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1.  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
2.  The Rac GTP Exchange Factor TIAM-1 Acts with CDC-42 and the Guidance Receptor UNC-40/DCC in Neuronal Protrusion and Axon Guidance 
PLoS Genetics  2012;8(4):e1002665.
The mechanisms linking guidance receptors to cytoskeletal dynamics in the growth cone during axon extension remain mysterious. The Rho-family GTPases Rac and CDC-42 are key regulators of growth cone lamellipodia and filopodia formation, yet little is understood about how these molecules interact in growth cone outgrowth or how the activities of these molecules are regulated in distinct contexts. UNC-73/Trio is a well-characterized Rac GTP exchange factor in Caenorhabditis elegans axon pathfinding, yet UNC-73 does not control CED-10/Rac downstream of UNC-6/Netrin in attractive axon guidance. Here we show that C. elegans TIAM-1 is a Rac-specific GEF that links CDC-42 and Rac signaling in lamellipodia and filopodia formation downstream of UNC-40/DCC. We also show that TIAM-1 acts with UNC-40/DCC in axon guidance. Our results indicate that a CDC-42/TIAM-1/Rac GTPase signaling pathway drives lamellipodia and filopodia formation downstream of the UNC-40/DCC guidance receptor, a novel set of interactions between these molecules. Furthermore, we show that TIAM-1 acts with UNC-40/DCC in axon guidance, suggesting that TIAM-1 might regulate growth cone protrusion via Rac GTPases in response to UNC-40/DCC. Our results also suggest that Rac GTPase activity is controlled by different GEFs in distinct axon guidance contexts, explaining how Rac GTPases can specifically control multiple cellular functions.
Author Summary
Axons extend great distances to make precise synaptic connections in the developing nervous system. Axons are guided to their targets by the growth cone, a dynamic structure at the axon distal tip that senses extracellular cues telling the axon where to go. In response to guidance cues, growth cones alter their shape and motility resulting in outgrowth and turning. The cytoskeleton (actin and microtubules) underlies growth cone motility and guidance. The signaling mechanisms linking guidance receptors to cytoskeletal change remain mysterious. Here, we define a new signaling mechanism downstream of the guidance receptor UNC-40/DCC involving the GTPases CDC-42 and Rac, which have long been known to control growth cone protrusion. We show that CDC-42 and Rac act in a linear pathway in axon guidance; CDC-42 acts upstream of the GTPase regulatory molecule TIAM-1, which is a GTP exchange factor specific for Rac and which activates Rac signaling. We also show that TIAM-1 acts with UNC-40/DCC signaling in protrusion and axon guidance. Our results imply that Rac GTPase function in axon guidance is complex and that distinct GEFs (TIAM-1 and UNC-73/Trio) might control Rac GTPases in different aspects of axon guidance.
doi:10.1371/journal.pgen.1002665
PMCID: PMC3343084  PMID: 22570618
3.  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
4.  Netrins and Wnts Function Redundantly to Regulate Antero-Posterior and Dorso-Ventral Guidance in C. elegans 
PLoS Genetics  2014;10(6):e1004381.
Guided migrations of cells and developing axons along the dorso-ventral (D/V) and antero-posterior (A/P) body axes govern tissue patterning and neuronal connections. In C. elegans, as in vertebrates, D/V and A/P graded distributions of UNC-6/Netrin and Wnts, respectively, provide instructive polarity information to guide cells and axons migrating along these axes. By means of a comprehensive genetic analysis, we found that simultaneous loss of Wnt and Netrin signaling components reveals previously unknown and unexpected redundant roles for Wnt and Netrin signaling pathways in both D/V and A/P guidance of migrating cells and axons in C. elegans, as well as in processes essential for organ function and viability. Thus, in addition to providing polarity information for migration along the axis of their gradation, Wnts and Netrin are each able to guide migrations orthogonal to the axis of their gradation. Netrin signaling not only functions redundantly with some Wnts, but also counterbalances the effects of others to guide A/P migrations, while the involvement of Wnt signaling in D/V guidance identifies Wnt signaling as one of the long sought mechanisms that functions in parallel to Netrin signaling to promote D/V guidance of cells and axons. These findings provide new avenues for deciphering how A/P and D/V guidance signals are integrated within the cell to establish polarity in multiple biological processes, and implicate broader roles for Netrin and Wnt signaling - roles that are currently masked due to prevalent redundancy.
