Getting neural circuits into shape with semaphorins - Nature

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Getting neural circuits into shape with semaphorins R. Jeroen Pasterkamp

Abstract | Semaphorins are key players in the control of neural circuit development. Recent studies have uncovered several exciting and novel aspects of neuronal semaphorin signalling in various cellular processes — including neuronal polarization, topographical mapping and axon sorting — that are crucial for the assembly of functional neuronal connections. This progress is important for further understanding the many neuronal and non-neuronal functions of semaphorins and for gaining insight into their emerging roles in the perturbed neural connectivity that is observed in some diseases. This Review discusses recent advances in semaphorin research, focusing on novel aspects of neuronal semaphorin receptor regulation and previously unexplored cellular functions of semaphorins in the nervous system. Growth cone The motile tip of growing neurites that senses molecular cues in the extracellular environment.

GTPase-activating proteins (GAPs). A family of proteins that bind and inactivate small GTP-binding proteins by increasing their rate of GTP hydrolysis.

Department of Neuroscience and Pharmacology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands. e‑mail: r.j.pasterkamp@ umcutrecht.nl doi:10.1038/nrn3302 Published online 16 August 2012

Neural circuit development is dependent on a precisely ordered series of cellular events including, but not limited to, the growth and guidance of neurites, the identification of target regions and cells, and the formation of synapses. Intriguingly, most, if not all, of the molecular signals controlling these events are used reiteratively throughout neural circuit development. That is, proteins that regulate early aspects of neural circuit formation, such as neuronal polarization, are often reused at later developmental stages, such as in neurite pruning. One group of proteins that are repeatedly used throughout development is the semaphorins (protein abbreviation SEMAs). The semaphorins are a family of membrane-associated and secreted proteins that were originally identified as neuronal growth cone-collapsing proteins involved in repulsive axon guidance1–8 (FIG. 1). It is now clear, however, that they have many roles beyond axon guidance in a variety of, often seemingly disparate, cellular processes that are required for the generation of precise neuronal connections. Several recent publications further strengthen this idea by implicating semaphorins in cellular events such as dendrite specification, axon sorting and synaptic specificity. Another emergent theme from work on various axon guidance cues is that the presentation and function of their receptors is under tight molecular control. Such control allows spatiotemporal regulation of the responsiveness of growth cones to guidance proteins and for the guidance cues themselves to have diverse effects, thereby enabling these cues to mediate a vast number

of wiring decisions. Although the regulation of semaphorin receptors remains incompletely understood, recent studies have begun to provide insight into the sophisticated molecular mechanisms that allow for the diversification and spatiotemporal control of semaphorin responsiveness. This Review focuses on these recent advances and is divided into two main sections. The first section discusses recent progress in our understanding of the molecular strategies used by neurons to control and amplify their semaphorin responses. Parallels are drawn to other guidance ligand–receptor systems, where appropriate, to highlight general regulatory principles. In the second section, recent insights into how semaphorins help to wire complex neuronal connections in the brain are examined. The focus here is on a number of previously unexplored neuronal functions of semaphorins.

Molecular control of receptor regulation In neurons, semaphorins signal predominantly through receptor proteins of the plexin (protein abbreviation PLXN, or Plex in flies) and neuropilin (protein abbreviation NRP) families9–21 (BOX 1; FIG. 1). The binding of semaphorins to these receptors induces the activation of the plexin GTPase-activating protein (GAP) domain22,23 and signalling through downstream molecules such as protein kinases, GTPases and cytoskeleton-associated proteins (for reviews see REFS 24–30). Despite considerable progress in identifying the building blocks of semaphorin receptors and downstream cytosolic signalling, how the expression and function of these proteins is

NATURE REVIEWS | NEUROSCIENCE

VOLUME 13 | SEPTEMBER 2012 | 605 © 2012 Macmillan Publishers Limited. All rights reserved

REVIEWS Invertebrates

Vertebrates

Intracellular Sema-1a Sema-2a Sema-1b Sema-2b

Sema-5c

Viral

SEMA3A SEMA3B SEMA3C SEMA3D SEMA3F SEMA3G

SEMA3E

SEMA4A SEMA4B SEMA4C SEMA4D SEMA4E SEMA4F SEMA4G

SEMA6A SEMA6B SEMA6C SEMA6D

SEMA5A SEMA5B

SEMA7A

SEMAV

Extracellular

?

