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1.  Neuropilins: A New Target for Cancer Therapy 
Cancers  2011;3(2):1899-1928.
Recent investigations highlighted strong similarities between neural crest migration during embryogenesis and metastatic processes. Indeed, some families of axon guidance molecules were also reported to participate in cancer invasion: plexins/semaphorins/neuropilins, ephrins/Eph receptors, netrin/DCC/UNC5. Neuropilins (NRPs) are transmembrane non tyrosine-kinase glycoproteins first identified as receptors for class-3 semaphorins. They are particularly involved in neural crest migration and axonal growth during development of the nervous system. Since many types of tumor and endothelial cells express NRP receptors, various soluble molecules were also found to interact with these receptors to modulate cancer progression. Among them, angiogenic factors belonging to the Vascular Endothelial Growth Factor (VEGF) family seem to be responsible for NRP-related angiogenesis. Because NRPs expression is often upregulated in cancer tissues and correlated with poor prognosis, NRPs expression might be considered as a prognostic factor. While NRP1 was intensively studied for many years and identified as an attractive angiogenesis target for cancer therapy, the NRP2 signaling pathway has just recently been studied. Although NRP genes share 44% homology, differences in their expression patterns, ligands specificities and signaling pathways were observed. Indeed, NRP2 may regulate tumor progression by several concurrent mechanisms, not only angiogenesis but lymphangiogenesis, epithelial-mesenchymal transition and metastasis. In view of their multiples functions in cancer promotion, NRPs fulfill all the criteria of a therapeutic target for innovative anti-tumor therapies. This review focuses on NRP-specific roles in tumor progression.
doi:10.3390/cancers3021899
PMCID: PMC3757396  PMID: 24212788
neuropilins; cancer; angiogenesis; lymphangiogenesis; targeted therapies
2.  Determination of Cell Adhesion Sites of Neuropilin-1 
The Journal of Cell Biology  2000;148(6):1283-1294.
Neuropilin-1 is a type 1 membrane protein with three distinct functions. First, it can mediate cell adhesion via a heterophilic molecular interaction. Second, in neuronal cells, neuropilin-1 binds the class 3 semaphorins, which are neuronal chemorepellents, and plays a role in the directional guidance of axons. Neuropilin-1 is expected to form complexes with the plexinA subfamily members and mediate the semaphorin-elicited inhibitory signals into neurons. Third, in endothelial cells, neuropilin-1 binds a potent endothelial cell mitogen, vascular endothelial growth factor (VEGF)165, and regulates vessel formation. Though the binding sites in neuropilin-1 for the class 3 semaphorins and VEGF165 have been analyzed, the sites involved in cell adhesion activity of the molecule have not been identified. In this study, we produced a variety of mutant neuropilin-1s and tested their cell adhesion activity. We showed that the b1 and b2 domains within the extracellular segment of neuropilin-1 were required for the cell adhesion activity, and peptides with an 18–amino acid stretch in the b1 and b2 domains were sufficient to induce the cell adhesion activity. In addition, we demonstrated that the cell adhesion ligands for neuropilin-1 were proteins and distributed in embryonic mesenchymal cells but distinct from the class 3 semaphorins, VEGF, or plexins.
PMCID: PMC2174302  PMID: 10725340
neuropilin-1; cell adhesion; cell aggregation; semaphorins; mutant protein
3.  Guidance molecules in lung cancer 
Cell Adhesion & Migration  2010;4(1):130-145.
Guidance molecules were first described in the nervous system to control axon outgrowth direction. They are also widely expressed outside the nervous system where they control cell migration, tissue development and establishment of the vascular network. In addition, they are involved in cancer development, tumor angiogenesis and metastasis. This review is primarily focused on their functions in lung cancer and their involvement in lung development is also presented. Five guidance molecule families and their corresponding receptors are described, including the semaphorins/neuropilins/plexins, ephrins and Eph receptors, netrin/DCC/UNC5, Slit/Robo and Notch/Delta. In addition, the possibility to target these molecules as a therapeutic approach in cancer is discussed.
PMCID: PMC2852570  PMID: 20139699
lung cancer; semaphorin; neuropilin; slit; robo; ephrin; netrin; DCC; UNC5; notch
4.  The VEGF-regulated transcription factor HLX controls the expression of guidance cues and negatively regulates sprouting of endothelial cells 
Blood  2011;117(9):2735-2744.