Author Summary
While ample information was gathered in past decades on identifying guidance cues and their downstream mediators, very little is known about how the information from multiple extracellular cues is integrated within the cell to generate normal patterning. Netrin and Wnt signaling pathways are both critical to multiple developmental processes and play key roles in normal development as well as in malignancies. The UNC-6/Netrin guidance cue has a conserved role in guiding cell and growth cone migrations along the dorso-ventral axis, whereas Wnts are critical for determining polarity and guidance along the antero-posterior axis. In this study we show that these two signaling pathways function redundantly in both antero-posterior and dorso-ventral guidance as well as in processes essential for viability. Furthermore, we demonstrate that a fine balance between Wnt and Netrin signaling pathways is critical for proper polarity establishment and identify Wnt signaling as one of the long sought mechanisms that signal in parallel to Netrin to promote dorso-ventral guidance of cells and axons in Caenorhabditis elegans. These findings pave the way to unraveling the broader roles of Wnt and Netrin signaling pathways and provide a conceptually novel view of how antero-posterior and dorso-ventral guidance mechanisms are orchestrated.
doi:10.1371/journal.pgen.1004381
PMCID: PMC4046927  PMID: 24901837
5.  The Caenorhabditis elegans Eph Receptor Activates NCK and N-WASP, and Inhibits Ena/VASP to Regulate Growth Cone Dynamics during Axon Guidance 
PLoS Genetics  2012;8(2):e1002513.
The Eph receptor tyrosine kinases (RTKs) are regulators of cell migration and axon guidance. However, our understanding of the molecular mechanisms by which Eph RTKs regulate these processes is still incomplete. To understand how Eph receptors regulate axon guidance in Caenorhabditis elegans, we screened for suppressors of axon guidance defects caused by a hyperactive VAB-1/Eph RTK. We identified NCK-1 and WSP-1/N-WASP as downstream effectors of VAB-1. Furthermore, VAB-1, NCK-1, and WSP-1 can form a complex in vitro. We also report that NCK-1 can physically bind UNC-34/Enabled (Ena), and suggest that VAB-1 inhibits the NCK-1/UNC-34 complex and negatively regulates UNC-34. Our results provide a model of the molecular events that allow the VAB-1 RTK to regulate actin dynamics for axon guidance. We suggest that VAB-1/Eph RTK can stop axonal outgrowth by inhibiting filopodia formation at the growth cone by activating Arp2/3 through a VAB-1/NCK-1/WSP-1 complex and by inhibiting UNC-34/Ena activity.
Author Summary
The correct wiring of the nervous system depends on the ability of axons to properly interpret extracellular cues that guide them to their targets. The Eph receptor tyrosine kinases (RTKs) have roles in guiding axons, but their signaling pathways are not completely understood. In this study, we used the nematode Caenorhabditis elegans to study how the VAB-1 Eph RTK regulates the growth cone structure for axon guidance. Genetic and molecular data show that VAB-1 regulates the conserved molecules NCK-1, WSP-1/N-WASP, and UNC-34/Ena. Our study provides a model of how the VAB-1 Eph RTK modulates the growth cone structure to inhibit axonal outgrowth. We show that activated VAB-1 can inhibit an NCK-1/UNC-34 interaction by binding to the NCK-1 SH2 domain. We also show that NCK-1 and WSP-1 can physically interact and that VAB-1/NCK-1 and WSP-1 form a complex in vitro. We suggest that the VAB-1 Eph RTK can contribute to the termination of axon outgrowth by two methods: 1) The VAB-1/NCK-1/WSP-1 complex activates ARP-2/3 to change the actin growth cone dynamics to that of a branched structure thus reducing the number of filopodia, and 2) VAB-1 inhibits axon extension by inhibiting UNC-34/Ena's function in actin polymerization.
doi:10.1371/journal.pgen.1002513
PMCID: PMC3285579  PMID: 22383893
6.  The netrin protein family 
Genome Biology  2009;10(9):239.
The netrins are a family of extracellular proteins that direct cell and axon migration during embryogenesis.
The name netrin is derived from the Sanskrit Netr, meaning 'guide'. Netrins are a family of extracellular proteins that direct cell and axon migration during embryogenesis. Three secreted netrins (netrins 1, 3 and 4), and two glycosylphosphatidylinositol (GPI)-anchored membrane proteins, netrins G1 and G2, have been identified in mammals. The secreted netrins are bifunctional, acting as attractants for some cell types and repellents for others. Receptors for the secreted netrins include the Deleted in Colorectal Cancer (DCC) family, the Down's syndrome cell adhesion molecule (DSCAM), and the UNC-5 homolog family: Unc5A, B, C and D in mammals. Netrin Gs do not appear to interact with these receptors, but regulate synaptic interactions between neurons by binding to the transmembrane netrin G ligands NGL1 and 2. The chemotropic function of secreted netrins has been best characterized with regard to axon guidance during the development of the nervous system. Extending axons are tipped by a flattened, membranous structure called the growth cone. Multiple extracellular guidance cues direct axonal growth cones to their ultimate targets where synapses form. Such cues can be locally derived (short-range), or can be secreted diffusible cues that allow target cells to signal axons from a distance (long-range). The secreted netrins function as short-range and long-range guidance cues in different circumstances. In addition to directing cell migration, functional roles for netrins have been identified in the regulation of cell adhesion, the maturation of cell morphology, cell survival and tumorigenesis.
doi:10.1186/gb-2009-10-9-239
PMCID: PMC2768972  PMID: 19785719
7.  UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph Polarize F-Actin during Embryonic Morphogenesis by Regulating the WAVE/SCAR Actin Nucleation Complex 
PLoS Genetics  2012;8(8):e1002863.
