The action of Semaphorin7A on thalamocortical axon branching

Developing axons form extensive branches to make synaptic contacts with their target cells. Despite the important role of axon branching in neural circuit formation, its underlying molecular mechanism is still largely unknown. In this study, we investigated the involvement of Semaphorin7A (Sema7A) in thalamocortical (TC) axon branching. In situ hybridization demonstrated that sema7a was expressed specifically in layer 4, the TC recipient layer, when TC axons form extensive arbors. A similar protein expression pattern was observed by immunohistochemistry with an anti-Sema7A antibody. The effect of Sema7A on axon branching was investigated in dissociated cell cultures from embryonic rat thalamus. TC axon branching increased dramatically on Sema7A-coated dishes. We further studied the activity of Sema7A in vivo using loss- and gain-of-function analyses. The number of vesicular glutamate transporter 2-positive puncta was markedly reduced in the Sema7A-deficient cortex. In contrast, their number increased significantly when Sema7A was over-expressed in layer 4 cells by in utero electroporation. Taken together, these findings suggest that Sema7A acts as a positive regulator for TC axon branching and/or pre-synaptic puncta formation.     

Invariant Sema5A inhibition serves an ensheathing function during optic nerve development

Retinal axon pathfinding from the retina into the optic nerve involves the growth promoting axon guidance molecules L1, laminin and netrin 1, each of which governs axon behavior at specific regions along the retinal pathway. In identifying additional molecules regulating this process during embryonic mouse development, we found that transmembrane Semaphorin5A mRNA and protein was specifically expressed in neuroepithelial cells surrounding retinal axons at the optic disc and along the optic nerve. Given that growth cone responses to a specific guidance molecule can be altered by co-exposure to a second guidance cue, we examined whether retinal axon responses to Sema5A were modulated by other guidance signals axons encountered along the retinal pathway. In growth cone collapse, substratum choice and neurite outgrowth assays, Sema5A triggered an invariant inhibitory response in the context of L1, laminin, or netrin 1 signaling, suggesting that Sema5A inhibited retinal axons throughout their course at the optic disc and nerve. Antibody-perturbation studies in living embryo preparations showed that blocking of Sema5A function led to retinal axons straying out of the optic nerve bundle, indicating that Sema5A normally helped ensheath the retinal pathway. Thus, development of some CNS nerves requires inhibitory sheaths to maintain integrity. Furthermore, this function is accomplished using molecules such as Sema5A that exhibit conserved inhibitory responses in the presence of co-impinging signals from multiple families of guidance molecules.     

On the turning of Xenopus retinal axons induced by ephrin-A5

The Eph family of receptor tyrosine kinases and their ligands, the ephrins, play important roles during development of the nervous system. Frequently they exert their functions through a repellent mechanism, so that, for example, an axon expressing an Eph receptor does not invade a territory in which an ephrin is expressed. Eph receptor activation requires membrane-associated ligands. This feature discriminates ephrins from other molecules sculpturing the nervous system such as netrins, slits and class 3 semaphorins, which are secreted molecules. While the ability of secreted molecules to guide axons, i.e. to change their growth direction, is well established in vitro, little is known about this for the membrane-bound ephrins. Here we set out to investigate--using Xenopus laevis retinal axons--the properties of substratum-bound and (artificially) soluble forms of ephrin-A5 (ephrin-A5-Fc) to guide axons. We find--as expected on the basis of chick experiments - that, when immobilised in the stripe assay, ephrin-A5 has a repellent effect such that retinal axons avoid ephrin-A5-Fc-containing lanes. Also, retinal axons react with repulsive turning or growth cone collapse when confronted with ephrin-A5-Fc bound to beads. However, when added in soluble form to the medium, ephrin-A5 induces growth cone collapse, comparable to data from chick. The analysis of growth cone behaviour in a gradient of soluble ephrin-A5 in the 'turning assay' revealed a substratum-dependent reaction of Xenopus retinal axons. On fibronectin, we observed a repulsive response, with the turning of growth cones away from higher concentrations of ephrin-A5. On laminin, retinal axons turned towards higher concentrations, indicating an attractive effect. In both cases the turning response occurred at a high background level of growth cone collapse. In sum, our data indicate that ephrin-As are able to guide axons in immobilised bound form as well as in the form of soluble molecules. To what degree this type of guidance is relevant for the in vivo situation remains to be shown.     