The HLX gene encoding a diverged homeobox transcription factor has been found to be up-regulated by vascular endothelial growth factor-A (VEGF-A) in endothelial cells. We have now investigated the gene repertoire induced by HLX and its potential biologic function. HLX strongly increased the transcripts for several repulsive cell-guidance proteins including UNC5B, plexin-A1, and semaphorin-3G. In addition, genes for transcriptional repressors such as HES-1 were up-regulated. In line with these findings, adenoviral overexpression of HLX inhibited endothelial cell migration, sprouting, and vessel formation in vitro and in vivo, whereas proliferation was unaffected. This inhibition of sprouting was caused to a significant part by HLX-mediated up-regulation of UNC5B as shown by short hairpin RNA (shRNA)–mediated down-modulation of the respective mRNA. VEGF-A stimulation of endothelial cells induced elevated levels of HLX over longer time periods resulting in especially high up-regulation of UNC5B mRNA as well as an increase in cells displaying UNC5B at their surface. However, induction of HLX was strongly reduced and UNC5B up-regulation completely abrogated when cells were exposed to hypoxic conditions. These data suggest that HLX may function to balance attractive with repulsive vessel guidance by up-regulating UNC5B and to down-modulate sprouting under normoxic conditions.
doi:10.1182/blood-2010-07-293209
PMCID: PMC3096761  PMID: 21224470
5.  Semaphorin Signaling in Vertebrate Neural Circuit Assembly 
Neural circuit formation requires the coordination of many complex developmental processes. First, neurons project axons over long distances to find their final targets and then establish appropriate connectivity essential for the formation of neuronal circuitry. Growth cones, the leading edges of axons, navigate by interacting with a variety of attractive and repulsive axon guidance cues along their trajectories and at final target regions. In addition to guidance of axons, neuronal polarization, neuronal migration, and dendrite development must be precisely regulated during development to establish proper neural circuitry. Semaphorins consist of a large protein family, which includes secreted and cell surface proteins, and they play important roles in many steps of neural circuit formation. The major semaphorin receptors are plexins and neuropilins, however other receptors and co-receptors also mediate signaling by semaphorins. Upon semaphorin binding to their receptors, downstream signaling molecules transduce this event within cells to mediate further events, including alteration of microtubule and actin cytoskeletal dynamics. Here, I review recent studies on semaphorin signaling in vertebrate neural circuit assembly, with the goal of highlighting how this diverse family of cues and receptors imparts exquisite specificity to neural complex connectivity.
doi:10.3389/fnmol.2012.00071
PMCID: PMC3368236  PMID: 22685427
semaphorin; plexin; neuropilin; axon guidance; synapse formation
6.  VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia 
The Journal of Cell Biology  2003;161(6):1163-1177.
Vascular endothelial growth factor (VEGF-A) is a major regulator of blood vessel formation and function. It controls several processes in endothelial cells, such as proliferation, survival, and migration, but it is not known how these are coordinately regulated to result in more complex morphogenetic events, such as tubular sprouting, fusion, and network formation. We show here that VEGF-A controls angiogenic sprouting in the early postnatal retina by guiding filopodial extension from specialized endothelial cells situated at the tips of the vascular sprouts. The tip cells respond to VEGF-A only by guided migration; the proliferative response to VEGF-A occurs in the sprout stalks. These two cellular responses are both mediated by agonistic activity of VEGF-A on VEGF receptor 2. Whereas tip cell migration depends on a gradient of VEGF-A, proliferation is regulated by its concentration. Thus, vessel patterning during retinal angiogenesis depends on the balance between two different qualities of the extracellular VEGF-A distribution, which regulate distinct cellular responses in defined populations of endothelial cells.
doi:10.1083/jcb.200302047
PMCID: PMC2172999  PMID: 12810700
VEGF; endothelial cell; filopodia; astrocyte; migration; proliferation
7.  Neuropilin-2 acts as a modulator of Sema3A-dependent glioma cell migration 
Cell Adhesion & Migration  2009;3(4):383-389.
