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In mammals, taste buds typically contain 50-100 tightly packed taste-receptor cells (TRCs), representing all five basic qualities: sweet, sour, bitter, salty and umami. Notably, mature taste cells have life spans of only 5-20 days and, consequently, are constantly replenished by differentiation of taste stem cells. Given the importance of establishing and maintaining appropriate connectivity between TRCs and their partner ganglion neurons (that is, ensuring that a labelled line from sweet TRCs connects to sweet neurons, bitter TRCs to bitter neurons, sour to sour, and so on), we examined how new connections are specified to retain fidelity of signal transmission. Here we show that bitter and sweet TRCs provide instructive signals to bitter and sweet target neurons via different guidance molecules (SEMA3A and SEMA7A). We demonstrate that targeted expression of SEMA3A or SEMA7A in different classes of TRCs produces peripheral taste systems with miswired sweet or bitter cells. Indeed, we engineered mice with bitter neurons that now responded to sweet tastants, sweet neurons that responded to bitter or sweet neurons responding to sour stimuli. Together, these results uncover the basic logic of the wiring of the taste system at the periphery, and illustrate how a labelled-line sensory circuit preserves signalling integrity despite rapid and stochastic turnover of receptor cells.

Optimal healing after myocardial infarction requires not only the induction of inflammation, but also its timely resolution. In patients, 30 days post myocardial infarction, circulating monocytes have increased expression of Semaphorin3A (Sema3A) as compared to directly after admission. This increased expression coincides with increased expression of Cx3CR1-a marker of non-classical monocytes that are important for immune resolution hence proper wound healing. In mice, the expression of Sema3A also increases in response to myocardial ischemia being expressed by infiltrating leukocytes. Comparing Sema3A heterozygote (HZ) and wild type (WT) mice post myocardial infarction, revealed increased presence of leukocytes in the cardiac tissues of HZ mice as compared to WT, with no differences in capillary density, collagen deposition, cardiomyocyte surface area, chemokine-or adhesion molecules expression. Whilst infarct sizes were similar 14 days after myocardial infarction in both genotypes, Sema3A HZ mice had thinner infarcts and reduced cardiac function as compared to their WT littermates. In vitro experiments were conducted to study the role of Sema3A in inflammation and resolution of inflammation as a potential explanation for the differences in leukocyte recruitment and cardiac function observed in our in vivo experiments. Here, recombinant Sema3A protein was able to affect the pro-inflammatory state of cultured bone marrow derived macrophages. First, the pro-inflammatory state was altered by the induced apoptosis of classical macrophages in the presence of Sema3A. Second, Sema3A promoted the polarization of classical macrophages to resolution-phase macrophages and enhanced their efferocytotic ability, findings that were reflected in the infarcted cardiac tissue of the Sema3A HZ mice. Finally, we demonstrated that besides promoting resolution of inflammation, Sema3A was also able to retard the migration of monocytes to the myocardium. Collectively our data demonstrate that Sema3A reduces cardiac inflammation and improves cardiac function after myocardial infarction by promoting the resolution of inflammation.

Recent studies show that guidance molecules that are known to regulate cell migration during development may also play an important role in adult pathophysiologic states. One such molecule, semaphorin3A (sema3A), is highly expressed after acute kidney injury (AKI) in mice and humans, but its pathophysiological role is unknown. Genetic inactivation of sema3A protected mice from ischemia-reperfusion-induced AKI, improved tissue histology, reduced neutrophil infiltration, prevented epithelial cell apoptosis, and increased cytokine and chemokine excretion in urine. Pharmacological-based inhibition of sema3A receptor binding likewise protected against ischemia-reperfusion-induced AKI. In vitro, sema3A enhanced toll-like receptor 4-mediated inflammation in epithelial cells, macrophages, and dendritic cells. Moreover, administration of sema3A-treated, bone marrow-derived dendritic cells exacerbated kidney injury. Finally, sema3A augmented cisplatin-induced apoptosis in kidney epithelial cells in vitro via expression of DFFA-like effector a (cidea). Our data suggest that the guidance molecule sema3A exacerbates AKI via promoting inflammation and epithelial cell apoptosis., (Copyright © 2014 the American Physiological Society.)