Many cells in a developing embryo, including neurons and their axons and growth cones, must integrate multiple guidance cues to undergo directed growth and migration. The UNC-6/netrin, SLT-1/slit, and VAB-2/Ephrin guidance cues, and their receptors, UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph, are known to be major regulators of cellular growth and migration. One important area of research is identifying the molecules that interpret this guidance information downstream of the guidance receptors to reorganize the actin cytoskeleton. However, how guidance cues regulate the actin cytoskeleton is not well understood. We report here that UNC-40/DCC, SAX-3/Robo, and VAB-1/Eph differentially regulate the abundance and subcellular localization of the WAVE/SCAR actin nucleation complex and its activator, Rac1/CED-10, in the Caenorhabditis elegans embryonic epidermis. Loss of any of these three pathways results in embryos that fail embryonic morphogenesis. Similar defects in epidermal enclosure have been observed when CED-10/Rac1 or the WAVE/SCAR actin nucleation complex are missing during embryonic development in C. elegans. Genetic and molecular experiments demonstrate that in fact, these three axonal guidance proteins differentially regulate the levels and membrane enrichment of the WAVE/SCAR complex and its activator, Rac1/CED-10, in the epidermis. Live imaging of filamentous actin (F-actin) in embryos developing in the absence of individual guidance receptors shows that high levels of F-actin are not essential for polarized cell migrations, but that properly polarized distribution of F-actin is essential. These results suggest that proper membrane recruitment and activation of CED-10/Rac1 and of WAVE/SCAR by signals at the plasma membrane result in polarized F-actin that permits directed movements and suggest how multiple guidance cues can result in distinct changes in actin nucleation during morphogenesis.
Author Summary
Neurons and other cells are most dynamic during embryonic development. Later in life, a diminished developmental potential in mature neurons makes injuries difficult to treat. Neuronal regeneration studies have benefited from genetic studies that identified the molecules that work during embryonic development to give neurons their plasticity and dynamism. Growth and regeneration require dynamic changes in the actin cytoskeleton. In this study we ask how signals that are known to guide migrations of neurons and other cells during development and regeneration are able to reorganize the actin cytoskeleton. Understanding how multiple signals are integrated to control actin dynamics is essential for understanding how cellular movements are regulated in embryos and adults. We find that the actin-nucleation regulating WAVE/SCAR complex molecules, which are conserved from the C. elegans soil nematodes to human beings, are localized and regulated by axonal guidance signals during embryonic development. These studies illustrate a mechanism by which different signals reorganize cellular F-actin through their regulation of the actin regulating WAVE/SCAR complex.
doi:10.1371/journal.pgen.1002863
PMCID: PMC3410845  PMID: 22876199
8.  The Axonal Guidance Cue Semaphorin 3C Contributes to Alveolar Growth and Repair 
PLoS ONE  2013;8(6):e67225.
Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD) in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C), contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O2-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.
doi:10.1371/journal.pone.0067225
PMCID: PMC3688622  PMID: 23840631
9.  Brain Endothelial Cells Control Fertility through Ovarian-Steroid–Dependent Release of Semaphorin 3A 
PLoS Biology  2014;12(3):e1001808.
Endothelial-cell–derived Sema3A promotes axonal outgrowth and plasticity and thereby regulates neurohormone release in the adult rodent brain in response to the ovarian cycle.
Neuropilin-1 (Nrp1) guides the development of the nervous and vascular systems, but its role in the mature brain remains to be explored. Here we report that the expression of the 65 kDa isoform of Sema3A, the ligand of Nrp1, by adult vascular endothelial cells, is regulated during the ovarian cycle and promotes axonal sprouting in hypothalamic neurons secreting gonadotropin-releasing hormone (GnRH), the neuropeptide controlling reproduction. Both the inhibition of Sema3A/Nrp1 signaling and the conditional deletion of Nrp1 in GnRH neurons counteract Sema3A-induced axonal sprouting. Furthermore, the localized intracerebral infusion of Nrp1- or Sema3A-neutralizing antibodies in vivo disrupts the ovarian cycle. Finally, the selective neutralization of endothelial-cell Sema3A signaling in adult Sema3aloxP/loxP mice by the intravenous injection of the recombinant TAT-Cre protein alters the amplitude of the preovulatory luteinizing hormone surge, likely by perturbing GnRH release into the hypothalamo-hypophyseal portal system. Our results identify a previously unknown function for 65 kDa Sema3A-Nrp1 signaling in the induction of axonal growth, and raise the possibility that endothelial cells actively participate in synaptic plasticity in specific functional domains of the adult central nervous system, thus controlling key physiological functions such as reproduction.