Semaphorin 3A (Sema3A) is a secreted guidance molecule initially described in the nervous system. This protein is able to control axon growth but also effects on endothelial cells migration. Here, we report that Sema3A acts as a chemorepellent factor for the rat C6 glioma cells and three different human glioma cell lines. Interestingly, Sema3A triggered a chemoattractive response in a fourth human glioma cell line. The nature of the receptor complex ensuring the appropriate signaling was dissected in C6 cells by using function blocking antibodies and gain- or loss-of function experiments using recombinant receptors. Our results demonstrate that neuropilin-1, neuropilin-2 and PlexinA1 are necessary to trigger cell repulsion. The selective blockade of neuropilin-1 or Plexin-A1 switched the chemorepulsive effect of Sema3A into a chemoattractive one. Strikingly, blocking Neuropilin-2 suppressed Sema3A-induced cell migration while overexpression of neuropilin-2 was able to convert the chemorepulsive effect of Sema3A into a chemoattractive one. Our results not only provide additional evidence for a biological function of Sema3A in glioma migration but also reveal part of the receptor complex involved. Hence, our study describes a receptor-based plasticity in cancer cells leading to opposite migration behavior in response to the same extracellular signal.
PMCID: PMC2802752  PMID: 19855168
semaphorin; neuropilin; glioma; cell migration; signalling; cancer
8.  Activation of ADF/cofilin mediates attractive growth cone turning toward nerve growth factor and netrin-1 
Developmental neurobiology  2010;70(8):565-588.
Proper neural circuitry requires that growth cones, motile tips of extending axons, respond to molecular guidance cues expressed in the developing organism. However, it is unclear how guidance cues modify the cytoskeleton to guide growth cone pathfinding. Here we show acute treatment with two attractive guidance cues, nerve growth factor (NGF) and netrin-1, for embryonic dorsal root ganglion and temporal retinal neurons, respectively, results in increased growth cone membrane protrusion, actin polymerization, and filamentous actin (F-actin). ADF/cofilin (AC) family proteins facilitate F-actin dynamics, and we found the inactive phosphorylated form of AC is decreased in NGF- or netrin-1-treated growth cones. Directly increasing AC activity mimics addition of NGF or netrin-1 to increase growth cone protrusion and F-actin levels. Extracellular gradients of NGF, netrin-1, and a cell-permeable AC elicit attractive growth cone turning and increased F-actin barbed ends, F-actin accumulation, and active AC in growth cone regions proximal to the gradient source. Reducing AC activity blunts turning responses to NGF and netrin. Our results suggest that gradients of NGF and netrin-1 locally activate AC to promote actin polymerization and subsequent growth cone turning toward the side containing higher AC activity.
doi:10.1002/dneu.20800
PMCID: PMC2908028  PMID: 20506164
guidance; neurotrophins; netrin; ADF/cofilin; actin; growth cone
9.  The vesicular SNARE Synaptobrevin is required for Semaphorin 3A axonal repulsion 
The Journal of Cell Biology  2012;196(1):37-46.
Semaphorin 3A-mediated signaling and axonal repulsion in the mouse brain require Synaptobrevin-dependent vesicular traffic.
Attractive and repulsive molecules such as Semaphorins (Sema) trigger rapid responses that control the navigation of axonal growth cones. The role of vesicular traffic in axonal guidance is still largely unknown. The exocytic vesicular soluble N-ethylmaleimide sensitive fusion protein attachment protein receptor (SNARE) Synaptobrevin 2 (Syb2) is known for mediating neurotransmitter release in mature neurons, but its potential role in axonal guidance remains elusive. Here we show that Syb2 is required for Sema3A-dependent repulsion but not Sema3C-dependent attraction in cultured neurons and in the mouse brain. Syb2 associated with Neuropilin 1 and Plexin A1, two essential components of the Sema3A receptor, via its juxtatransmembrane domain. Sema3A receptor and Syb2 colocalize in endosomal membranes. Moreover, upon Sema3A treatment, Syb2-deficient neurons failed to collapse and transport Plexin A1 to cell bodies. Reconstitution of Sema3A receptor in nonneuronal cells revealed that Sema3A further inhibited the exocytosis of Syb2. Therefore, Sema3A-mediated signaling and axonal repulsion require Syb2-dependent vesicular traffic.
doi:10.1083/jcb.201106113
PMCID: PMC3255983  PMID: 22213797
10.  Expression patterns of plexins and neuropilins are consistent with cooperative and separate functions during neural development 
Background
Plexins are a family of transmembrane proteins that were shown to act as receptors for Semaphorins either alone or in a complex together with Neuropilins. Based on structural criteria Plexins were subdivided into 4 classes, A through D. PlexinAs are mainly thought to act as mediators of repulsive signals in cell migration and axon guidance. Their functional role in vertebrates has been studied almost exclusively in the context of Semaphorin signaling, i.e. as co-receptors for class 3 Semaphorins. Much less is known about Plexins of the other three classes. Despite the fact that Plexins are involved in the formation of neuronal circuits, the temporal changes of their expression patterns during development of the nervous system have not been analyzed in detail.
