In mammals, Nerve growth factor (NGF) responsive neurons that terminate in the dorsal spinal cord carry pain and temperature signals. Semaphorin III functions as a selective chemorepellent to these neural projections, while Neurotropin-3 responsive large muscle afferents that terminate in the ventral cord are not affected. Responsible for the chemorepellent action, Semaphorin III is produced by ventral spinal cord cells, the targets of the large muscle afferents. In addition to the Semaphorin III cue, NGF injections in utero can cause these NGF responsive neurons to grow further than their normal termination site. The axons can reach or even cross the midline, but they never project into the ventral spinal cords. It is concluded that multiple factors, acting both positively and negatively, can direct the NGF responsive neurons (Messersmith, 1995).

Collapsin-1 is a member of the semaphorin family of signaling molecules that acts as a repellent for growing spinal sensory axons. A chimeric collapsin-1/alkaline phosphatase probe has been constructed to visualize putative collapsin-1 receptors in vitro and in situ. As predicted by the activity profile of collapsin-1, the probe binds spinal sensory tracts, ventral spinal roots, and the sympathetic chain, but does not bind retinal axons. The probe also binds sensory axons arising from the olfactory epithelium and some, but not all, cranial sensory nerves. In vitro assays demonstrate that primary olfactory sensory, trigeminal, and jugular ganglion growth cones collapse in the presence of soluble collapsin-1. Comparing the expression pattern of collapsin-1 with the trajectories of collapsin-1 responsive axons suggests that in both the spinal cord and the olfactory bulb, collapsin-1 prevents premature entry of sensory axons into their target and helps determine the final location of sensory terminations (Kobayashi, 1997).

During embryogenesis, different subclasses of sensory neurons extend central projections from the periphery to specific locations in the spinal cord. Muscle and cutaneous afferents initially project to the same location in the dorsal cord. Later, specific muscle afferents leave other afferents behind and project into the ventral cord. Previous studies have shown that ventral spinal cord explants secrete a repellent for sensory neurites. Antibodies to collapsin-1, a semaphorin family member, neutralize this repellent activity. All afferents respond to collapsin-1 when they are first confined to the dorsal cord, but ventrally projecting muscle afferents become collapsin-1 insensitive as they project into the ventral cord. These results suggest that the transient dorsal expression of collapsin-1 prevents all efferents from entering the cord early and sustained ventral expression prevents dorsally terminating afferents from entering the ventral cord later. Muscle afferents lose much of their responsiveness to collapsin-1, thereby permitting them to invade ventrally (Shepherd, 1997). Temporal changes in gene expression patterns of axons as they grow to their targets will undoubtedly be one of the major strategies governing complex patterns of innervation.

Chick collapsin-1/human semaphorin III/mouse semaphorin D is believed to guide the extension of specific axons by a repellent mechanism. It has a role in the guidance of axons of the ganglion of Remak in the developing chick intestine. The ganglionated nerve of Remak, termed 'Remak' in this study, runs parallel to the intestine within the mesentery and the mesorectum from the duodeno-jejunal junction to the cloaca. Early in embryogenesis, Remak axons extend parallel to, but do not enter, the intestine when collapsin-1 is expressed in the adjacent rectal wall. Remak axons later penetrate the peripheral portions of the rectal wall when collapsin-1 expression retreats from the outer muscle layer to the more internal submucosal and mucosal layers of the rectum. Extension of Remak neurites is repelled in vitro by rectum explants and also by 293T cells expressing collapsin-1. The rectal chemorepellent activity is blocked by anti-collapsin-1 antibodies. These results suggest that collapsin-1 may help prevent Remak axons from projecting into the intestinal wall at early developmental times and later restricts Remak axon trajectories to the outer part of the intestinal muscle layer (Shepherd, 1999).

Collapsin-1 or semaphorin III(D) inhibits axonal outgrowth by collapsing the lamellipodial and filopodial structures of the neuronal growth cones. Because growth cone collapse is associated with actin depolymerization, the possibility that small GTP-binding proteins of the rho subfamily might participate in collapsin-1 signal transduction was examined. Recombinant rho, rac1, and cdc42 proteins were triturated into embryonic chick (DRG) neurons. Constitutively active rac1 increases the proportion of collapsed growth cones, and dominant negative rac1 inhibits collapsin-1-induced collapse of growth cones and collapsin-1 inhibition of neurite outgrowth. DRG neurons treated with dominant negative rac1 remain sensitive to myelin-induced growth cone collapse. Similar mutants of cdc42 do not alter growth cone structure, neurite elongation, or collapsin-1 sensitivity. Whereas the addition of activated rho has no effect, the inhibition of rho with Clostridium botulinum C3 transferase stimulates the outgrowth of DRG neurites. C3 transferase-treated growth cones exhibit little or no lamellipodial spreading and are minimally responsive to collapsin-1 and myelin. These data demonstrate a prominent role for rho and rac1 in modulating growth cone motility and indicate that rac1 may mediate collapsin-1 action (Jin, 1997).

In order to examine whether semaphorins may be involved in guiding the formation of the reciprocal thalamocortical connections in the rat, analysis has been carried out on the spatial and temporal expression of five recently identified rodent semaphorins (semB, C, D, F and G) using in situ hybridization. Transcripts of all five genes are present throughout the period examined (E15-P7) and display highly specific spatiotemporal distributions. The spatiotemporal expression patterns are compatible with a role as chemorepellants in several developmental events. Specifically, semaphorins are in the position to: (1) prevent neurite extension into the ventricular neuroepithelium throughout the brain; (2) confer non-permissive properties to the embryonic cortical plate, hence regulating the radial invasion of corticopetal afferents; (3) confine axonal extension to the intermediate zone and subplate; (4) maintain the fasciculated state of thalamocortical and corticothalamic axons, and prevent them from branching while they grow through the striatum; and (5) restrict the terminal arborizations of thalamic afferents to layer IV. The evidence that different semaphorin genes are often co-expressed further suggests that the various molecules might interact in synergistic ways. Taken together, these results support the hypothesis that semaphorins could act as guidance signals in the development of the thalamocortical projections and suggest that innervation specificity is achieved through the combined action of multiple guidance cues (Skaliora, 1998)