Sema-1a

NRP

NRP IgCAM ERBB2

OTK PlexA

PlexB

PLXNA

VEGFR2 PLXND1

PLXNB

PLXNA

SEMA6 HSPGs CSPGs

Integrins PLXNA

PLXNC1

Figure 1 | Semaphorins and their neuronal receptor complexes. Twenty seven semaphorin proteins have been 1 identified that can be categorized into eight classes on the basis of structural and amino acid sequence similarities . Nature Reviews | Neuroscience Semaphorins exist as secreted (SEMA2s, SEMA3s and SEMAV), membrane-spanning (SEMA1s, SEMA4s, SEMA5s and SEMA6s) or glycosylphosphatidylinositol (GPI)-anchored proteins (SEMA7A). Sema‑1as and Sema‑2as, along with Sema‑5c, comprise the invertebrate semaphorins, whereas SEMAV is found in the genomes of certain non-neurotrophic DNA viruses. The other semaphorin classes are found in vertebrates. In neurons, most semaphorins signal through plexins. Nine plexins have been identified in vertebrates (PLXNA1–PLXNA4, PLXNB1–PLXNB3, PLXNC1 and PLXND1) and two plexins have been identified in invertebrates (plexin A (PlexA) and plexin B (PlexB))173. Sema‑1s and Sema‑2s signal through PlexA and PlexB, respectively, and these two plexins can form heteromultimeric receptor complexes. Neuronal semaphorin holoreceptor complexes can contain numerous co‑receptors that modulate receptor function (for example, off-track (OTK) in PlexA receptors). Interestingly, Sema‑1a may act as a receptor for Sema‑2s. In vertebrates, SEMA3s, SEMA5s and SEMA6s signal through PLXNAs, with SEMA3s also requiring neuropilins (NRP1 or NRP2) for such signalling11. SEMA3E binds directly to PLXND1 and elicits an attractant signal in the presence of NRP1 and vascular endothelial growth factor receptor 2 (VEGFR2). In the absence of NRP1, SEMA3E–PLXND1 interactions elicit repulsive responses. Some SEMA3s may signal directly through immunoglobulin super family cell adhesion molecules (IgCAMs) independently of plexins. SEMA4s associate and signal through PLXNBs. Proteoglycans modulate SEMA5 signalling through an unidentified signal-transducing receptor. Interestingly, some membrane-spanning semaphorins, including SEMA6s and Sema‑1a, can function both as ligands and as receptors. SEMA7A, which is GPI-linked, binds neuronal integrin receptors to promote axon outgrowth and PLXNC1 to influence the immune system. SEMAVs also bind PLXNC1. SEMA6s and PLXNAs can negatively influence each other through cis inhibitory interactions. CSPGs, chondroitin sulphate proteoglycans; HSPGs, heparan sulphate. Figure 1 is modified, with permission, from REF. 6 © (2009) Elsevier Science.

regulated to diversify and spatiotemporally control semaphorin responses is only beginning to be understood. The following section considers our current knowledge of these regulatory mechanisms in neurons, focusing on semaphorin receptor regulation. Diversification of semaphorin–plexin signalling. An intriguing aspect of neural circuit development is that a relatively small number of proteins set up the wiring of a vast number of neuronal connections. Accumulating evidence indicates that mechanisms

exist to diversify the effects of guidance proteins, allowing them to mediate this large number of wiring events. How do neurons diversify and expand their semaphorin signalling capacities? One way this goal is achieved is through the ability of individual semaphorins to function as attractive and repulsive guidance cues. Various extrinsic and intrinsic signals have been identified that modulate the nature of semaphorin responses 30, although the most extensively studied modulators of these responses have been semaphorin co‑receptor subunits.

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REVIEWS Proteins in all eight semaphorin classes bind receptor complexes that contain plexins, which function as signaltransduction subunits. Semaphorins either bind plexins directly or, in case of class 3 semaphorins (SEMA3s), bind neuropilins, which act as ligand-binding semaphorin co‑receptors. The inclusion of additional modulatory co‑ receptors in the semaphorin receptor complex can provide these receptors with unique signalling properties31–38 (FIG. 1). Indeed, the presence of a single additional co‑receptor can determine the response to a specific semaphorin. For example, SEMA5A acts as an attractant for axons expressing heparan sulphate proteoglycans alongside as yet unidentified signal transduction receptor subunits. By contrast, for axons expressing chondroitin sulphate proteoglycans, SEMA5A functions as a repellent36. Thus, semaphorin receptors seem to fit a model of multisubunit receptors that applies to many other neurite growth and guidance proteins, such as neurotrophic factors, netrins, WNTs and NOGO39–42. For example, whether netrin 1 acts as an attractant or repellent depends on the expression levels of uncoordinated 5 (UNC5), deleted in colorectal cancer (DCC) and Down syndrome cell adhesion molecule (DSCAM). Expression of DCC and DSCAM mediate the attractive effects of netrin 1,