Semaphorin 3A (Sema3A) axon repellant serves multiple developmental functions. Sema3A mRNAs are expressed in epithelial and mesenchymal components of the developing incisor in a dynamic manner. Here, we investigate the functions of Sema3A during development of incisors using Sema3A-deficient mice. We analyze histomorphogenesis and innervation of mandibular incisors using immunohistochemistry as well as computed tomography and thick tissue confocal imaging. Whereas no apparent disturbances in histomorphogenesis or hard tissue formation of Sema3A (-/-) incisors were observed, nerve fibers were prematurely seen in the presumptive dental mesenchyme of the bud stage Sema3A (-/-) tooth germ. Later, nerves were ectopically present in the Sema3A (-/-) dental papilla mesenchyme during the cap and bell stages, whereas in the Sema3A (+/+) mice the first nerve fibers were seen in the pulp after the onset of dental hard tissue formation. However, no apparent topographic differences in innervation pattern or nerve fasciculation were seen inside the pulp between postnatal and adult Sema3A (+/+) or Sema3A (-/-) incisors. In contrast, an abnormally large number of nerves and arborizations were observed in the Sema3A (-/-) developing dental follicle target field and periodontium and, unlike in the wild-type mice, nerve fibers were abundant in the labial periodontium. Of note, the observed defects appeared to be mostly corrected in the adult incisors. The expressions of Ngf and Gdnf neurotrophins and their receptors were not altered in the Sema3A (-/-) postnatal incisor or trigeminal ganglion, respectively. Thus, Sema3A is an essential, locally produced chemorepellant, which by creating mesenchymal exclusion areas, regulates the timing and patterning of the dental nerves during the development of incisor tooth germ.

Astrocytes, the most abundant cells in the central nervous system, promote synapse formation and help to refine neural connectivity. Although they are allocated to spatially distinct regional domains during development, it is unknown whether region-restricted astrocytes are functionally heterogeneous. Here we show that postnatal spinal cord astrocytes express several region-specific genes, and that ventral astrocyte-encoded semaphorin 3a (Sema3a) is required for proper motor neuron and sensory neuron circuit organization. Loss of astrocyte-encoded Sema3a leads to dysregulated α-motor neuron axon initial segment orientation, markedly abnormal synaptic inputs, and selective death of α- but not of adjacent γ-motor neurons. In addition, a subset of TrkA(+) sensory afferents projects to ectopic ventral positions. These findings demonstrate that stable maintenance of a positional cue by developing astrocytes influences multiple aspects of sensorimotor circuit formation. More generally, they suggest that regional astrocyte heterogeneity may help to coordinate postnatal neural circuit refinement.

Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated that Sema3a(-/-) mice have multiple developmental defects due to abnormal neuronal innervations. Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a(-/-) mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice (Sema3acol1(-/-) and Sema3aosx(-/-) mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons (Sema3asynapsin(-/-) and Sema3anestin(-/-) mice) had low bone mass, similar to Sema3a(-/-) mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3asynapsin(-/-) mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3anestin(-/-) mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a(-/-) mice, such as olfactory development, were identified in Sema3asynasin(-/-) mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a(-/-) mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