Results
Only seven plexins are found in the chicken genome in contrast to mammals, where nine plexins have been identified. Here, we describe the dynamic expression patterns of all known plexin family members in comparison to the neuropilins in the developing chicken spinal cord.
Conclusion
Our in situ hybridization study revealed that the expression patterns of plexins and neuropilins are only partially overlapping, especially during early and intermediate stages of spinal cord development, supporting both cooperative and separate functions of plexins and neuropilins in neural circuit formation.
doi:10.1186/1471-213X-6-32
PMCID: PMC1543641  PMID: 16846494
11.  Common Cues Regulate Neural and Vascular Patterning 
Summary
Nerves and blood vessels often follow parallel trajectories as they course through the body to their distal targets. Proteins that regulate the process of axon guidance have likewise been shown to play a critical role in blood vessel migration. With the recent description of the endothelial tip cell as an analog of the axonal growth cone, the nerve-vessel analogy seems complete. Notwithstanding these considerable similarities, one critical difference remains between neural and vascular guidance. While a navigating axon is but a single cell, a sprouting vessel is composed of multiple cells that must be coordinately regulated. Recent studies of the Dll4-Notch1 signaling pathway have provided valuable insight into how the vasculature accomplishes this critical task.
doi:10.1016/j.gde.2007.07.004
PMCID: PMC2279096  PMID: 17692512
angiogenesis; vascular guidance; axon guidance cues; blood vessels
12.  Signaling from Axon Guidance Receptors 
Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues—netrins, semaphorins, ephrins, and slits—have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance.
Binding of axon guidance molecules to their receptors activates numerous downstream signals, including Rho GTPases that regulate cytoskeletal organization and receptor location.
doi:10.1101/cshperspect.a001941
PMCID: PMC2857166  PMID: 20452961
13.  Semaphorin-4D (Sema-4D), the Plexin-B1 ligand, is involved in mouse ovary follicular development 
Background
Human Plexin-B1 is expressed in two truncated forms. The long form encodes a trans-membranal protein, while the short form, which is bound to the cell surface and partially secreted, possibly serves as a decoy receptor. Plexin receptors are trans-membrane proteins. The sema domain, found in the extracellular region, is common to all plexins, semaphorins, and the scatter factor receptors and is crucial for the biological activity and plexin receptor specificity. Semaphorin-4D/Plexin-B1 binding provides attractive and repulsive cues for the navigation of axonal growth cones, and new studies suggest that this system also plays a role in the regulation of the biological functions of endothelial cells, specifically in the control of angiogenesis. In a previous study, we have demonstrated the expression and possible role of Plexin-B1 in the mouse ovary. The present study was designed to test the hypothesis that Plexin-B1 effects are mediated by Semaphorin-4D.
Methods
In vivo expression and localization of mouse ovarian Sema-4D were tested by immunohisto-chemistry. The role of Sema-4D in follicular development was examined by in vitro growth of preantral follicles in the presence or absence of Semaphorin-4D, with or without neutralizing antibodies against Plexin-B1. Follicular growth and steroid hormone secretion rates were tested.
Results
Semaphorin-4D is expressed in the mouse ovary in vivo mostly in the granulosa cells and and its expression is modulated by PMSG and hCG. In the presence of Semaphorin-4D, in-vitro constant growth was observed as indicated by follicular diameter during the culture period and elevated steroid hormone secretion rates compared with control. These effects were abolished after addition of neutralizing antibodies against Plexin-B1.
Conclusion
In the ovarian follicle, the effect of Plexin-B1 is mediated by sema-4D.
doi:10.1186/1477-7827-5-12
PMCID: PMC1838422  PMID: 17376242
14.  FAK and Src kinases are required for netrin-induced tyrosine phosphorylation of UNC5 
Journal of cell science  2006;119(Pt 1):47-55.