Semaphorin3A regulates axon growth independently of growth cone repulsion via modulation of TrkA signaling

Regulation of axon growth is a critical event in neuronal development. Nerve growth factor (NGF) is a strong inducer of axon growth and survival in the dorsal root ganglia (DRG). Paradoxically, high concentrations of NGF are present in the target region where axon growth must slow down for axons to accurately identify their correct targets. Semaphorin3A (Sema3A), a powerful axonal repellent molecule for DRG neurons, is also situated in their target regions. NGF is a modulator of Sema3A-induced repulsion and death. We show that Sema3A is a regulator of NGF-induced neurite outgrowth via the TrkA receptor, independent of its growth cone repulsion activity. First, neurite outgrowth of DRG neurons is more sensitive to Sema3A than repulsion. Second, at concentrations sufficient to significantly inhibit Sema3A-induced repulsion, NGF has no effect on Sema3A-induced axon growth inhibition. Third, Sema3A-induced outgrowth inhibition, but not repulsion activity, is dependent on NGF stimulation. Fourth, Sema3A attenuates TrkA-mediated growth signaling, but not survival signaling, and over-expression of constitutively active TrkA blocks Sema3A-induced axon growth inhibition, suggesting that Sema3A activity is mediated via regulation of NGF/TrkA-induced growth. Finally, quantitative analysis of axon growth in vivo supports the possibility that Sema3A affects axon growth, in addition to its well-documented role in axon guidance. We suggest a model whereby NGF at high concentrations in the target region is important for survival, attraction and inhibition of Sema3A-induced repulsion, while Sema3A inhibits its growth-promoting activity. The combined and cross-modulatory effects of these two signaling molecules ensure the accuracy of the final stages in axon targeting.     

Semaphorin 5A is a bifunctional axon guidance cue for axial motoneurons in vivo

Semaphorins are a large class of proteins that function throughout the nervous system to guide axons. It had previously been shown that Semaphorin 5A (Sema5A) was a bifunctional axon guidance cue for mammalian midbrain neurons. We found that zebrafish sema5A was expressed in myotomes during the period of motor axon outgrowth. To determine whether Sema5A functioned in motor axon guidance, we knocked down Sema5A, which resulted in two phenotypes: a delay in motor axon extension into the ventral myotome and aberrant branching of these motor axons. Both phenotypes were rescued by injection of full-length rat Sema5A mRNA. However, adding back RNA encoding the sema domain alone significantly rescued the branching phenotype in sema5A morphants. Conversely, adding back RNA encoding the thrombospondin repeat (TSR) domain alone into sema5A morphants exclusively rescued delay in ventral motor axon extension. Together, these data show that Sema5A is a bifunctional axon guidance cue for vertebrate motor axons in vivo. The TSR domain promotes growth of developing motor axons into the ventral myotome whereas the sema domain mediates repulsion and keeps these motor axons from branching into surrounding myotome regions.     

DRG Axon Elongation and Growth Cone Collapse Rate Induced by Sema3A are Differently Dependent on NGF Concentration

Regeneration of embryonic and adult dorsal root ganglion (DRG) sensory axons is highly impeded when they encounter neuronal growth cone-collapsing factor semaphorin3A (Sema3A). On the other hand, increasing evidence shows that DRG axon’s regeneration can be stimulated by nerve growth factor (NGF). In this study, we aimed to evaluate whether increased NGF concentrations can counterweight Sema3A-induced inhibitory responses in 15-day-old mouse embryo (E15) DRG axons. The DRG explants were grown in Neurobasal-based medium with different NGF concentrations ranging from 0 to 100 ng/mL and then treated with Sema3A at constant 10 ng/mL concentration. To evaluate interplay between NGF and Sema3A number of DRG axons, axon outgrowth distance and collapse rate were measured. We found that the increased NGF concentrations abolish Sema3A-induced inhibitory effect on axon outgrowth, while they have no effect on Sema3A-induced collapse rate.     