The semaphorins constitute a large gene family of transmembrane and secreted molecules, many of which are expressed in the nervous system. Genetic studies in Drosophila have revealed a role for semaphorins in axon guidance and synapse formation, and several in vitro studies in mice have demonstrated semaphorin III's (Sema III) dramatic chemorepellent effect on the axons of several populations of neurons. To investigate the function of Sema III during in vivo axon guidance in the mammalian CNS, the development of axonal projections was studied in mutant mice lacking Sema III. Projections were studied for which either the in vitro evidence suggests a role for Sema III in axon guidance (e.g., cerebellar mossy fibers, thalamocortical axons, or cranial motor neurons) or the in vivo expression suggests a role for Sema III in axon guidance (e.g., cerebellar Purkinje cells, neocortex). Many major axonal projections, including climbing fiber, mossy fiber, thalamocortical, and basal forebrain projections and cranial nerves, are found to develop normally in the absence of Sema III. Despite its in vitro function and in vivo expression, it appears as if Sema III is not absolutely required for the formation of many major CNS tracts. Such data are consistent with recent models suggesting that axon guidance is controlled by a balance of forces resulting from multiple guidance cues. It is suggested that if Sema III functions in part to guide the formation of major axonal projections, then it does so in combination with both other semaphorins and other families of guidance molecules (Catalano, 1998).

The semaphorin/collapsin gene family encodes secreted and transmembrane proteins, several of which can repulse growth cones. Although the in vitro activity of Semaphorin III/D/Collapsin 1 is clear, recent analyses of two different strains of semaphorin III/D/collapsin 1 knockout mice have generated conflicting findings. In order to clarify the in vivo action of this molecule, sema Z1a, a zebrafish homolog of semaphorin III/D/collapsin 1 was analyzed. The expression pattern of sema Z1a in zebrafish suggests that it delimited the pathway of the growth cones of a specific set of sensory neurons, the posterior ganglion of the lateral line. To examine the in vivo action of this molecule, the following were analyzed: (1) the pathways followed by lateral line growth cones in mutants in which the expression of sema Z1a is altered; (2) response of lateral line growth cones to exogenous Sema Z1a in living embryos, and (3) the pathway followed by lateral line growth cones when Sema Z1a is misexpressed by cells along their normal route. The results suggest that a repulsive action of Sema Z1a collapses specific growth cones and helps to guide the growth cones of the lateral line along their normal pathway (Shoji, 1998).

Semaphorin III, a secretable semaphorin, is expressed by the ventral spinal cord at the time corresponding to projection of sensory afferents from the dorsal root ganglion (DRG) into the spinal cord. The inhibitory effect of E14 ventral cord is active only on nerve growth factor (NGF)-responsive sensory afferents (small-diameter A-delta and C fibers subserving sensations of temperature and pain). Similarly, COS cells secreting recombinant semaphorin III are able to selectively repel DRG afferents whose growth is stimulated by NGF and not NT-3. However, it is not known whether these molecules can exert a functional role in the fully developed adult peripheral nervous system. Transfection and production of semaphorin III in corneal epithelial cells in adult rabbits in vivo can cause repulsion of established A-delta and C fiber trigeminal sensory afferents. In addition, it is shown that, following epithelial wounding and denervation, semaphorin III is able to inhibit collateral nerve sprouts from innervating the reepithelialized tissue. These findings are significant in that they provide direct evidence that small-diameter adult sensory neurons retain the ability to respond to semaphorin III (Tanelian, 1997).

Semaphorin III/D has been shown to repel dorsal root ganglion (DRG) axons in vitro, and has been implicated in the patterning of sensory afferents in the spinal cord. Although semaphorin III/D mRNA is expressed in a wide variety of neural and nonneural tissues in vivo, the role played by semaphorin III/D in regions other than the spinal cord is not known. Mice homozygous for a targeted mutation in semaphorin III/D show severe abnormality in peripheral nerve projection. This abnormality is seen in the trigeminal, facial, vagus, accessory, and glossopharyngeal nerves but not in the oculomotor nerve. These results suggest that semaphorin III/D functions as a selective repellent in vivo (Taniguchi, 1997).

Semaphorins D and E are members of the class III semaphorin secreted proteins. A study was carried out to see if these semaphorins influence the growth of cortical axons, since these axons develop in cultured cortical explants. Recombinant Semaphorin D can collapse cortical axon growth cones, functions as a chemorepellent, and, in addition, inhibits cortical axonal branching. In contrast, semaphorin E acts as an attractive guidance signal for cortical axons. Attractive effects are observed only when growth cones encounter increasing concentrations or a patterned distribution of Semaphorin E, but not when they are exposed to uniform concentrations of this molecule. Specific binding sites for Semaphorin D and Semaphorin E are present on cortical fibers both in vitro and in vivo at the time when corticofugal projections are established. In situ hybridization analysis reveals that the population of cortical neurons used in these experiments express neuropilin-1 and neuropilin-2, which are essential components of receptors for the class III semaphorins. Moreover, semD mRNA is detected in the ventricular zone of the neocortex whereas semE mRNA is restricted to the subventricular zone. Taken together, these results indicate that semaphorins are bifunctional molecules whose effects depend on their spatial distribution. Growing corticofugal axons, that is axons leaving the cortex to travel deep into the intermediate zone, might require attractant signals driving them into the intermediate zone. These attractive signals, provided by Semaphorin E, work in combination with repulsive molecules (SemD) that prevent these fibers from growing into the ventricular zone. Because SemE mRNA can be detected in the subventricular zone (located between the intermediate zone and the more ventral ventricular zone) and SemD transcripts are expressed in the ventricular zone, it is suggested that cortical axons are directed by a gradient of SemE into the intermediate zone until they encounter SemD, a repulsive cue that keeps them out of the ventricular and subventricular regions. The coordinated expression of different semaphorins, together with their specific activities on cortical axons, suggests that multiple guidance signals contribute to the formation of precise corticofugal pathways (Bagnard, 1998).