Summary
During neuronal development, netrin and its receptors UNC5 and DCC (deleted in colorectal cancer) guide axonal growth cones in navigating to their targets. Netrin also plays important roles in the regulation of cell migration, tissue morphogenesis and tumor growth. Here, we show that netrin induces UNC5 tyrosine phosphorylation and that this effect of netrin is dependent on its co-receptor DCC. UNC5 tyrosine phosphorylation is known to be important for netrin to induce cell migration and axonal repulsion. Src tyrosine kinase activity is required for netrin to stimulate UNC5 tyrosine phosphorylation in neurons and transfected cells. The SH2 domain of Src kinase directly interacts with the cytosolic domain of UNC5 in a tyrosine-phosphorylation-dependent manner. Furthermore, the tyrosine kinase focal adhesion kinase (FAK) is also involved in netrin-induced UNC5 tyrosine phosphorylation. Both Src and FAK can phosphorylate UNC5. Our data suggest a model in which netrin stimulates UNC5 tyrosine phosphorylation and signaling in a manner dependent on the co-receptor DCC, through the recruitment of Src and FAK kinases.
doi:10.1242/jcs.02697
PMCID: PMC2248276  PMID: 16371650
Src family tyrosine kinases; Focal adhesion kinase; FAK; Deleted in colorectal cancer; DCC; UNC5 receptor; Netrin signaling; Tyrosine phosphorylation
15.  Neuropilin-2 mediates VEGF-C–induced lymphatic sprouting together with VEGFR3 
The Journal of Cell Biology  2010;188(1):115-130.
If neuropilin-2 and the growth factor VEGF-C don’t come together, lymphatic vessels don’t branch apart.
Vascular sprouting is a key process-driving development of the vascular system. In this study, we show that neuropilin-2 (Nrp2), a transmembrane receptor for the lymphangiogenic vascular endothelial growth factor C (VEGF-C), plays an important role in lymphatic vessel sprouting. Blocking VEGF-C binding to Nrp2 using antibodies specifically inhibits sprouting of developing lymphatic endothelial tip cells in vivo. In vitro analyses show that Nrp2 modulates lymphatic endothelial tip cell extension and prevents tip cell stalling and retraction during vascular sprout formation. Genetic deletion of Nrp2 reproduces the sprouting defects seen after antibody treatment. To investigate whether this defect depends on Nrp2 interaction with VEGF receptor 2 (VEGFR2) and/or 3, we intercrossed heterozygous mice lacking one allele of these receptors. Double-heterozygous nrp2vegfr2 mice develop normally without detectable lymphatic sprouting defects. In contrast, double-heterozygote nrp2vegfr3 mice show a reduction of lymphatic vessel sprouting and decreased lymph vessel branching in adult organs. Thus, interaction between Nrp2 and VEGFR3 mediates proper lymphatic vessel sprouting in response to VEGF-C.
doi:10.1083/jcb.200903137
PMCID: PMC2812843  PMID: 20065093
16.  Semaphorin Signaling in the Development and Function of the Gonadotropin Hormone-Releasing Hormone System 
The semaphorin proteins are among the best-studied families of guidance cues, contributing to morphogenesis and homeostasis in a wide range of tissue types. The major semaphorin receptors are plexins and neuropilins, however other receptors and co-receptors are capable to mediate signaling by semaphorins. These guidance proteins were originally identified as growth cone “collapsing factors” or as inhibitory signals, crucial for nervous system development. Since those seminal discoveries, the list of functions of semaphorins has rapidly grown. Over the past few years, a growing body of data indicates that semaphorins are involved in the regulation of the immune and vascular systems, in tumor growth/cancer cell metastasis and in neural circuit formation. Recently there has been increasing emphasis on research to determine the potential influence of semaphorins on the development and homeostasis of hormone systems and how circulating reproductive hormones regulate their expression and functions. Here, we focus on the emerging role of semaphorins in the development, differentiation and plasticity of unique neurons that secrete gonadotropin-releasing hormone (GnRH), which are essential for the acquisition and maintenance of reproductive competence in all vertebrates. Genetic evidence is also provided showing that insufficient semaphorin signaling contributes to some forms of reproductive disorders in humans, characterized by the reduction or failure of sexual competence. Finally, we will review some studies with the goal of highlighting how the expression of semaphorins and their receptors might be regulated by gonadal hormones in physiological and pathological conditions.
doi:10.3389/fendo.2013.00133
PMCID: PMC3779810  PMID: 24065959
gonadotropin-releasing hormone; reproduction; neuronal plasticity; cell migration; development
17.  Role of the neuropilin ligands VEGF164 and SEMA3A in neuronal and vascular patterning in the mouse 
Novartis Foundation symposium  2007;283:230-241.