Author Summary
In the developing embryo, endothelial cells release chemotropic signals such as Semaphorin 3A (Sema3A) that, upon activation of its receptor Neuropilin-1 (Nrp1), regulate neuronal migration and axon guidance. However, whether endothelial cells in the adult brain retain the ability to secrete molecules that influence neuronal function is unknown. Here we show in the adult brain of rodents that vascular endothelial cells release Sema3A and that the amount released is regulated by the ovulatory cycle. Sema3A, in turn, promotes the outgrowth of axons of hypothalamic neurons that express Neuropilin-1 towards the endothelial wall of portal blood vessels. These neurons release there the neuropeptide that controls reproduction: gonadotropin-releasing hormone (GnRH). Notably, this endothelial-cell-mediated sprouting of GnRH axons regulates neuropeptide release at a key stage of the estrous cycle, the proestrus, when the surge of GnRH triggers ovulation. Thus, by promoting GnRH axonal growth in the adult brain, Sema3A/Neuropilin-1 plays a pivotal role in orchestrating the central control of reproduction. Our results suggest a model in which vascular endothelial cells are dynamic signaling components that relay peripheral information to the brain to control key physiological functions, including species survival.
doi:10.1371/journal.pbio.1001808
PMCID: PMC3949669  PMID: 24618750
10.  MIG-10 Functions with ABI-1 to Mediate the UNC-6 and SLT-1 Axon Guidance Signaling Pathways 
PLoS Genetics  2012;8(11):e1003054.
Extracellular guidance cues steer axons towards their targets by eliciting morphological changes in the growth cone. A key part of this process is the asymmetric recruitment of the cytoplasmic scaffolding protein MIG-10 (lamellipodin). MIG-10 is thought to asymmetrically promote outgrowth by inducing actin polymerization. However, the mechanism that links MIG-10 to actin polymerization is not known. We have identified the actin regulatory protein ABI-1 as a partner for MIG-10 that can mediate its outgrowth-promoting activity. The SH3 domain of ABI-1 binds to MIG-10, and loss of function of either of these proteins causes similar axon guidance defects. Like MIG-10, ABI-1 functions in both the attractive UNC-6 (netrin) pathway and the repulsive SLT-1 (slit) pathway. Dosage sensitive genetic interactions indicate that MIG-10 functions with ABI-1 and WVE-1 to mediate axon guidance. Epistasis analysis reveals that ABI-1 and WVE-1 function downstream of MIG-10 to mediate its outgrowth-promoting activity. Moreover, experiments with cultured mammalian cells suggest that the interaction between MIG-10 and ABI-1 mediates a conserved mechanism that promotes formation of lamellipodia. Together, these observations suggest that MIG-10 interacts with ABI-1 and WVE-1 to mediate the UNC-6 and SLT-1 guidance pathways.
Author Summary
To form neural circuits, axons must navigate through the developing nervous system to reach their correct targets. Axon navigation is led by the growth cone, a structure at the tip of the growing axon that responds to extracellular guidance cues. Many of these guidance cues and their receptors have been identified. However, much less is known about the internal signaling events that give rise to the structural changes required for growth cone steering. A key component of the internal response is MIG-10, a protein that becomes asymmetrically localized in response to the extracellular cues. MIG-10 is thought to serve as a scaffold that can spatially control outgrowth-promoting proteins within the growth cone. However, we do not know the identity of the outgrowth-promoting proteins that associate with MIG-10. Here we report that MIG-10 associates physically with the actin regulatory protein ABI-1. We present genetic evidence indicating that ABI-1 functions downstream of MIG-10 to mediate its outgrowth-promoting activity. Additional genetic evidence indicates that these proteins function in both attractive and repulsive guidance signaling pathways. We also present evidence suggesting that the connection between MIG-10 and ABI-1 represents a phylogenetically conserved mechanism for the control of cellular outgrowth.
doi:10.1371/journal.pgen.1003054
PMCID: PMC3510047  PMID: 23209429
11.  Ephrin-A5 Suppresses Neurotrophin Evoked Neuronal Motility, ERK Activation and Gene Expression 
PLoS ONE  2011;6(10):e26089.
During brain development, growth cones respond to attractive and repulsive axon guidance cues. How growth cones integrate guidance instructions is poorly understood. Here, we demonstrate a link between BDNF (brain derived neurotrophic factor), promoting axonal branching and ephrin-A5, mediating axonal repulsion via Eph receptor tyrosine kinase activation. BDNF enhanced growth cone filopodial dynamics and neurite branching of primary neurons. We show that ephrin-A5 antagonized this BDNF-evoked neuronal motility. BDNF increased ERK phosphorylation (P-ERK) and nuclear ERK entry. Ephrin-A5 suppressed BDNF-induced ERK activity and might sequester P-ERK in the cytoplasm. Neurotrophins are well established stimulators of a neuronal immediate early gene (IEG) response. This is confirmed in this study by e.g. c-fos, Egr1 and Arc upregulation upon BDNF application. This BDNF-evoked IEG response required the transcription factor SRF (serum response factor). Notably, ephrin-A5 suppressed a BDNF-evoked neuronal IEG response, suggesting a role of Eph receptors in modulating gene expression. In opposite to IEGs, long-term ephrin-A5 application induced cytoskeletal gene expression of tropomyosin and actinin. To uncover specific Eph receptors mediating ephrin-As impact on neurotrophin signaling, EphA7 deficient mice were analyzed. In EphA7 deficient neurons alterations in growth cone morphology were observed. However, ephrin-A5 still counteracted neurotrophin signaling suggesting that EphA7 is not required for ephrin and BDNF crosstalk. In sum, our data suggest an interaction of ephrin-As and neurotrophin signaling pathways converging at ERK signaling and nuclear gene activity. As ephrins are involved in development and function of many organs, such modulation of receptor tyrosine kinase signaling and gene expression by Ephs might not be limited to the nervous system.