Spinal nerve segmentation in the chick embryo: analysis of distinct axon-repulsive systems

In higher vertebrates, the segmental organization of peripheral spinal nerves is established by a repulsive mechanism whereby sensory and motor axons are excluded from the posterior half-somite. A number of candidate axon repellents have been suggested to mediate this barrier to axon growth, including Sema3A, Ephrin-B, and peanut agglutinin (PNA)-binding proteins. We have tested the candidacy of these factors in vitro by examining their contribution to the growth cone collapse-inducing activity of somite-derived protein extracts on sensory, motor, and retinal axons. We find that Sema3A is unlikely to play a role in the segmentation of sensory or motor axons and that Ephrin-B may contribute to motor but not sensory axon segmentation. We also provide evidence that the only candidate molecule(s) that induces the growth cone collapse of both sensory and motor axons binds to PNA and is not Sema3A or Ephrin-B. By grafting primary sensory, motor, and quail retinal neurons into the chick trunk in vivo, we provide further evidence that the posterior half-somite represents a universal barrier to growing axons. Taken together, these results suggest that the mechanisms of peripheral nerve segmentation should be considered in terms of repellent molecules in addition to the identified molecules.     

Dissociation of corticothalamic and thalamocortical axon targeting by an EphA7-mediated mechanism

Molecular mechanisms generating the topographic organization of corticothalamic (CT) circuits, which comprise more than three-quarters of the synaptic inputs onto sensory relay neurons, and their interdependence with thalamocortical (TC) axon development are unknown. Using in utero electroporation-mediated gene transfer, we show that EphA7-mediated signaling on neocortical axons controls the within-nucleus topography of CT projections in the thalamus. Notably, CT axons that mis-express EphA7 do not shift the relative positioning of their pathway within the subcortical telencephalon (ST), indicating that they do not depend upon EphA7/ephrin-A signaling in the ST for establishing this topography. Moreover, mis-expression of cortical EphA7 results in disrupted topography of CT projections, but unchanged inter- and intra-areal topography of TC projections. Our results support a model in which EphA/ephrin-A signaling controls independently the precision with which CT and TC projections develop, yet is essential for establishing their topographic reciprocity.     

Semaphorin 3E/collapsin-5 inhibits growing retinal axons

During development, the formation of neural networks is reflected by the oriented extension of neurites. Using retinal ganglion cells (RGCs) as a model, we identified the yet uncharacterized chick semaphorin Sema3E/collapsin-5 as a repulsive cue for outgrowing axons. Sema3E/collapsin-5 was highly regulated during retinal histogenesis, with peak expression during the period of intraretinal axon growth. Polymerase chain reaction analysis demonstrated Sema3E/collapsin-5 mRNA in retina layers, from which RGC axons are excluded. Neither isolated RGCs nor purified retinal Müller glia cells synthesized Sema3E/collapsin-5. Sema3E/collapsin-5 receptor sites were visualized by alkaline phosphatase fusion proteins in the axon-rich optic fiber layer. Time-lapse video recording of chick in vitro cultures revealed a growth cone collapsing activity of recombinant Sema3E/collapsin-5. This effect was specific for RGCs, since dorsal root ganglia (DRG) neurons of the peripheral nervous system were not affected. Comparison with Sema3A/collapsin-1 displayed a reciprocal specificity, because Sema3A/collapsin-1 hampered exclusively DRG but not RGC growth cones. The collapsing effect was mediated by low cGMP levels, but not cAMP, as revealed by a set of agonists. In summary, the data suggest a possible role of chick Sema3E/collapsin-5 in restricting growth of retinal ganglion cell axons to the optic fiber layer.     

Semaphorin 5A is a bifunctional axon guidance cue regulated by heparan and chondroitin sulfate proteoglycans

The response of neuronal growth cones to axon guidance cues depends on the developmental context in which these cues are encountered. We show here that the transmembrane protein semaphorin 5A (Sema5A) is a bifunctional guidance cue exerting both attractive and inhibitory effects on developing axons of the fasciculus retroflexus, a diencephalon fiber tract associated with limbic function. The thrombospondin repeats of Sema5A physically interact with the glycosaminoglycan portion of both chondroitin sulfate proteoglycans (CSPGs) and heparan sulfate proteoglycans (HSPGs). CSPGs function as precisely localized extrinsic cues that convert Sema5A from an attractive to an inhibitory guidance cue. Therefore, glycosaminoglycan bound guidance cues provide a molecular mechanism for CSPG-mediated inhibition of axonal extension. Further, axonal HSPGs are required for Sema5A-mediated attraction, suggesting that HSPGs are components of functional Sema5A receptors. Thus, neuronal responses to Sema5A are proteoglycan dependent and interpreted according to the biological context in which this membrane bound guidance cue is presented.     

Characterization of factors regulating lamina-specific growth of thalamocortical axons

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer-specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina-specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol-linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers.     

Distinct roles for secreted semaphorin signaling in spinal motor axon guidance

Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.     