Blood vessels and neurons use similar guidance cues to control their behaviour during embryogenesis. The semaphorin SEMA3A was originally identified as a repulsive cue for developing axons that acts by signalling through receptor complexes containing NRP1 and A-type plexins. SEMA3A also competes with the VEGF164 isoform of vascular endothelial growth factor for binding to NRP1 to modulate the migration of endothelial cells in vitro. Surprisingly, we have found that SEMA3A and semaphorin-signalling through NRP1 were not required for blood vessel development in the mouse. Moreover, we found that there was no genetic interaction between SEMA3A and VEGF164 during vasculogenesis or angiogenesis. Our observations suggest that in vivo vascular NRP1 preferentially confers VEGF164 signals, whilst axonal NRP1 preferentially transmits SEMA3A signals.
PMCID: PMC2953635  PMID: 18300426
semaphorin; VEGF; VEGF164; neuropilin; angiogenesis; vasculogenesis; blood vessel; nerve; axon
18.  Plasma membrane-derived microvesicles released from tip endothelial cells during vascular sprouting 
Angiogenesis  2012;15(4):761-769.
During human foetal brain vascularization, activated CD31+/CD105+ endothelial cells are characterized by the emission of filopodial processes which also decorate the advancing tip of the vascular sprout. Together with filopodia, both the markers also reveal a number of plasma membrane-derived microvesicles (MVs) which are concentrated around the tip cell tuft of processes. At this site, MVs appear in tight contact with endothelial filopodia and follow these long processes, advancing into the surrounding neuropil to a possible cell target. These observations suggest that, like shedding vesicles of many other cell types that deliver signalling molecules and play a role in cell-to-cell communication, MVs sent out from endothelial tip cells could be involved in tip cell guidance and/or act on target cells, regulating cell-to-cell mutual recognition during vessel sprouting and final anastomosis. The results also suggest a new role for tip cell filopodia as conveyor processes for transporting MVs far from the cell of origin in a controlled microenvironment. Additional studies focused on the identification of MV content are needed to ultimately clarify the significance of tip cell MVs during human brain vascularization.
Electronic supplementary material
The online version of this article (doi:10.1007/s10456-012-9292-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s10456-012-9292-y
PMCID: PMC3496552  PMID: 22886085
Neuroangiogenesis; Tip cells; Endothelial filopodia; Plasma membrane-derived microvesicles; Human foetal brain; Immunofluorescence confocal microscopy
19.  Gating of Sema3E/PlexinD1 Signaling by Neuropilin-1 Switches Axonal Repulsion to Attraction during Brain Development 
Neuron  2007;56(5):807-822.
Summary
The establishment of functional neural circuits requires the guidance of axons in response to the actions of secreted and cell-surface molecules such as the semaphorins and their neuropilin and plexin receptors. Semaphorin 3E, acting through its receptor PlexinD1, elicits repulsion in non-neuronal systems. Sema3E and PlexinD1 are expressed in the brain but their functions are unknown. Here, we show that Sema3E/PlexinD1 signaling plays an important role in initial development of descending axon tracts in the forebrain. Early errors in axonal projections are reflected in behavioral deficits in Sema3E null-mutant mice. Two distinct signaling mechanisms can be distinguished downstream of Sema3E. Some corticofugal and striatonigral axons express PlexinD1 but not neuropilin-1, and for these neurons Sema3E acts as a repellent. In contrast, subiculo-mammillary neurons co-express neuropilin-1 and PlexinD1, and for these Sema3E acts as an attractant. The extracellular domain of neuropilin-1 is sufficient to convert repulsive signaling by PlexinD1 to attraction. Our data therefore reveal a novel “gating” function of neuropilins in semaphorin-plexin signaling during the assembly of forebrain neuronal circuits.
doi:10.1016/j.neuron.2007.10.019
PMCID: PMC2700040  PMID: 18054858
axon guidance; semaphorin; plexin; neuropilin; brain development
20.  Furin Processing of Semaphorin 3F Determines its Anti-Angiogenic Activity by Regulating Direct Binding and Competition for Neuropilin† 
Biochemistry  2010;49(19):4068-4075.