During development, growth cones can be guided at a distance by diffusible factorsthat may be attractants and/or repellents. The semaphorins are the largest family of repulsive axon guidance molecules. Secreted semaphorins bind neuropilin receptors and repel sensory, sympathetic, motor, and forebrain axons. In rat embryos, the olfactory epithelium releases a diffusible factor that repels olfactory bulb axons. In addition, Sema A and Sema IV, but not Sema III, Sema E, or Sema H, are able to orient in vitro the growth of olfactory bulb axons; Sema IV has a strong repulsive action, whereas Sema A appears to attract those axons. The expression patterns of sema A and sema IV in the developing olfactory system confirm that they may play a cooperative role in the formation of the lateral olfactory tract. This also represents further evidence for the chemoattractive function of secreted semaphorins (Castro, 1999).

In humans, defects of the corticospinal tract have been attributed to mutations in the gene encoding L1 CAM, a phenotype that is reproduced in L1-deficient mice. Using coculture assays, Sema3A secreted from the ventral spinal cord is reported to repel cortical axons from wild-type but not from L1-deficient mice. L1 and neuropilin-1 (NP-1) form a stable complex, and their extracellular domains can directly associate. Thus, L1 is a component of the Sema3A receptor complex, and L1 mutations may disrupt Sema3A signaling in the growth cone, leading to guidance errors. Addition of soluble L1Fc chimeric molecules does not restore Sema3A responsiveness of L1-deficient axons; instead, it converts the repulsion of wild-type axons into an attraction, further supporting a function for L1 in the Sema3A transducing pathways within the growth cone (Castellani, 2000).

To date, L1 activity in the guidance of neuronal projections has been associated with axonal fasciculation and neurite extension. Accordingly, L1 interacts with a number of cell adhesion and extracellular matrix molecules, including members of the Ig superfamily, integrins, and chondroitin sulfate proteoglycans. The finding that L1 is required for Sema3A signaling expands its range of actions and assigns it as a player in mechanisms of guidance at a distance. This raises the possibility that other fiber tracts depending upon Sema3A for their guidance might also be impaired in L1-deficient mice (Castellani, 2000).

NP-1 and NP-2 are the first proteins identified as receptors for the semaphorin family of guidance cues. Only NP-1 is necessary for Sema3A, and probably Sema3E, whereas binding sites for Sema3B and Sema3C are formed by either NP-2 homodimers or NP-1-NP-2 heterodimers. Both the coculture and collapse assays reported here demonstrate that L1 is required for the growth cone to respond to Sema3A-induced chemorepulsion but is not necessary for Sema3B, -3C, or -3E biological activity. Thus, the functional coupling of L1 to Sema3A may confer an additional level of specificity to the functions of semaphorins in the guidance of neuronal projections (Castellani, 2000).

The coimmunoprecipitation and binding experiments have showen that L1 and NP-1, but not NP-2, extracellular domains are able to interact and that L1 participates in the formation of a Sema3A receptor multimolecular complex. There are precedents for the participation of NP-1 in receptor heterocomplexes. For example, NP-1 binds to vascular endothelial growth factor receptor (VEGFR), thereby enhancing the affinity of the VEGF ligand for its receptor. Furthermore, NP-1 forms a stable complex with the transmembrane protein plexin-A1. Within this complex, plexin-A1 serves as a signal tranducer for Sema3A-induced growth cone collapse of sensory neurons. The coculture and collapse assays indicate that L1 is also necessary for neurons to respond to Sema3A. Application of L1Fc could not restore the response of L1-deficient axons to Sema3A, indicating that the intracellular domain of L1 is essential for this response. Furthermore, L1Fc does not simply block Sema3A chemorepulsion of wild-type neurons but switches it into attraction. It is therefore likely that L1-L1 homophilic interactions contribute to the outcome of Sema3A intracellular signaling in the growth cone (Castellani, 2000).

L1 could achieve this function via different interactions. (1) Its intracellular domain associates with tyrosine kinases and two serine/threonine kinases, including casein kinase II, which has been implicated in neurite fasciculation. (2) L1 stimulation is assumed to activate second-messenger systems that are common to the pathways used by fibroblast growth factor receptor (FGFR) tyrosine kinases. (3) The intracytoplasmic region of L1 interacts with the ankyrin family of spectrin binding proteins. This coupling may provide a molecular linkage between Sema3A and actin cytoskeletal dynamics within the growth cone. Alternatively, L1 could regulate the Sema3A signaling by influencing the clustering of NP-1 within the heterocomplex. As first shown for ephrins, another family of repulsive signals, and their receptors, the Eph tyrosine kinases, the oligomeric status of receptors and ligands determines their biological activity. Recent work has expanded this idea to the semaphorin family with the demonstration that monomeric, dimeric, and oligomeric forms of Sema1A confer distinct functional properties during axonal guidance in Drosophila embryos (Castellani, 2000 and references therein).

The molecular mechanisms by which L1Fc switches the Sema3A-induced response are not clear. However, two results suggest that it is achieved through activation of L1 signaling in the growth cone: (1) L1Fc alone induces a strong increase in neurite outgrowth, a process assumed to result from L1-L1 homophilic interaction; (2) the response of L1-deficient axons to Sema3A is not modulated by L1Fc, indicating that even if L1Fc can interact with NP1, transmembrane L1 in the receptor heterocomplex is crucial for the growth cone to undergo Sema3A-induced chemoattraction. L1Fc may modify the composition of the Sema3A receptor, for example, by diverting L1 into another functionally distinct transduction unit. Such changes in the interactions contracted by the different components in the complex could initiate an attractive response to Sema3A. Likewise, recent work has demonstrated that the cytoplasmic domain of axon guidance receptors and the formation of heterocomplexes are crucial in determining the type of response of the growth cone to chemotropic cues (Castellani, 2000).