The sympathetic nervous system (SNS) arises from neural crest (NC) cells during embryonic development and innervates the internal organs of vertebrates to modulate their stress response. NRP1 and NRP2 are receptors for guidance cues of the class 3 semaphorin (SEMA) family and are expressed in partially overlapping patterns in sympathetic NC cells and their progeny. By comparing the phenotypes of mice lacking NRP1 or its ligand SEMA3A with mice lacking NRP1 in the sympathetic versus vascular endothelial cell lineages, we demonstrate that SEMA3A signalling through NRP1 has multiple cell-autonomous roles in SNS development. These roles include neuronal cell body positioning, neuronal aggregation and axon guidance, first during sympathetic chain assembly and then to regulate the innervation of the heart and aorta. Loss of NRP2 or its ligand SEMA3F impaired sympathetic gangliogenesis more mildly than loss of SEMA3A/NRP1 signalling, but caused ectopic neurite extension along the embryonic aorta. The analysis of compound mutants lacking SEMA3A and SEMA3F or NRP1 and NRP2 in the SNS demonstrated that both signalling pathways cooperate to organise the SNS. We further show that abnormal sympathetic development in mice lacking NRP1 in the sympathetic lineage has functional consequences, as it causes sinus bradycardia, similar to mice lacking SEMA3A., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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

The bony skeleton is maintained by local factors that regulate bone-forming osteoblasts and bone-resorbing osteoclasts, in addition to hormonal activity. Osteoprotegerin protects bone by inhibiting osteoclastic bone resorption, but no factor has yet been identified as a local determinant of bone mass that regulates both osteoclasts and osteoblasts. Here we show that semaphorin 3A (Sema3A) exerts an osteoprotective effect by both suppressing osteoclastic bone resorption and increasing osteoblastic bone formation. The binding of Sema3A to neuropilin-1 (Nrp1) inhibited receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation by inhibiting the immunoreceptor tyrosine-based activation motif (ITAM) and RhoA signalling pathways. In addition, Sema3A and Nrp1 binding stimulated osteoblast and inhibited adipocyte differentiation through the canonical Wnt/β-catenin signalling pathway. The osteopenic phenotype in Sema3a−/− mice was recapitulated by mice in which the Sema3A-binding site of Nrp1 had been genetically disrupted. Intravenous Sema3A administration in mice increased bone volume and expedited bone regeneration. Thus, Sema3A is a promising new therapeutic agent in bone and joint diseases.

During development, the axons of retinal ganglion cell (RGC) neurons must decide whether to cross or avoid the midline at the optic chiasm to project to targets on both sides of the brain. By combining genetic analyses with in vitro assays, we show that neuropilin 1 (NRP1) promotes contralateral RGC projection in mammals. Unexpectedly, the NRP1 ligand involved is not an axon guidance cue of the class 3 semaphorin family, but VEGF164, the neuropilin-binding isoform of the classical vascular growth factor VEGF-A. VEGF164 is expressed at the chiasm midline and is required for normal contralateral growth in vivo. In outgrowth and growth cone turning assays, VEGF164 acts directly on NRP1-expressing contralateral RGCs to provide growth-promoting and chemoattractive signals. These findings have identified a permissive midline signal for axons at the chiasm midline and provide in vivo evidence that VEGF-A is an essential axon guidance cue., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

Mutations in the gene encoding the neural recognition molecule L1 result in hypoplasia of the corticospinal tract and path finding errors of corticospinal axons at the pyramidal decussation. Candidate molecules that have been implicated in L1-dependent guidance of corticospinal axons from the ventral medullary pyramids to the contralateral dorsal columns of the cervical spinal cord include Semaphorin3A and CD24. In the present study, we anterogradely labeled corticospinal axons from the sensorimotor cortex of young postnatal Semaphorin3A- and CD24-deficient mice to elucidate potential functions of both proteins during formation of this long axon projection. Our results indicate that elongation, collateralization, fasciculation and path finding of corticospinal axons in mice proceed normally in the absence of Semaphorin3A or CD24.