Topography of thalamic projections requires attractive and repulsive functions of Netrin-1 in the ventral telencephalon

Recent studies have demonstrated that the topography of thalamocortical (TC) axon projections is initiated before they reach the cortex, in the ventral telencephalon (VTel). However, at this point, the molecular mechanisms patterning the topography of TC projections in the VTel remains poorly understood. Here, we show that a long-range, high-rostral to low-caudal gradient of Netrin-1 in the VTel is required in vivo for the topographic sorting of TC axons to distinct cortical domains. We demonstrate that Netrin-1 is a chemoattractant for rostral thalamic axons but functions as a chemorepulsive cue for caudal thalamic axons. In accordance with this model, DCC is expressed in a high-rostromedial to low-caudolateral gradient in the dorsal thalamus (DTh), whereas three Unc5 receptors (Unc5A–C) show graded expression in the reverse orientation. Finally, we show that DCC is required for the attraction of rostromedial thalamic axons to the Netrin-1–rich, anterior part of the VTel, whereas DCC and Unc5A/C receptors are required for the repulsion of caudolateral TC axons from the same Netrin-1–rich region of the VTel. Our results demonstrate that a long-range gradient of Netrin-1 acts as a counteracting force from ephrin-A5 to control the topography of TC projections before they enter the cortex.     

Characterization of factors regulating lamina‐specific growth of thalamocortical axons

During development, most thalamocortical axons extend through the deep layers to terminate in layer 4 of neocortex. To elucidate the molecular mechanisms that underlie the formation of layer‐specific thalamocortical projections, axon outgrowth from embryonic rat thalamus onto postnatal neocortical slices which had been fixed chemically was used as an experimental model system. When the thalamic explant was juxtaposed to the lateral edge of fixed cortical slice, thalamic axons extended farther in the deep layers than the upper layers. Correspondingly, thalamic axons entering from the ventricular side extended farther than those from the pial side. In contrast, axons from cortical explants cultured next to fixed cortical slices tended to grow nearly as well in the upper as in the deep layers. Biochemical aspects of lamina‐specific thalamic axon growth were studied by applying several enzymatic treatments to the cortical slices prior to culturing. Phosphatidylinositol phospholipase C treatment increased elongation of thalamic axons in the upper layers without influencing growth in the deep layers. Neither chondroitinase, heparitinase, nor neuraminidase treatment influenced the overall projection pattern, although neuraminidase slightly decreased axonal elongation in the deep layers. These findings suggest that glycosylphosphatidylinositol‐linked molecules in the cortex may contribute to the laminar specificity of thalamocortical projections by suppressing thalamic axon growth in the upper cortical layers. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 56–68, 2000     

Denervation-induced spatiotemporal upregulation of ephrin-A2 in the mouse hippocampus after transections of the perforant path

Transections of the entorhinal afferent fibers to hippocampus, perforant path (PP), result in the denervation in specific hippocampal subregions, which is followed by a series of plastic events including axon sprouting and reactive synaptogenesis. Many growth-associated molecules are thought to participate in these events. In the present study, we proved the upregulation of ephrin-A2 in the denervated areas of the ipsilateral hippocampus following PP transections. Interestingly, when the elevation of ephrin-A2 reached the maximum axon sprouting in the denervated areas almost finished, implying the possible inhibitory effect of ephrin-A2 on sprouting. In addition, ephrin-A2 expression was observed in synapses during reactive synaptogenesis, suggesting that this molecule might also be implicated in the formation and maturation of synapses in the denervated areas.     

Semaphorin 3d guides laterality of retinal ganglion cell projections in zebrafish

The optic chiasm is an important choice point at which retinal ganglion cell (RGC) axons either cross the midline to innervate the contralateral brain or turn back to innervate the ipsilateral brain. Guidance cues that regulate this decision, particularly those directing the midline crossing of contralateral axons, are still not well understood. Here we show that Sema3d, a secreted semaphorin expressed at the midline, guides the crossing of RGC axons in zebrafish. Both Sema3d knockdown and ubiquitous overexpression induced aberrant ipsilateral projections, suggesting that Sema3d normally guides axons into the contralateral optic tract. Live imaging in vivo showed that RGC growth cones responded to ubiquitous Sema3d overexpression by pausing for extended periods and increasing their exploratory behavior at the midline, suggesting that Sema3d overexpression causes the midline environment to become less favorable for RGC axon extension. Interestingly, Sema3d overexpression did not affect growth cone behaviors before the midline, suggesting that RGC axons normally respond to Sema3d only upon reaching the midline. After Sema3d knockdown, growth cones grew across the midline but then paused or repeatedly retracted, impairing their ability to leave the midline region. Our results indicate that a proper balance of Sema3d is needed at the midline for the progression of RGC axons from the chiasm midline into the contralateral optic tract.     