It has also been shown that cyclic nucleotide levels are pivotal for the decision of the growth cone to be attracted or repelled by secreted guidance signals. Sema3A chemorepulsion can be converted into attraction by an increase in cGMP levels in the growth cone, driven by nitric oxide-dependent intracellular pathways. Accordingly, it is possible that L1Fc induces the switch by activating the synthesis of cGMP. Consistent with this, preliminary data suggest that blockade of soluble guanylate cyclase prevents the L1Fc-induced switch in the Sema3A response (Castellani, 2000).

In the present experiments, L1Fc may mimic L1-L1 trans-interaction of the growth cone with another cell or axonal surface. This implies that during the development of neuronal projections, changes in L1-dependent interactions may be able to switch growth cone responses to Sema3A from repulsion to attraction. Finally, the present study raises the more general view that specific cross-talk between cell contact and chemotropic guidance cues could be a potent mechanism to coordinate the events required for guiding axon projections toward their appropriate targets (Castellani, 2000).

Semaphorin3A (previously known as Semaphorin III, Semaphorin D, or collapsin-1) is a member of the semaphorin gene family, many of which have been shown to guide axons during nervous system development. Semaphorin3A is a diffusible chemorepulsive molecule for axons of selected neuronal populations in vitro. Analysis of embryogenesis in two independent lines of Semaphorin3A knockout mice supports the hypothesis that this molecule is an important guidance signal for neurons of the peripheral nervous system. Surprisingly, newborn Semaphorin3A null mutant mice exhibit no significant abnormalities. In this study the hypothesis is tested that guidance abnormalities that occurred during early stages of Semaphorin3A null mice development are corrected later in development. The extensive abnormalities formed during early developmental stages in the peripheral nervous system are largely eliminated by embryonic day 15.5. At least in one distinct anatomical location these abnormalities are mainly the result of aberrant projections. Elimination of aberrant projections is most probably due to mechanisms based on programmed cell death and/or selective axonal pruning. In conclusion, these findings suggest the existence of correction mechanisms that eliminate most sensory axon pathfinding errors early in development (White, 2000).

Plexins are receptors implicated in mediating signaling by semaphorins, a family of axonal chemorepellents. The role of specific plexins in mediating semaphorin function in vivo has not, however, yet been examined in vertebrates. Plexin-A3 is the most ubiquitously expressed plexin family member within regions of the developing mammalian nervous system known to contain semaphorin-responsive neurons. Using a chimeric receptor construct, evidence has been provided that plexin-A3 can transduce a repulsive signal in growth cones in vitro. Analysis of plexin-A3 knockout mice shows that plexin-A3 contributes to Sema3F and Sema3A signaling and that plexin-A3 regulates the development of hippocampal axonal projections in vivo (Cheng, 2001).

To test directly for a plexin-A3 signaling function, a gain-of-function approach was taken, asking whether the plexin-A3 cytoplasmic domain could mediate a repulsive response in the context of a chimeric receptor in which the extracellular domain of plexin-A3 is replaced by that of Met, a receptor for hepatocyte growth factor (HGF). This Met-plexin-A3 chimera was then introduced into embryonic Xenopus spinal neurons, which are repelled by Sema3A. Wild-type neurons do not respond to HGF but are attracted to HGF when wild-type Met is introduced in these cells. In contrast, when the chimeric Met-plexin-A3 receptor is introduced into these neurons, HGF elicites a robust repulsive response. This result provides evidence that plexin-A3 can function directly in signaling repulsion (Cheng, 2001).

Tracing studies have shown that developing hippocampal afferent axons invade their appropriate domains and layers in a highly specific fashion. Such stereotyped growth suggests the involvement of short-range cues providing layer-specific targeting information. Studies of reeler mutant mice deficient in reelin implicate a chemorepellent associated with Cajal-Retzius cells that inhibits commissural axon outgrowth into stratum lacunosum moleculare. Moreover, it has been suggested that Sema3F may play an important role in lamina-specific projections of hippocampal afferents. Plexin-A3 mutant commissural axons project to inappropriate laminae within the hippocampus, supporting the idea that the loss of plexin-A3 causes a reduction or loss in response to this Sema3F-based cue. Additionally, the observed switch in laminar termination zones also suggests the unmasking of an attractive cue within SLM. Laminar termination defects were not observed in spinal cord or cerebellum in plexin-A3 mutant mice, suggesting the importance of cues other than Sema3F in directing these terminations (Cheng, 2001).

During development of the amniote peripheral nervous system, the initial trajectory of primary sensory axons is determined largely by the action of axon repellents. Tissues flanking dorsal root ganglia, the notochord (lying medially) and the dermamyotomes (lying laterally), are sources of secreted molecules that prevent axons from entering inappropriate territories. Although there is evidence suggesting that SEMA3A contributes to the repellent activity of the dermamyotome, the nature of the activity secreted by the notochord remains undetermined. An expression cloning strategy has been employed to search for axon repellents secreted by the notochord, and SEMA3A has been defined as a candidate repellent. Using a spectrum of different axon populations to assay the notochord activity, together with neuropilin/Fc receptor reagents to block semaphorin activity in collagen gel assays, it has been shown that SEMA3A probably contributes to notochord-mediated repulsion. Sympathetic axons that normally avoid the midline in vivo are also repelled, in part, by a semaphorin-based notochord activity. Although these results implicate semaphorin signalling in mediating repulsion by the notochord, repulsion of early dorsal root ganglion axons is only partially blocked when using neuropilin/Fc reagents. Moreover, retinal axons, which are insensitive to SEMA3A, are also repelled by the notochord. It is concluded that multiple factors act in concert to guide axons in this system, and that further notochord repellents remain to be identified (Anderson, 2003).