Newborn neurons elaborate an axon that undertakes a complicated journey to find its ultimate target in the brain or periphery. Although major progress in the study of this process has been made by analysis of dissociated neurons in vitro, one would like to observe and manipulate axonal outgrowth and pathfinding as it occurs in situ, as fasciculated nerves growing within the tissue itself. Here, we present a simple technique to do this, through cultivation of embryonic mouse slices expressing enhanced green fluorescent protein (EGFP) specifically in newborn neurons. This system allows for imaging of outgrowth of peripheral nerves into structures such as the developing limb. We demonstrate a reproduction of normal innervation patterns by spinal nerves derived from spinal cord motor neurons and sensory neurons of the dorsal root ganglia. The slices can be manipulated pharmacologically as well as genetically, by crossing the EGFP-expressing line with lines containing targeted mutations in genes of interest., (2007 Wiley-Liss, Inc)

The p75 neurotrophin receptor (p75(NTR)) interacts with multiple ligands and coreceptors. It is thought to mediate myelin growth inhibition as part of the Nogo receptor complex, in addition to its other roles. Paradoxically, however, peripheral axons of p75(ExonIII-/-) mutant embryos are severely stunted. This inhibition of axon growth may be a result of neurite elongation defects in p75(NTR) mutant neurons. Here, we show that p75(ExonIII-/-) DRG neurons are hypersensitive to the repellent molecule Semaphorin3A (Sema3A). NGF modulates Sema3A activity equally well in both the p75(NTR) mutant and wild-type neurons, indicating that the hypersensitivity of p75(NTR) mutant neurons is probably not related to their NGF receptor activity. Neuropilin1 and p75(NTR) partially colocalize in DRG growth cones. After Sema3A stimulation, the degree of colocalization is dramatically increased, particularly in clusters associated with Sema3A receptor complex activation. Coimmunoprecipitation studies show that p75(NTR) interacts directly with the Sema3A receptors Neuropilin1 and PlexinA4. When coexpressed with both Neuropilin1 and PlexinA4, p75(NTR) reduces the interaction between these two receptor components. Finally, p75(NTR)/Sema3A double-mutant embryos show growth similar to that observed in Sema3A-null mice. These data indicate that p75(NTR) is an important functional modulator of Sema3A activity and that, in the absence of p75(NTR), oversensitivity to Sema3A leads to severe reduction in sensory innervation. Our results also suggest that while inhibition of p75(NTR) in CNS injury may enhance nerve regeneration resulting from the inhibition of myelin-associated protein, it may also inhibit nerve regeneration through its modulation of Sema3A.

In the early stages, the dorsal root ganglion neurons extend their axons toward the dorsal spinal cord. We previously showed that surround repulsion by semaphorin 3A prevents sensory axons from straying from their paths. The finding, however, that sensory trajectories toward the dorsal spinal cord are almost normal in semaphorin 3A-deficient littermates raises the possibility that a chemoattraction-based mechanism also contributes to the formation of sensory axonal projections. By employing culture assays, we show that the dorsal spinal cord secretes chemoattractants for the dorsal root ganglion axons. Furthermore, we demonstrate that the activity of a dorsal spinal cord-derived cue is specific for early sensory axons. These results suggest that dorsal spinal cord-derived chemoattractants contribute to the formation of the initial trajectories of sensory axons.

Axon guidance cues are critical for neuronal circuitry formation. Guidance molecules may repel or attract axons directly by effecting growth cone motility, or by impinging on neuronal polarity. In Semaphorin3A null mice, many axonal errors are detected, most prominently in DRG neurons. It has been generally assumed the repellent properties of Semaphorin3A are the cause of these erroneous axonal projections. Here we show that, in semaphorin3A-null mice, the initial trajectory of neurons in the DRG is abnormal, suggesting that Semaphorin3A may instruct neuronal polarity. In corroboration, in vitro Semaphorin3A dramatically increases neuronal polarization, as indicated by GSK3beta and Rac1 sub-cellular localization in DRG neurons. Polarization effects of Semaphorin3A are regulated by activated MAPK, as indicated by p-MAPK 42/44 polarization and the need for its activity for Rac1 and GSK3beta polarization. Taken together, our findings suggest that Semaphorin3A plays a role in the formation of neuronal polarity, in addition to its classic repellent role.