doi:10.1371/journal.pone.0026089
PMCID: PMC3191169  PMID: 22022520
12.  Guidance Molecules in Axon Regeneration 
The regenerative capacity of injured adult mammalian central nervous system (CNS) tissue is very limited. Disease or injury that causes destruction or damage to neuronal networks typically results in permanent neurological deficits. Injury to the spinal cord, for example, interrupts vital ascending and descending fiber tracts of spinally projecting neurons. Because neuronal structures located proximal or distal to the injury site remain largely intact, a major goal of spinal cord injury research is to develop strategies to reestablish innervation lost as a consequence of injury. The growth inhibitory nature of injured adult CNS tissue is a major barrier to regenerative axonal growth and sprouting. An increasing complexity of molecular players is being recognized. CNS inhibitors fall into three general classes: members of canonical axon guidance molecules (e.g., semaphorins, ephrins, netrins), prototypic myelin inhibitors (Nogo, MAG, and OMgp) and chondroitin sulfate proteoglycans (lecticans, NG2). On the other end of the spectrum are molecules that promote neuronal growth and sprouting. These include growth promoting extracellular matrix molecules, cell adhesion molecules, and neurotrophic factors. In addition to environmental (extrinsic) growth regulatory cues, cell intrinsic regulatory mechanisms exist that greatly influence injury-induced neuronal growth. Various degrees of growth and sprouting of injured CNS neurons have been achieved by lowering extrinsic inhibitory cues, increasing extrinsic growth promoting cues, or by activation of cell intrinsic growth programs. More recently, combination therapies that activate growth promoting programs and at the same time attenuate growth inhibitory pathways have met with some success. In experimental animal models of spinal cord injury (SCI), mono and combination therapies have been shown to promote neuronal growth and sprouting. Anatomical growth often correlates with improved behavioral outcomes. Challenges ahead include testing whether some of the most promising treatment strategies in animal models are also beneficial for human patients suffering from SCI.
Growth inhibition mechanisms active in the adult CNS limit our ability to repair spinal chord injuries. Promising new therapies aim to attenuate these mechanisms and activate growth-promoting programs.
doi:10.1101/cshperspect.a001867
PMCID: PMC2890195  PMID: 20519341
13.  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
14.  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
15.  Dorsal turning of motor corticospinal axons at the pyramidal decussation requires plexin signaling 
Neural Development  2008;3:21.
Background
The development of the corticospinal tract (CST) in higher vertebrates relies on a series of axon guidance decisions along its long projection pathway. Several guidance molecules are known to be involved at various decision points to regulate the projection of CST axons. However, previous analyses of the CST guidance defects in mutant mice lacking these molecules have suggested that there are other molecules involved in CST axon guidance that are yet to be identified. In this study, we investigate the role of plexin signaling in the guidance of motor CST axons in vivo.
Results
Expression pattern studies show that plexin-A3, plexin-A4, and neuropilin-1 are expressed in the developing cerebral cortex when the motor CST axons originating from layer V cortical neurons are guided down to the spinal cord. By analyzing mutant mice, we show that motor CST axons that turn dorsally to cross the midline at the pyramidal decussation require plexin-A3 and plexin-A4 signaling. Although other CST guidance defects are found in neuropilin-1 mutants, this dorsal turning defect is not observed in either neuropilin-1 or neuropilin-2 mutants, suggesting that the local cues that activate plexin signaling at the dorsal turning point are membrane-bound semaphorins. Further expression pattern study and mutant analysis indicate that Sema6A is one of the local cues for motor CST axon turning at the pyramidal decussation.
Conclusion
Dorsal turning and midline crossing at the pyramidal decussation is a crucial step to properly direct CST axons into the dorsal spinal cord. We show that the signaling of plexin-A3, plexin-A4, and Sema6A is at least partially required for dorsal turning of the CST axons, while neuropilin-1 is required for proper fasciculation of the tract at midline crossing. Together with previous reports, these results demonstrate that several guidance cues are specifically utilized to regulate the dorsal turning and midline crossing of developing CST axons.
doi:10.1186/1749-8104-3-21
PMCID: PMC2532682  PMID: 18727829
16.  Endothelial Expression of Guidance Cues in Vessel Wall Homeostasis: Dysregulation under pro-atherosclerotic conditions 
Objective
Emerging evidence suggests that neuronal guidance cues, typically expressed during development, are involved in both physiological and pathological immune responses. We hypothesized that endothelial expression of such guidance cues may regulate leukocyte trafficking into the vascular wall during atherogenesis.