Chemorepulsion by semaphorins plays a critical role during the development of neuronal projections. Although semaphorin-induced chemoattraction has been reported in vitro, the contribution of this activity to axon pathfinding is still unclear. Using genetic and culture models, evidence is provided that both attraction and repulsion by Sema3B, a secreted semaphorin, are critical for the positioning of a major brain commissural projection, the anterior commissure (AC). NrCAM, an immunoglobulin superfamily adhesion molecule of the L1 subfamily, associates with neuropilin-2 and is a component of a receptor complex for Sema3B and Sema3F. Finally, it is shown that activation of the FAK/Src signaling cascade distinguishes Sema3B-mediated attractive from repulsive axonal responses of neurons forming the AC, revealing a mechanism underlying the dual activity of this guidance cue (Falk, 2005).

Commissural axon guidance requires complex modulations of growth cone sensitivity to midline-derived cues, but underlying mechanisms in vertebrates remain largely unknown. By using combinations of ex vivo and in vivo approaches, a molecular pathway was uncovered controlling the gain of response to a midline repellent, Semaphorin3B (Sema3B). Evidence is provided that Semaphorin3B/Plexin-A1 signaling participates in the guidance of commissural projections at the vertebrate ventral midline. At the precrossing stage, commissural neurons synthesize the Neuropilin-2 and Plexin-A1 Semaphorin3B receptor subunits, but Plexin-A1 expression is prevented by a calpain1-mediated processing, resulting in silencing commissural responsiveness. During floor plate (FP) in-growth, calpain1 activity is suppressed by local signals, allowing Plexin-A1 accumulation in the growth cone and sensitization to Sema3B. The FP cue NrCAM mediates the switch of Plexin-A1 processing underlying growth cone sensitization to Sema3B. This reveals pathway-dependent modulation of guidance receptor processing as a novel mechanism for regulating guidance decisions at intermediate targets (Nawabi, 2010).

In Drosophila, responsiveness of precrossing commissural axons to Slit is silenced through coupling of the Slit receptor Robo to Commissureless, and sorting for proteasome degradation. In vertebrates, a spliced variant of one of the three Robo gene products, Robo-3.1, appears as a functional equivalent of Drosophila Commissureless, preventing through a yet-undetermined mechanism Robo1 and Robo2 from mediating responsiveness to Slits at the precrossing stage. The present study highlights a novel mechanism by which precrossing commissural responses are silenced, based on processing of guidance receptors (Nawabi, 2010).

An interesting aspect of this pathway is that it does not prevent ligand/receptor interaction as for the Robo/Slit pair in Drosophila and possibly in vertebrates, since Nrp2 sorting to the growth cone surface is not prevented. Rather, it precludes accumulation of full-length signaling moiety of the receptor complex specifically transducing Sema3B in these neurons. This mechanism is advantageous and well-suited to the Semaphorin signaling, as it enables the other receptor subunit, Nrp2, to engage in other complexes (Nawabi, 2010).

Calpains are calcium-dependent cysteine proteases, regulating various processes. For example, they play pivotal roles in cell motility and synaptic functions by cleaving components of adhesion complexes and neurotransmitter receptors. Their functions during neuronal development are less characterized, but calpains are present in neuronal growth cones, are activated by intracellular calcium transients to reduce growth cone motility, and also are found downstream from Semaphorin5B. Interestingly, calpains process rather than degrade proteins, and are seen as regulators of protein functions, modulating protein-protein interactions, phosphorylation state, distribution, and traffic. The current data indicate that calpain1 is active in commissural neurons and maintains the integral form of Plexin-A1 at very low levels at the precrossing stage by cleaving the Plexin-A1 extracellular domain, generating two fragments that can be detected in Western blot. This processing could take place before protein sorting to the cell membrane. Likewise, previous studies reported detection of calpain in the lumen of endoplasmic reticulum and golgi vesicles, thus being at an appropriate location for cleaving neosynthesized target proteins during their intracellular traffic. Alternatively, Plexin-A1 could be cleaved at the cell surface, since increasing evidence indicates that calpains are externalized and can be retained to the membrane surface through association with proteoglycans. Several of the results support an important role for calpain activity during precrossing commissural axon guidance. First, in the spinal cord sections, calpains were found active in spinal neurons and precrossing commissural axon segments. Second, inhibition of calpain activity in vivo resulted in strong defects of commissural axon behaviors before FP crossing: axons failing to enter the FP, turning before FP crossing, or even not reaching the FP. Interestingly, all of these defects were also induced by Plexin-A1 overexpression in the chick embryo, thus supporting that calpain-1 activity prevents Plexin-A1 expression at the precrossing stage. Whether this protease suppresses precrossing commissural responses to other midline repellents by processing guidance receptors other than Plexin-A1, such as Robo1/2 or Eph receptors, is an intriguing possibility that will be assessed in future studies (Nawabi, 2010).

The results showed that suppression of calpain activity in the FP is instrumental for the gain of commissural responsiveness to Sema3B. Several data support this conclusion. First, in unfixed spinal cord sections, calpain were not active in FP cells or in the crossing axon tract. Second, it was found that the FP tissue released signals that inhibit calpain activity in spinal tissue. Third, suppression of calpain activity could increase integral Plexin-A1 levels in acute dorsal tissues and cultured commissural neurons and induce responsiveness to Sema3B (Nawabi, 2010).

Commissural axons were found to acquire responsiveness to a variety of repellents, but whether this occurs through a pathway-dependent or pathway-independent mechanism remains unclear. In the Xenopus visual system, the temporal switch from attractive to repulsive behavior of retinal axons to Netrin1 does not depend on pathway experience. The current data do not support the view that such an experience-independent mechanism operates to confer responsiveness to Sema3B at the midline. First, dorsal spinal neurons isolated from early E11 to late E13.5 were equally unresponsive to exogenous Sema3B application. Second, in open book preparations, removing the FP was sufficient to abolish the repulsive behavior of commissural neurons to a focal source of Sema3B. Finally, neurons became sensitive to Sema3B upon exposure to FP^cm (control supernatant), independent from the developmental stage at which they were collected. The data thus support that signals emanating from intermediate target cells play pivotal roles in the switch of responsiveness (Nawabi, 2010).