Sympathetic innervation is critical for effective cardiac function. However, the developmental and regulatory mechanisms determining the density and patterning of cardiac sympathetic innervation remain unclear, as does the role of this innervation in arrhythmogenesis. Here we show that a neural chemorepellent, Sema3a, establishes cardiac sympathetic innervation patterning. Sema3a is abundantly expressed in the trabecular layer in early-stage embryos but is restricted to Purkinje fibers after birth, forming an epicardial-to-endocardial transmural sympathetic innervation patterning. Sema3a(-/-) mice lacked a cardiac sympathetic innervation gradient and exhibited stellate ganglia malformation, which led to marked sinus bradycardia due to sympathetic dysfunction. Cardiac-specific overexpression of Sema3a in transgenic mice (SemaTG) was associated with reduced sympathetic innervation and attenuation of the epicardial-to-endocardial innervation gradient. SemaTG mice demonstrated sudden death and susceptibility to ventricular tachycardia, due to catecholamine supersensitivity and prolongation of the action potential duration. We conclude that appropriate cardiac Sema3a expression is needed for sympathetic innervation patterning and is critical for heart rate control.

Blood vessels and neurons use similar guidance cues to control their behaviour during embryogenesis. The semaphorin SEMA3A was originally identified as a repulsive cue for developing axons that acts by signalling through receptor complexes containing NRP1 and A-type plexins. SEMA3A also competes with the VEGF164 isoform of vascular endothelial growth factor for binding to NRP1 to modulate the migration of endothelial cells in vitro. Surprisingly, we have found that SEMA3A and semaphorin signalling through NRP1 were not required for blood vessel development in the mouse. Moreover, we found that there was no genetic interaction between SEMA3A and VEGF164 during vasculogenesis or angiogenesis. Our observations suggest that in vivo vascular NRP1 preferentially confers VEGF164 signals, whilst axonal NRP1 preferentially transmits SEMA3A signals.

Vascular endothelial growth factor165 (VEGF165) and semaphorin3A (SEMA3A) elicit pro- and antiangiogenic signals respectively in endothelial cells (ECs) by binding to their receptors VEGFR-2, neuropilin-1 (NRP1), and plexin-A1. Here we show that the VEGF165-driven angiogenic potential of multiple myeloma (MM) ECs is significantly higher than that of monoclonal gammopathy of undetermined significance (MGUS) ECs (MGECs) and human umbilical vein (HUV) ECs. This is probably due to a constitutive imbalance of endogenous VEGF165/SEMA3A ratio, which leans on VEGF165 in MMECs but on SEMA3A in MGECs and HUVECs. Exogenous VEGF165 induces SEMA3A expression in MGECs and HUVECs, but not in MMECs. Moreover, by counteracting VEGF165 activity as efficiently as an anti-VEGFR-2 antibody, exogenous SEMA3A restrains the over-angiogenic potential of MMECs. Our data indicate that loss of endothelial SEMA3A in favor of VEGF165 could be responsible for the angiogenic switch from MGUS to MM.

A member of semaphorin family, semaphorin3A (Sema3A), acts as a chemorepellent or chemoattractant on a wide variety of axons and dendrites in the development of the nervous systems. We here show that Sema3A induces clustering of both postsynaptic density-95 (PSD-95) and presynaptic synapsin I in cultured cortical neurons without changing the density of spines or filopodia. Neuropilin-1 (NRP-1), a receptor for Sema3A, is present on both axons and dendrites. When the cultured neurons are exposed to Sema3A, the cluster size of PSD-95 is markedly enhanced, and an extensive colocalization of PSD-95 and NRP-1 or actin-rich protrusion is seen. The effects of Sema3A on spine morphology are blocked by PP2, an Src type tyrosine kinase inhibitor, but not by the PP3, the inactive-related compound. In the cultured cortical neurons from fyn(-/-) mice, dendrites bear few spines, and Sema3A does not induce PSD-95 cluster formation on the dendrites. Sema3A and its receptor genes are highly expressed during the synaptogenic period of postnatal days 10 and 15. The cortical neurons in layer V, but not layer III, show a lowered density of synaptic bouton-like structure on dendrites in sema3A- and fyn-deficient mice. The neurons of the double-heterozygous mice show the lowered spine density, whereas those of single heterozygous mice show similar levels of the spine density as the wild type. These findings suggest that the Sema3A signaling pathway plays an important role in the regulation of dendritic spine maturation in the cerebral cortex neurons.