Neuropilin is an essential cell surface receptor that functions in both semaphorin dependent axon guidance and vascular endothelial growth factor (VEGF) dependent angiogenesis. The interplay between these two seemingly distinct pathways is a source of considerable interest. Indeed, several semaphorin family members have been shown to have potent anti-angiogenic activity in vivo. However, reports conflict as to whether semaphorin and VEGF competitively bind to neuropilin. Previous work has demonstrated that all known ligands and inhibitors of neuropilin interact with the b1 domain of neuropilin via a C-terminal arginine. No semaphorin family member possesses a C-terminal arginine, leading to uncertainty as to the physical mechanism of interaction between the C-terminal domain of semaphorin and the b1 domain of neuropilin. Semaphorin 3F (Sema3F) possesses an RXRR furin recognition site in its C-terminus and we demonstrate that it is proteolytically processed. This processing is found to be essential for the interaction of the C-terminus of Sema3F with the b1 domain of neuropilin. We further demonstrate that furin activation of the C-terminus of Sema3F produces a species that potently inhibits the binding of VEGF to neuropilin. These studies provide a mechanistic basis for understanding the anti-angiogenic activity of semaphorin as well as the physical interaction and competition between neuropilin ligands.
doi:10.1021/bi100327r
PMCID: PMC2868107  PMID: 20387901
21.  RACK-1 Acts with Rac GTPase Signaling and UNC-115/abLIM in Caenorhabditis elegans Axon Pathfinding and Cell Migration 
PLoS Genetics  2010;6(11):e1001215.
Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones.
Author Summary
In the developing nervous system, the growth cone guides axons of neurons to their correct targets in the organism. The growth cone is a highly dynamic specialization at the tip of the axon that senses cues and responds by crawling toward its target, leaving the axon behind. Key to growth cone motility are dynamic cellular protrusions called lamellipodia and filopodia. These protrusions are required for growth cone movement and steering. The genes that are involved in lamellipodia and filopodia formation in the growth cone are still being discovered, and studies to understand how these genes act together in cell signaling events that control growth cone movement are in their infancy. Here we report discovery of a new gene necessary for growth cone movement in Caenorhabditis elegans called rack-1. This gene is conserved in vertebrates and is involved in cellular signaling. We show that it interacts in a novel manner with other cell signaling genes (the Rac GTPase genes) and a gene involved in lamellipodia and filopodia formation, called unc-115/abLIM. We think that rack-1 is involved in a novel cellular signaling event involving Rac GTPases that regulates lamellipodia and filopodia protrusion in the growth cone during nervous system development.
doi:10.1371/journal.pgen.1001215
PMCID: PMC2987834  PMID: 21124943
22.  Endothelial Tip Cells in Ocular Angiogenesis 
Endothelial tip cells are leading cells at the tips of vascular sprouts coordinating multiple processes during angiogenesis. In the developing retina, tip cells play a tightly controlled, timely role in angiogenesis. In contrast, excessive numbers of tip cells are a characteristic of the chaotic pathological blood vessels in proliferative retinopathies. Tip cells control adjacent endothelial cells in a hierarchical manner to form the stalk of the sprouting vessel, using, among others, the VEGF-DLL-Notch signaling pathway, and recruit pericytes. Tip cells are guided toward avascular areas by signals from the local extracellular matrix that are released by cells from the neuroretina such as astrocytes. Recently, tip cells were identified in endothelial cell cultures, enabling identification of novel molecular markers and mechanisms involved in tip cell biology. These mechanisms are relevant for understanding proliferative retinopathies. Agents that primarily target tip cells can block pathological angiogenesis in the retina efficiently and safely without adverse effects. A striking example is platelet-derived growth factor, which was recently shown to be an efficacious additional target in the treatment of retinal neovascularization. Here we discuss these and other tip cell-based strategies with respect to their potential to treat patients with ocular diseases dominated by neovascularization.
doi:10.1369/0022155412467635
PMCID: PMC3636692  PMID: 23092791
angiogenesis; endothelial tip cell; proliferative retinopathy; anti-angiogenesis therapy; retinal neovascularization; vascular sprouts; endothelial stalk cell; molecular mediators of angiogenesis; pericytes
23.  Slit2–Robo4 signalling promotes vascular stability by blocking Arf6 activity 
Nature cell biology  2009;11(11):1325-1331.
