion proteins, Src family members, p53, and Pyk2. In contrast, in FAK ⌬/⌬ fibroblasts and endothelial cells, spreading and lamellipodia formation were altered with an increased size and number of focal adhesions, enriched in FAK⌬. FAK mutation also decreased fibroblast proliferation. These results show that the physiological functions of FAK in vivo are achieved through both autophosphorylation-independent and autophosphorylation-dependent mechanisms.Focal adhesion kinase (FAK) 2 is a nonreceptor tyrosine kinase critical for processes ranging from embryo development (1) to cancer invasiveness and metastasis (2). FAK activation following integrin engagement or stimulation of a variety of transmembrane receptors triggers its phosphorylation on tyrosine and the formation of multimolecular signaling complexes (3). FAK is enriched in focal adhesions, controlling their turnover and consequently adhesion-related processes such as spreading, migration, survival, and proliferation (1).The important physiological role of FAK is demonstrated by the lethality of its null mutation at embryonic day (E) 8.5 (4, 5). Further studies using conditional deletion showed that FAK regulates the development of the nervous system (6 -9), morphogenesis of the vascular network (5, 10, 11), and cardiac development (12-15). These reports clearly established that FAK is necessary for essential processes in vivo.In vitro studies have shown that, following its recruitment to focal adhesions, FAK autophosphorylation on Tyr-397 creates a high affinity binding site for multiple signaling proteins, including the Src family kinases (SFKs) (3). Following their binding to phospho-Tyr-397 and activation, SFKs phosphorylate other FAK residues inducing its complete activation, its interaction with other signaling proteins, and the stimulation of downstream signaling cascades (16). ...

Netrin-1 is a bifunctional axonal guidance cue, capable of attracting or repelling developing axons via activation of receptors of the deleted in colorectal cancer (DCC) and uncoordinated 5 (UNC5) families, respectively. In addition to its role in axon guidance, Netrin-1 has been implicated in angiogenesis, where it may also act as a bifunctional cue. Attractive effects of Netrin-1 on endothelial cells appear to be mediated by an as yet unknown receptor, while repulsion of developing blood vessels in mouse embryos is mediated by the UNC5B receptor. To explore evolutionary conservation of vascular UNC5B expression and function, we have cloned the chick unc5b homologue. Chick and quail embryos showed unc5b expression in arterial EC and sprouting angiogenic capillaries. To test if Netrin-1 displayed pro- or anti-angiogenic activities in the avian embryo, we grafted cell lines expressing recombinant chick or human Netrin-1 at different stages of development. Netrin-1 expressing cells inhibited angiogenic sprouting of unc5b expressing blood vessels, but had no pro-angiogenic activity at any stage of development examined. Netrin-1 also had no effect on the recruitment of circulating endothelial precursor cells. Taken together, these data indicate that vascular unc5b expression and function is conserved between chick and mice.

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