Microglia exist under physiological conditions in a resting state but become activated after neuronal injury. Recent studies have highlighted the reciprocal role of neurons in controlling both the number and activity of microglia. In this study, microglia derived from newborn rat cortices were cultured and activated by interferon-gamma (IFNgamma) treatment, then exposed to recombinant Sema3A or conditioned medium derived from stressed embryonic cortical neurons. We found that activation of microglia by IFNgamma induced differential upregulation of the semaphorin receptors Plexin-A1 and Neuropilin-1. This result was confirmed by Northern blotting, reverse transcription-PCR, and Western blotting. Furthermore, recombinant Sema3A induced apoptosis of microglia when added to the in vitro culture, and a similar result was obtained on activated microglia when Sema3A was produced by stressed neurons. Using an in vivo model of microglia activation by striatal injection of lipopolysaccharide demonstrated a corresponding upregulation of Plexin-A1 and Neuropilin-1 in activated microglia and enhanced production of Sema3A by stressed adult neurons. These results suggest a novel semaphorin-mediated mechanism of neuroprotection whereby stressed neurons can protect themselves from further damage by activated microglia.

The molecular mechanisms that control dendritic development are still unclear. Semaphorin 3A, a class III semaphorin, has been shown to regulate the radial orientation of pyramidal neurons in the developing neocortex. Here, we investigate the effects of Sema3A on the development of dendritic topology. Neocortical slices from Sema3A null mutant mice were cultured and neurons were transfected with GFP, reconstructed, and compared with neurons from wild-type and heterozygote littermates. We also added exogenous Sema3A to cultured wild-type neocortical slices to further test its effects on dendritic development. We document reductions in dendritic length and branching in Sema3A null mice and increases in dendritic length and branching after the addition of exogenous Sema3A to wild-type neurons. We conclude that Sema3A is necessary for the elaboration of second and third order dendritic branches in pyramidal neurons., (Copyright 2003 Wiley Periodicals, Inc. J Neurobiol 58: 403-412, 2004)

The neural scar that forms after injury to the mammalian central nervous system is a barrier to sprouting and regenerating axons. In addition to reactive astrocytes that are present throughout the lesion site, leptomeningeal fibroblasts invade the lesion core. When isolated in vitro, these cells form a very poor substrate for growing neurites, even more so than reactive astrocytes. Nevertheless the molecular mechanisms involved in this growth inhibition are not well understood. Semaphorins have been reported to be upregulated in meningeal cells (MCs) on mechanical injury to the brain and spinal cord. In the present study, we show that Sema3A mRNA and active protein are produced by cultured meningeal cells. A protein extract from these cells induces the collapse of embryonic dorsal root ganglion (DRG) growth cones. This collapsing activity is partially blocked by neuropilin-1 antibodies and is absent in meningeal cells derived from Sema3A-knockout mice. In addition to growth cone collapse, recombinant Sema3A but not Sema3C inhibits neurite outgrowth of embryonic DRGs. Consistent with this result we find that the inhibitory effect of meningeal cells on neurite outgrowth is partially overcome on Sema3A-deficient MCs. Furthermore we show that the inhibitory effect of MC-derived Sema3A on neurite outgrowth is modulated by nerve growth factor. Our results show that Sema3A, a chemorepellent during nervous system development, is a major neurite growth-inhibitory molecule in meningeal fibroblasts and is therefore likely to contribute to the inhibitory properties of the neural scar.