Consistently, it was shown that the Ig superfamily cell adhesion molecule NrCAM is an active FP component, regulating the Plexin-A1 level and acquisition of responsiveness to Sema3B. High levels of NrCAM transcripts were detected in the FP, and the protein was present in the FP^cm. In the neuronal assay, soluble NrCAM mimicked the FP^cm, triggering responsiveness to Sema3B and increase of the PlexinA1 level. In contrast, neither Netrin-1 nor Shh could recapitulate the gain of responsiveness to Sema3B conferred by the FP^cm. Moreover, the biochemical or genetic depletion of NrCAM strongly altered the properties of the FP^cm. In vivo, strong decrease of the Plexin-A1 level in the FP of NrCAM-null embryos was found and significant amounts of axon tracts abnormally stalled in the FP. NrCAM is expressed by various developing neuronal projections, and several previous studies implicated it in the regulation of axon navigation, as receptors or coreceptors for environmental guidance cues. Likewise, NrCAM interaction with Nrp2 was found to be required for axons to normally form the anterior commissure in the brain, and NrCAM expression is required by RGC axons to form proper patterns of ipsilateral/contralateral commissures in the visual system. Interestingly, NrCAM is highly expressed in specialized glial structures in the ventral midline -- not only in the spinal cord, but also in all upper floors of the CNS, but how NrCAM glial sources contribute to axon pathfinding remains unknown. The present work establishes a novel function for NrCAM as an intermediate target cue regulating the expression level of guidance receptors in the growth cones to control pathway choices at the ventral midline (Nawabi, 2010).

The rate and direction of axon and dendrite growth depend on multiple guidance signals and growth factors. Semaphorin 3A (Sema3A) acts as a repellent for axons and attractant for dendrites. This study shows that the requirement for integrin engagement distinguishes the response of axons and dendrites to Sema3A in hippocampal neurons. Sema3A promotes the extension of hippocampal dendrites by a pathway that requires focal adhesion kinase (FAK). The stimulation of dendrite growth and FAK phosphorylation by Sema3A depend on integrin engagement. Unlike their function as a target of Sema3A during the collapse of axonal growth cones, integrins facilitate the stimulation of dendrite extension. Conditional inactivation of the genes encoding β1 integrin or FAK blocks the growth-promoting effect of Sema3A but not the collapse of axonal growth cones. These results demonstrate that different pathways mediate the stimulation of dendrite growth and the collapse of axonal growth cones by Sema3A (Schlomann, 2009).

These results reveal a bidirectional interaction of integrins and semaphorin signalling. β1 integrins facilitate the effect of Sema3A on dendrites, whereas integrins are inhibited by Sema3A during growth-cone collapse and in endothelial cells. Sema3A stimulates dendrite extension only when neurons are cultured on the integrin ligand fibronectin. Fibronectin could be substituted by Mn2+ ions, which stabilise the active conformation of integrins, allowing Sema3A to promote dendrite extension on the non-specific adhesive PO. A soluble RGD peptide that blocks the interaction with integrins and deletion of the Itgb1 gene abrogated the stimulatory effect of Sema3A. By contrast, the collapse of axonal growth cones was independent of integrin engagement. Unlike the effect of cyclic nucleotides on the response to axon-guidance signals, β1-integrin engagement does not modulate the response of dendrites because Sema3A had no effect on dendrite length after inactivation of Itgb1 or Fak. The results show that the effect of Sema3A on dendrite growth requires a signalling pathway that is distinct from that involved in collapse of the growth cone (Schlomann, 2009).

Neurovascular congruency does not arise from interdependence between peripheral nerves and blood vessels: the hypothesis is supported that such congruency arises by a shared patterning mechanism that utilizes semaphorin3A. Peripheral nerves and blood vessels have similar patterns in quail forelimb development. Usually, nerves extend adjacent to existing blood vessels, but in a few cases, vessels follow nerves. Nerves have been proposed to follow vascular smooth muscle, endothelium, or their basal laminae. Focusing on the major axial blood vessels and nerves, it was found that when nerves grow into forelimbs at E3.5-E5, vascular smooth muscle is not detectable by smooth muscle actin immunoreactivity. Additionally, transmission electron microscopy at E5.5 confirms that early blood vessels lack smooth muscle and show that the endothelial cell layer lacks a basal lamina, and physical contact is observed between peripheral nerves and these endothelial cells. To test more generally whether lack of nerves affected blood vessel patterns, forelimb-level neural tube ablations were performed at E2 to produce aneural limbs; these have completely normal vascular patterns up to at least E10. To test more generally whether vascular perturbation affected nerve patterns, VEGF₁₆₅, VEGF₁₂₁, Ang-1, and soluble Flt-1/Fc proteins singly and in combination were focally introduced via beads implanted into E4.5 forelimbs. These produce significant alterations to the vascular patterns, which include the formation of neo-vessels and the creation of ectopic avascular spaces at E6, but in both under- and overvascularized forelimbs, the peripheral nerve pattern is normal. The spatial distribution of semaphorin3A protein immunoreactivity is consistent with a negative regulation of neural and/or vascular patterning. Semaphorin3A bead implantations into E4.5 forelimbs causes failure of nerves and blood vessels to form and to deviate away from the bead. Conversely, semaphorin3A antibody bead implantation is associated with a local increase in capillary formation. Furthermore, neural tube electroporation at E2 with a construct for the soluble form of neuropilin-1 causes vascular malformations and hemorrhage as well as altered nerve trajectories and peripheral nerve defasciculation at E5-E6 (Bates, 2003).