Approach/Results
We demonstrate that members of the Netrin, Semaphorin and Ephrin family of guidance molecules are differentially regulated under conditions that promote or protect from atherosclerosis. Netrin-1 and Semaphorin3A are expressed by coronary artery endothelial cells and potently inhibit chemokine-directed migration of human monocytes. Endothelial expression of these negative guidance cues is down-regulated by pro-atherogenic factors, including oscillatory shear stress and pro-inflammatory cytokines associated with monocyte entry into the vessel wall. Furthermore, we show using intravital microscopy that inhibition of Netrin-1 or Semaphorin3A using blocking peptides increases leukocyte adhesion to the endothelium. Unlike Netrin-1 and Semaphorin3A, the guidance cue EphrinB2 is up-regulated under pro-atherosclerotic flow conditions and functions as a chemoattractant, increasing leukocyte migration in the absence of additional chemokines.
Conclusions
The concurrent regulation of negative and positive guidance cues may facilitate leukocyte infiltration of the endothelium through a balance between chemoattraction and chemorepulsion. These data indicate a previously unappreciated role for axonal guidance cues in maintaining the endothelial barrier and regulating leukocyte trafficking during atherogenesis.
doi:10.1161/ATVBAHA.112.301155
PMCID: PMC3647028  PMID: 23430612
axonal guidance; migration; atherosclerosis; endothelial-leukocyte interaction
17.  Signaling Mechanisms in Cortical Axon Growth, Guidance, and Branching 
Precise wiring of cortical circuits during development depends upon axon extension, guidance, and branching to appropriate targets. Motile growth cones at axon tips navigate through the nervous system by responding to molecular cues, which modulate signaling pathways within axonal growth cones. Intracellular calcium signaling has emerged as a major transducer of guidance cues but exactly how calcium signaling pathways modify the actin and microtubule cytoskeleton to evoke growth cone behaviors and axon branching is still mysterious. Axons must often pause their extension in tracts while their branches extend into targets. Some evidence suggests a competition between growth of axons and branches but the mechanisms are poorly understood. Since it is difficult to study growing axons deep within the mammalian brain, much of what we know about signaling pathways and cytoskeletal dynamics of growth cones comes from tissue culture studies, in many cases, of non-mammalian species. Consequently it is not well understood how guidance cues relevant to mammalian neural development in vivo signal to the growth cone cytoskeleton during axon outgrowth and guidance. In this review we describe our recent work in dissociated cultures of developing rodent sensorimotor cortex in the context of the current literature on molecular guidance cues, calcium signaling pathways, and cytoskeletal dynamics that regulate growth cone behaviors. A major challenge is to relate findings in tissue culture to mechanisms of cortical development in vivo. Toward this goal, we describe our recent work in cortical slices, which preserve the complex cellular and molecular environment of the mammalian brain but allow direct visualization of growth cone behaviors and calcium signaling. Findings from this work suggest that mechanisms regulating axon growth and guidance in dissociated culture neurons also underlie development of cortical connectivity in vivo.
doi:10.3389/fnana.2011.00062
PMCID: PMC3202218  PMID: 22046148
axon outgrowth; axon guidance; axon branching; calcium signaling; Wnt5a; CaMKII; corpus callosum; microtubules
18.  The UNC-6/Netrin receptors UNC-40/DCC and UNC-5 inhibit growth cone filopodial protrusion via UNC-73/Trio, Rac-like GTPases and UNC-33/CRMP 
Development (Cambridge, England)  2014;141(22):4395-4405.
UNC-6/Netrin is a conserved axon guidance cue that can mediate both attraction and repulsion. We previously discovered that attractive UNC-40/DCC receptor signaling stimulates growth cone filopodial protrusion and that repulsive UNC-40–UNC-5 heterodimers inhibit filopodial protrusion in C. elegans. Here, we identify cytoplasmic signaling molecules required for UNC-6-mediated inhibition of filopodial protrusion involved in axon repulsion. We show that the Rac-like GTPases CED-10 and MIG-2, the Rac GTP exchange factor UNC-73/Trio, UNC-44/Ankyrin and UNC-33/CRMP act in inhibitory UNC-6 signaling. These molecules were required for the normal limitation of filopodial protrusion in developing growth cones and for inhibition of growth cone filopodial protrusion caused by activated MYR::UNC-40 and MYR::UNC-5 receptor signaling. Epistasis studies using activated CED-10 and MIG-2 indicated that UNC-44 and UNC-33 act downstream of the Rac-like GTPases in filopodial inhibition. UNC-73, UNC-33 and UNC-44 did not affect the accumulation of full-length UNC-5::GFP and UNC-40::GFP in growth cones, consistent with a model in which UNC-73, UNC-33 and UNC-44 influence cytoskeletal function during growth cone filopodial inhibition.
doi:10.1242/dev.110437
PMCID: PMC4302909  PMID: 25371370
UNC-40/DCC; UNC-5; UNC-6; Axon repulsion; Filopodia; Growth cone; Caenorhabditis elegans
19.  Extension of the Caenorhabditis elegans Pharyngeal M1 Neuron Axon Is Regulated by Multiple Mechanisms 
G3: Genes|Genomes|Genetics  2013;3(11):2015-2029.