Motoneuronal Sema3C is essential for setting stereotyped motor tract positioning in limb-derived chemotropic semaphorins

The wiring of neuronal circuits requires complex mechanisms to guide axon subsets to their specific target with high precision. To overcome the limited number of guidance cues, modulation of axon responsiveness is crucial for specifying accurate trajectories. We report here a novel mechanism by which ligand/receptor co-expression in neurons modulates the integration of other guidance cues by the growth cone. Class 3 semaphorins (Sema3 semaphorins) are chemotropic guidance cues for various neuronal projections, among which are spinal motor axons navigating towards their peripheral target muscles. Intriguingly, Sema3 proteins are dynamically expressed, forming a code in motoneuron subpopulations, whereas their receptors, the neuropilins, are expressed in most of them. Targeted gain- and loss-of-function approaches in the chick neural tube were performed to enable selective manipulation of Sema3C expression in motoneurons. We show that motoneuronal Sema3C regulates the shared Sema3 neuropilin receptors Nrp1 and Nrp2 levels in opposite ways at the growth cone surface. This sets the respective responsiveness to exogenous Nrp1- and Nrp2-dependent Sema3A, Sema3F and Sema3C repellents. Moreover, in vivo analysis revealed a context where this modulation is essential. Motor axons innervating the forelimb muscles are exposed to combined expressions of semaphorins. We show first that the positioning of spinal nerves is highly stereotyped and second that it is compromised by alteration of motoneuronal Sema3C. Thus, the role of the motoneuronal Sema3 code could be to set population-specific axon sensitivity to limb-derived chemotropic Sema3 proteins, therefore specifying stereotyped motor nerve trajectories in their target field.     

p75(NTR) mediates ephrin-A reverse signaling required for axon repulsion and mapping

Reverse signaling by ephrin-As upon binding EphAs controls axon guidance and mapping. Ephrin-As are GPI-anchored to the membrane, requiring that they complex with transmembrane proteins that transduce their signals. We show that the p75 neurotrophin receptor (NTR) serves this role in retinal axons. p75(NTR) and ephrin-A colocalize within caveolae along retinal axons and form a complex required for Fyn phosphorylation upon binding EphAs, activating a signaling pathway leading to cytoskeletal changes. In vitro, retinal axon repulsion to EphAs by ephrin-A reverse signaling requires p75(NTR), but repulsion to ephrin-As by EphA forward signaling does not. Constitutive and retina-specific p75(NTR) knockout mice have aberrant anterior shifts in retinal axon terminations in superior colliculus, consistent with diminished repellent activity mediated by graded ephrin-A reverse signaling induced by graded collicular EphAs. We conclude that p75(NTR) is a signaling partner for ephrin-As and the ephrin-A- p75(NTR) complex reverse signals to mediate axon repulsion required for guidance and mapping.     

Ephrin-A5 inhibits growth of embryonic sensory neurons

EphA-ephrin signaling has recently been implicated in the establishment of motor innervation patterns, in particular in determining whether motor axons project into dorsal versus ventral nerve trunks in the limb. We investigated whether sensory axons, which grow out together with and can be guided by motor axons, are also influenced by Eph-ephrin signaling. We show that multiple EphA receptors are expressed in DRGs when limb innervation is being established, and EphA receptors are present on growth cones of both NGF-dependent (predominantly cutaneous) and NT3-dependent (predominantly proprioceptive) afferents. Both soluble and membrane-attached ephrin-A5 inhibited growth of approximately half of each population of sensory axons in vitro. On average, growth cones that collapsed in response to soluble ephrin-A5 extended more slowly than those that did not, and ephrin-A5 significantly slowed the extension of NGF-dependent growth cones that did not collapse. Finally, we show that ectopic expression of ephrin-A5 in ovo reduced arborization of cutaneous axons in skin on the limb. Together these results suggest that sensory neurons respond directly to A-class ephrins in the limb. Thus, ephrins appear to pattern sensory axon growth in two ways-both directly, and indirectly via their inhibitory effects on neighboring motor axons.