Slit–Roundabout (Robo) signalling has a well-understood role in axon guidance1–5. Unlike in the nervous system, however, Slitdependent activation of an endothelial-specific Robo, Robo4, does not initiate a guidance program. Instead, Robo4 maintains the barrier function of the mature vascular network by inhibiting neovascular tuft formation and endothelial hyperpermeability induced by pro-angiogenic factors 6. In this study, we used cell biological and biochemical techniques to elucidate the molecular mechanism underlying the maintenance of vascular stability by Robo4. Here, we demonstrate that Robo4 mediates Slit2-dependent suppression of cellular protrusive activity through direct interaction with the intracellular adaptor protein paxillin and its paralogue, Hic-5. Formation of a Robo4–paxillin complex at the cell surface blocks activation of the small GTPase Arf6 and, consequently, Rac by recruitment of Arf-GAPs (ADP-ribosylation factor- directed GTPase-activating proteins) such as GIT1. Consistent with these in vitro studies, inhibition of Arf6 activity in vivo phenocopies Robo4 activation by reducing pathologic angiogenesis in choroidal and retinal vascular disease and VEGF-165 (vascular endothelial growth factor-165)-induced retinal hyperpermeability. These data reveal that a Slit2–Robo4–paxillin–GIT1 network inhibits the cellular protrusive activity underlying neovascularization and vascular leak, and identify a new therapeutic target for ameliorating diseases involving the vascular system.
doi:10.1038/ncb1976
PMCID: PMC2854659  PMID: 19855388
24.  Selective requirements for NRP1 ligands during neurovascular patterning 
Development (Cambridge, England)  2007;134(10):1833-1843.
Blood vessels and neurons share several types of guidance cues and cell surface receptors to control their behaviour during embryogenesis. The transmembrane protein NRP1 is present on blood vessels and nerves. NRP1 binds two structurally diverse ligands, the semaphorin SEMA3A and the VEGF164 isoform of vascular endothelial growth factor. SEMA3A was originally identified as a repulsive cue for developing axons that acts by signalling through receptor complexes containing NRP1 and plexins. In vitro, SEMA3A also inhibits integrin function and competes with VEGF164 for binding to NRP1 to modulate the migration of endothelial cells. These observations resulted in a widely accepted model of vascular patterning in which the balance of VEGF164 and SEMA3A determines endothelial cell behaviour. However, we now demonstrate that SEMA3A is not required for angiogenesis in the mouse, which instead is controlled by VEGF164. We find that SEMA3A, but not VEGF164, is required for axon patterning of limb nerves, even though the competition between VEGF164 and SEMA3A for NRP1 affects the migration of neuronal progenitor cells in vitro and has been hypothesised to control axon guidance. Moreover, we show that there is no genetic interaction between SEMA3A and VEGF164 during vasculogenesis, angiogenesis or limb axon patterning, suggesting that ligand competition for NRP1 binding cannot explain neurovascular congruence, as previously suggested. We conclude that NRP1 contributes to both neuronal and vascular patterning by preferentially relaying SEMA3A signals in peripheral axons and VEGF164 signals in blood vessels.
doi:10.1242/dev.002402
PMCID: PMC2702678  PMID: 17428830
VEGF; Neuropilin; Semaphorin; Mouse
25.  VEGF-C induced angiogenesis preferentially occurs at a distance from lymphangiogenesis 
Cardiovascular research  2007;78(2):315-323.
Aims
Vascular endothelial growth factor-C (VEGF-C) has been shown to stimulate both angiogenesis and lymphangiogenesis in some but not all models where VEGF-C is over-expressed. Our aim was to investigate the interaction between lymphangiogenesis and angiogenesis in adult tissues regulated by VEGF-C and identify evidence of polarized growth of lymphatics driven by specialized cells at the tip of the growing sprout.
Methods and results
We used an adult model of lymphangiogenesis in the rat mesentery. The angiogenic effect of VEGF-C was markedly attenuated in the presence of a growing lymphatic network. Furthermore, we show that this growth of lymphatic vessels can occur both by recruitment of isolated lymphatic islands to a connected network and by filopodial sprouting. The latter is independent of polarized tip cell differentiation that can be generated all along lymphatic capillaries, independently of the proliferation status of the lymphatic endothelial cells.
Conclusion
These results both demonstrate a dependence of VEGF-C-mediated angiogenesis on lymphatic vascular networks and indicate that the mechanism of VEGF-C-mediated lymphangiogenesis is different from that of classical angiogenic mechanisms.
doi:10.1093/cvr/cvm094
PMCID: PMC2613351  PMID: 18065770
Angiogenesis; Lymphangiogenesis; VEGF-C; Sprouting

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