The guidance of axons to their correct targets is a critical step in development. The C. elegans pharynx presents an attractive system to study neuronal pathfinding in the context of a developing organ. The worm pharynx contains relatively few cells and cell types, but each cell has a known lineage and stereotyped developmental patterns. We found that extension of the M1 pharyngeal axon, which spans the entire length of the pharynx, occurs in two distinct phases. The first proximal phase does not require genes that function in axon extension (unc-34, unc-51, unc-115, and unc-119), whereas the second distal phase does use these genes and is guided in part by the adjacent g1P gland cell projection. unc-34, unc-51, and unc-115 had incompletely penetrant defects and appeared to act in conjunction with the g1P cell for distal outgrowth. Only unc-119 showed fully penetrant defects for the distal phase. Mutations affecting classical neuronal guidance cues (Netrin, Semaphorin, Slit/Robo, Ephrin) or adhesion molecules (cadherin, IgCAM) had, at best, weak effects on the M1 axon. None of the mutations we tested affected the proximal phase of M1 elongation. In a forward genetic screen, we isolated nine mutations in five genes, three of which are novel, showing defects in M1, including axon overextension, truncation, or ectopic branching. One of these mutations appeared to affect the generation or differentiation of the M1 neuron. We conclude that M1 axon extension is a robust process that is not completely dependent on any single guidance mechanism.
doi:10.1534/g3.113.008466
PMCID: PMC3815062  PMID: 24048649
Caenorhabditis elegans; neuron; axon; genetics; pharynx
20.  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
21.  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
22.  Subcellular Profiling Reveals Distinct and Developmentally Regulated Repertoire of Growth Cone mRNAs 
Cue-directed axon guidance depends partly on local translation in growth cones. Many mRNA transcripts are known to reside in developing axons, yet little is known about their subcellular distribution or, specifically, which transcripts are in growth cones. Here laser capture microdissection (LCM) was used to isolate the growth cones of retinal ganglion cell (RGC) axons of two vertebrate species, mouse and Xenopus, coupled with unbiased genomewide microarray profiling. An unexpectedly large pool of mRNAs defined predominant pathways in protein synthesis, oxidative phosphorylation, cancer, neurological disease, and signaling. Comparative profiling of “young” (pathfinding) versus “old” (target-arriving) Xenopus growth cones revealed that the number and complexity of transcripts increases dramatically with age. Many presynaptic protein mRNAs are present exclusively in old growth cones, suggesting that functionally related sets of mRNAs are targeted to growth cones in a developmentally regulated way. Remarkably, a subset of mRNAs was significantly enriched in the growth cone compared with the axon compartment, indicating that mechanisms exist to localize mRNAs selectively to the growth cone. Furthermore, some receptor transcripts (e.g., EphB4), present exclusively in old growth cones, were equally abundant in young and old cell bodies, indicating that RNA trafficking from the soma is developmentally regulated. Our findings show that the mRNA repertoire in growth cones is regulated dynamically with age and suggest that mRNA localization is tailored to match the functional demands of the growing axon tip as it transforms into the presynaptic terminal.
doi:10.1523/JNEUROSCI.1800-10.2010
PMCID: PMC3683943  PMID: 21084603
23.  Anesthetics Interfere with Axon Guidance in Developing Mouse Neocortical Neurons In Vitro via a γ-Aminobutyric Acid Type A Receptor Mechanism 
Anesthesiology  2013;118(4):825-833.
Background
The finding that exposure to general anesthetics (GAs) in childhood may increase rates of learning disabilities has raised a concern that anesthetics may interfere with brain development. The generation of neuronal circuits, a complex process in which axons follow guidance cues to dendritic targets, is an unexplored potential target for this type of toxicity.
Methods
GA exposures were conducted in developing neocortical neurons in culture and in early postnatal neocortical slices overlaid with fluorescently labeled neurons. Axon targeting, growth cone collapse, and axon branching were measured using quantitative fluorescence microscopy.
Results
Isoflurane exposure causes errors in Semaphorin 3A dependent axon targeting (n = 77 axons) and a disruption of the response of axonal growth cones to Semaphorin 3A (n = 2,358 growth cones). This effect occurs at clinically relevant anesthetic doses of numerous GAs with allosteric activity at γ-aminobutyric acid type A receptors, and it was reproduced with a selective agonist. Isoflurane also inhibits growth cone collapse induced by Netrin-1, but does not interfere branch induction by Netrin-1. Insensitivity to guidance cues caused by isoflurane is seen acutely in growth cones in dissociated culture, and errors in axon targeting in brain slice culture occur at the earliest point at which correct targeting is observed in controls.