The identification of new signaling pathways critical for cardiac morphogenesis will contribute to understanding of congenital heart disease (CHD), which remains a leading cause of mortality in newborn children worldwide. Signals mediated by semaphorin ligands and plexin receptors contribute to the intricate patterning of axons in the central nervous system. A related signaling pathway involves secreted class 3 semaphorins, neuropilins, and a plexin receptor, PlexinD1, expressed by endothelial cells. Interruption of this pathway in mice results in CHD and vascular patterning defects. The type of CHD caused by inactivation of PlexinD1 has been previously attributed to abnormalities of neural crest. This study shows that this form of CHD can be caused by cell-autonomous endothelial defects. Thus, molecular programs that mediate axon guidance in the central nervous system also function in endothelial cells to orchestrate critical aspects of cardiac morphogenesis (Gitler, 2004).

The closely related phenotypes involving the heart outflow tract (OT) and aortic arch defects of sema3C, plexinD1, and Npn-1 knockout mice, coupled with biochemical data, strongly suggest that semaphorin signaling mediated by endothelial cells (ECs) expressing PlexinD1 and neuropilins compose a receptor-ligand paracrine signaling pathway that orchestrates septation of the OT and development of aortic arch artery derivatives. Congenital heart defects in humans frequently involve OT and aortic arch defects, and SEMA3C, PLEXIND1, and NPN-1 are candidate genes for congenital heart disease. The ability of neuropilin to bind distinct ligands (VEGF¹⁶⁵ and SEMA3 proteins), coupled with elegant genetic studies, has suggested that neuropilin facilitates VEGF signaling in endothelium and semaphorin signaling in non-ECs including cardiac neural crest. Endothelial-specific loss of Npn-1 has been interpreted in terms of loss of VEGF¹⁶⁵ signaling in endothelium. However, biochemical and genetic data identify a direct role for neuropilin-mediated semaphorin signaling in endothelium. This result demands reinterpretation of existing data concerning the role of neuropilin, VEGF, and semaphorin signaling in cardiovascular development and led to the proposal of a unifying model. It is suggested that neuropilin, in ECs, functions in both VEGF and semaphorin signaling and that both pathways are required for proper cardiac OT development. Either Npn-1 or Npn-2 is able to cooperate with PlexinD1 to bind Sema3C. This explains why specific inhibition of Npn-1-dependent semaphorin signaling does not result in OT defects unless the redundant Npn-2 is also inactivated, whereas inactivating either the ligand Sema3C or the coreceptor PlexinD1 is sufficient to produce OT defects (Gitler, 2004 and references therein).

Major vessels of the vertebrate circulatory system display evolutionarily conserved and reproducible anatomy, but the cues guiding this stereotypic patterning remain obscure. In the nervous system, axonal pathways are shaped by repulsive cues provided by ligands of the semaphorin family that are sensed by migrating neuronal growth cones through plexin receptors. Proper blood vessel pathfinding requires the endothelial receptor PlexinD1 and semaphorin signals, and mutations have been identified in plexinD1 in the zebrafish vascular patterning mutant out of bounds. These results reveal the fundamental conservation of repulsive patterning mechanisms between axonal migration in the central nervous system and vascular endothelium during angiogenesis (Torres-Vazquez, 2004).

Chick collapsin-1 has been implicated in axonal pathfinding as a repulsive guidance cue. Collapsin-1 induces growth cone collapse via a pathway that may include CRMP-62 and heterotrimeric G proteins. CRMP-62 protein is related to UNC-33, a nematode neuronal protein required for appropriately directed axonal extension. Mutations in unc-33 affect neural microtubules, the basic cytoskeletal elements for axoplasmic transport. Using computer-assisted video-enhanced differential interference contrast microscopy, it is demonstrated that collapsin-1 potently promotes axoplasmic transport. Collapsin-1 doubles the number of antero- and retrograde-transported organelles but not their velocity. Collapsin-1 decreases the number of stationary organelles, suggesting that the fraction of time during which a particle is moving is increased. Collapsin-1-stimulated transport occurs by a mechanism distinct from that causing growth cone collapse. Pertussis toxin (PTX) but not its B oligomer blocks collapsin-induced growth cone collapse. The holotoxin does not affect collapsin-stimulated axoplasmic transport. Mastoparan and a myelin protein NI-35 induce PTX-sensitive growth cone collapse but do not stimulate axoplasmic transport. These results provide evidence that collapsin has a unique property to activate axonal vesicular transport systems (Goshima, 1997).

Precise growth cone guidance is the consequence of a continuous reorganization of actin filament structures within filopodia and lamellipodia in response to inhibitory and promoting cues. The small GTPases rac1, cdc42, and rhoA are critical for regulating distinct actin structures in non-neuronal cells and presumably in growth cones. Collapse, a retraction of filopodia and lamellipodia, is a typical growth cone behavior on contact with inhibitory cues and is associated with depolymerization and redistribution of actin filaments. An examination was carried out to see whether small GTPases mediate the inhibitory properties of CNS myelin or collapsin-1, a soluble semaphorin, in chick embryonic motor neuron cultures. As demonstrated for collapsin-1, CNS myelin-evoked growth cone collapse is accompanied by a reduction of rhodamine-phalloidin staining most prominent in the growth cone periphery, suggesting actin filament disassembly. Specific mutants of small GTPases are capable of desensitizing growth cones to CNS myelin or collapsin-1. Adenoviral-mediated expression of constitutively active rac1 or rhoA abolishes CNS myelin-induced collapse and allows remarkable neurite extension on a CNS myelin substrate. In contrast, expression of dominant negative rac1 or cdc42 negated collapsin-1 induces growth cone collapse and promotes neurite outgrowth on a collapsin-1 substrate. These findings suggest that small GTPases can modulate the signaling pathways of inhibitory stimuli and, consequently, allow the manipulation of growth cone behavior. However, the fact that opposite mutants of rac1 are effective against different inhibitory stimuli speaks against a universal signaling pathway underlying growth cone collapse (Kuhn, 1999).