Conclusion
Our results demonstrate a generalized inhibitory effect of GAs on repulsive growth cone guidance in the developing neocortex that may occur via a γ-aminobutyric acid type A receptor mechanism. The finding that GAs interfere with axon guidance, and thus potentially with circuit formation, represents a novel form of anesthesia neurotoxicity in brain development.
doi:10.1097/ALN.0b013e318287b850
PMCID: PMC3605271  PMID: 23364597
24.  Ephrin-B2 elicits differential growth cone collapse and axon retraction in retinal ganglion cells from distinct retinal regions 
Developmental neurobiology  2010;70(11):781-794.
The circuit for binocular vision and stereopsis is established at the optic chiasm, where retinal ganglion cell (RGC) axons diverge into the ipsilateral and contralateral optic tracts. In the mouse retina, ventrotemporal (VT) RGCs express the guidance receptor EphB1, which interacts with the repulsive guidance cue ephrin-B2 on radial glia at the optic chiasm to direct VT RGC axons ipsilaterally. RGCs in the ventral retina also express EphB2, which interacts with ephrin-B2, whereas dorsal RGCs express low levels of EphB receptors. To investigate how growth cones of RGCs from different retinal regions respond upon initial contact with ephrin-B2, we utilized time-lapse imaging to characterize the effects of ephrin-B2 on growth cone collapse and axon retraction in real time. We demonstrate that bath application of ephrin-B2 induces rapid and sustained growth cone collapse and axon retraction in VT RGC axons, whereas contralaterally-projecting dorsotemporal RGCs display moderate growth cone collapse and little axon retraction. Dose response curves reveal that contralaterally-projecting ventronasal axons are less sensitive to ephrin-B2 treatment compared to VT axons. Additionally, we uncovered a specific role for Rho kinase signaling in the retraction of VT RGC axons but not in growth cone collapse. The detailed characterization of growth cone behavior in this study comprises an assay for the study of Eph signaling in RGCs, and provides insight into the phenomena of growth cone collapse and axon retraction in general.
doi:10.1002/dneu.20821
PMCID: PMC2930402  PMID: 20629048
Retinal ganglion cells; ipsilateral projection; growth cones; Eph receptors and ephrins; Rho kinase
25.  Reduction in endothelial tip cell filopodia corresponds to reduced intravitreous but not intraretinal vascularization in a model of ROP 
Experimental eye research  2009;89(5):718-727.
Purpose
To determine the effect of a vascular endothelial growth factor receptor 2 tyrosine kinase (VEGFR2) inhibitor on intravitreous neovascularization (IVNV), endothelial tip cell filopodia, and intraretinal vascularization in a rat model of retinopathy of prematurity (ROP).
Methods
Within 4 hours of birth, newborn Sprague-Dawley rats pups and their mothers were cycled between 50% and 10% oxygen daily until postnatal day (p)12. Pups were given intravitreous injections of VEGFR2 inhibitor, SU5416, or control (dimethyl sulfoxide, DMSO) and returned to oxygen cycling until p14, then placed into room air. Intravitreous neovascularization (IVNV), avascular/total retinal areas, and endothelial tip cell filopodial number and length were determined in lectin-labeled neurosensory retinal flat mounts. Cryosections or fresh tissue were analyzed for phospho-VEGFR1, phospho-VEGFR2, activated caspase 3, or phospho-β3 integrin,. Human umbilical venous (HUVECs) and human choroidal endothelial cells (ECs) were treated with VEGFR2 inhibitor to determine effect on VEGFR2 phosphorylation and on directed EC migration toward a VEGF gradient. Filopodial length and number of migrated ECs were also measured.
Results
Compared to control, the VEGFR2 inhibitor reduced VEGFR2 phosphorylation in HUVECs in vitro and clock hours and areas of IVNV but not percent avascular retina in vivo. Filopodial length and number of filopodia /EC tip cell were reduced in retinal flat mounts at doses that inhibited IVNV, whereas at lower doses, only a reduction in filopodial length/ EC tip cell was found. There was no difference in phosphorylated β3 integrin and cleaved caspase-3 labeling in VEGFR2 inhibitor-treated compared to control in vivo. Doses of the VEGFR2 inhibitor that reduced filopodial length and number of filopodia/migrating EC corresponded to reduced EC migration in in vitro models.
Conclusions
VEGFR2 inhibitor reduced IVNV and filopodial number and length/EC tip cell without interfering with intraretinal vascularization. Reducing the number and length of filopodia/endothelial tip cell may reduce guidance cues for endothelial cells to migrate into the vitreous without interfering with migration into the retina toward a VEGF gradient.
doi:10.1016/j.exer.2009.06.011
PMCID: PMC2757474  PMID: 19576214
vascular endothelial growth factor (VEGF); retinopathy of prematurity (ROP); intravitreous neovascularization; intraretinal neovascularization; SU5416; filopodia

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