In the chick dorsal mesencephalon, the optic tectum, the developing axons must choose between remaining on the same side of the midline or growing across it. The ipsilaterally projecting axons, forming the tectobulbar tract, course circumferentially toward the ventrally situated floor plate but before reaching the basal mesencephalon (the tegmentum), they gradually turn caudally. Here, they follow the course of the medial longitudinal fasciculus (MLF), located parallel to the floor plate. By in vivo labeling of tectal axons, it could be demonstrated that these axons arise primarily in the dorsal tectum. To test the idea that chemorepellent molecules are involved in guidance of the nondecussating axons, coculture experiments were performed employing tectal explants from various positions along the dorso-ventral axis. Axons emanating from dorsal tectal explants were strongly repelled by diencephalic tissue containing the neurons that give rise to the MLF, whereas ventral tectal axons showed only a moderate response. This inhibitory effect was substantially neutralized by the addition of anti-neuropilin-1 antibodies. A similar differential response of axons was observed when tectal explants were cocultured with cell aggregates secreting the chemorepellent Semaphorin 3A (Sema3A). Sema3B and Sema3C, respectively, do not inhibit growth of tectal axons. In addition, neither the floor plate nor Slit2-secreting cell aggregates influenced outgrowth of dorsal fibers. In Sema3A-deficient mice, DiI-labeling revealed that dorsal mesencephalic axons cross the MLF instead of turning posteriorly upon reaching the fiber tract, thus behaving like the ventrally originating contralaterally projecting axons. A differential responsiveness of tectal axons to Sema3A most likely released by the MLF thus contributes to pathfinding in the ventral mesencephalon (Henke-Fahle, 2001).

Developing neurons accurately position their somata within the neural tube to make contact with appropriate neighbors and project axons to their preferred targets. Taking advantage of a collection of genetically engineered mouse mutants, the behavior of somata and axons of the facial nerve is shown to be regulated independently by two secreted ligands for the transmembrane receptor neuropilin 1 (Nrp1), the semaphorin Sema3A and the VEGF164 isoform of Vascular Endothelial Growth Factor. Although Sema3A is known to control the guidance of facial nerve axons, it is not required for the pathfinding of their somata. Vice versa, it is found that VEGF164 is not required for axon guidance of facial motor neurons, but is essential for the correct migration of their somata. These observations demonstrate that VEGF contributes to neuronal patterning in vivo, and that different compartments of one cell can be co-ordinately patterned by structurally distinct ligands for a shared receptor (Schwarz, 2004).

Cyclic nucleotide-gated channels (CNGCs) transduce external signals required for sensory processes, e.g., photoreception, olfaction, and taste. Nerve growth cone guidance by diffusible attractive and repulsive molecules is regulated by differential growth cone Ca2+ signaling. However, the Ca2+-conducting ion channels that transduce guidance molecule signals are largely unknown. This study shows that rod-type CNGC-like channels function in the repulsion of cultured Xenopus spinal neuron growth cones by Sema3A, which triggers the production of the cGMP that activates the Xenopus CNGA1 (xCNGA1) subunit-containing channels in interneurons. Downregulation of xCNGA1 or overexpression of a mutant xCNGA1 incapable of binding cGMP abolished CNG currents and converted growth cone repulsion to attraction in response to Sema3A. Ca2+ entry through xCNGCs is required to mediate the repulsive Sema3A signal. These studies extend or knowledge of the function of CNGCs by demonstrating their requirement for signal transduction in growth cone guidance (Togashi, 2008).

A model is proposed describing the early signaling events induced by a repellent Sema3A gradient at the growth cone: activation of a Sema3A receptor complex upon binding of its ligand, Sema3A, triggers cGMP production by sGC, regulated by a mechanism that is likely independent of Ca2+ entry. Subsequent binding of cGMP to the CNB of xCNGA1 subunits (and presumably to the CNB of xCNGB1 as well) gates xCNGCs, resulting in entry of the Ca2+ responsible for Sema3A-induced growth cone repulsion. Inactivation of xCNGCs converts the Sema3A-induced repulsion to PKG-dependent attraction, suggesting that CNGC activity is crucial for the maintenance of the Sema3A repulsive signal. Sema3A triggers growth cone [cGMP]i increases above the micromolar range, which should be sufficient to activate PKG. How a repulsive Sema3A signal predominantly affects the cGMP-CNGC pathway, rather than the PKG-dependent signaling pathway, which would otherwise result in attraction, remains to be determined. Different levels of CNGC expression, of cGMP production, or of cGMP compartmentalization may all result in different growth cone turning behaviors in response to Sema3A (Togashi, 2008).

In the vertebrate retina, establishment of precise synaptic connections among distinct retinal neuron cell types is critical for processing visual information and for accurate visual perception. Retinal ganglion cells (RGCs), amacrine cells and bipolar cells establish stereotypic neurite arborization patterns to form functional neural circuits in the inner plexiform layer (IPL), a laminar region that is conventionally divided into five major parallel sublaminae. However, the molecular mechanisms governing distinct retinal subtype targeting to specific sublaminae within the IPL remain to be elucidated. This study shows that the transmembrane semaphorin Sema6A signals through its receptor PlexinA4 (PlexA4) to control lamina-specific neuronal stratification in the mouse retina. Expression analyses demonstrate that Sema6A and PlexA4 proteins are expressed in a complementary fashion in the developing retina: Sema6A in most ON sublaminae and PlexA4 in OFF sublaminae of the IPL, ON and OFF referring to observations separate channels encoding light increments (ON) and decrements (OFF) are spawned in the outer retina and relayed to different sublaminas of the IPL. Mice with null mutations in PlexA4 or Sema6A exhibit severe defects in stereotypic lamina-specific neurite arborization of tyrosine hydroxylase (TH)-expressing dopaminergic amacrine cells, intrinsically photosensitive RGCs (ipRGCs) and calbindin-positive cells in the IPL. Sema6A and PlexA4 genetically interact in vivo for the regulation of dopaminergic amacrine cell laminar targeting. Therefore, neuronal targeting to subdivisions of the IPL in the mammalian retina is directed by repulsive transmembrane guidance cues present on neuronal processes (Matsuoka, 2011).