whereas the expression of UNC5–DCC heterodimers mediate netrin 1 repulsion42. Exactly how co‑receptors influence neuronal plexin-mediated semaphorin signalling is largely unknown. Studies on other multisubunit receptors with related functions highlight specific roles for co‑receptors in ligand presentation, recruitment of signalling molecules and modulation of downstream signalling 39–42. These studies provide an excellent conceptual framework for the further dissection of the molecular basis of bifunctional semaphorin signalling. A second strategy used by neurons to expand and diversify their semaphorin signalling is plexin receptor heterodimerization. Plexins not only bind structurally unrelated co‑receptor proteins such as neuropilins, but can also engage in heterodimeric interactions with other plexins43,44 (FIG. 1). This ability provides plexins with access to the co‑receptors and signal transduction pathways of their plexin binding partner. For example, work in Drosophila melanogaster shows that the Sema‑1a receptor PlexA binds and activates the cytosolic signalling cue Mical (molecule interacting with CasL), which is a flavoprotein monooxygenase that disassembles filamentous actin (BOX 2) to induce repulsive axon guidance. PlexB, the other plexin found in flies and other invertebrates,

Box 1 | Structural basis of semaphorin–plexin binding Ligand binding

Semaphorin

Oligomerization?

Intracellular Extracellular

Cis interaction

Plexin

Extracellular

Monomer

Intracellular

Dimer Signalling

Signalling

Three recent studies present crystal structures and mutational analyses of phylogenetically distinct semaphorin–plexin | Neuroscience Nature Reviews complexes, namely SEMA4D–PLXNB1, SEMA6A–PLXNA2, SEMAVA–PLXNC1 and SEMA7A–PLXNC1 (REFS 14,16,17). These studies, together with previous structural work on SEMA3A and SEMA4D162,163, provide invaluable insights into ligand–receptor binding and activation of plexin signalling. In general, semaphorins exist as homodimers, both in an unbound state and when interacting with plexins. During receptor binding, semaphorin homodimers bring together two plexin monomers to form a symmetrical complex (see the figure). Both plexin monomers and homodimers have been reported to exist in the absence of ligand. This finding suggests that semaphorin dimers may collect plexin monomers into the complex and/or disrupt existing plexin homodimers in favour of heterodimeric interactions with semaphorin molecules. The interaction between semaphorins and plexins is mediated by conserved residues in the plexin and semaphorin sema domains. The bivalent nature of semaphorin–plexin complexes is necessary for sufficiently tight plexin binding and a prerequisite for cellular plexin signalling. As weak plexin–plexin cis interactions have been reported, semaphorin-stabilized plexin dimerization may seed further oligomerization of semaphorin–plexin complexes. In the future, further structural work can be expected on the thus far uncharacterized parts of plexins, such as the stalk region, and on more-complete structures under different activational states. SEMA3A and SEMA6A both use PLXNAs as their signal-transducing receptors. However, whereas SEMA6A binds these plexins directly, SEMA3A requires NRP1 for plexin binding. Nevertheless, SEMA6A and NRP1‑bound SEMA3A seem to share the same plexin binding interface for plexin activation. Mutagenesis of orthologous interface residues in SEMA3A that are known to abolish SEMA6A–PLXNA2 binding leads to the loss of the collapse-inducing activity of SEMA3A. However, these mutants display normal SEMA3A–NRP1 binding17. Thus, neuropilins may not act as a direct bridge between PLXNAs and SEMA3s but may function, for example, in the positioning and presentation of the plexin-binding surface of SEMA3A.



REVIEWS Box 2 | Redox signalling provides a link to F‑actin and microtubules Although regulators of the actin and microtubule cytoskeleton have been implicated in the signalling pathways downstream of plexins and other axon guidance receptors30,164, the MICAL (molecule interacting with CasL) family of proteins provide plexins with the unique ability to directly redox modify the cytoskeleton. MICALs belong to a small family of multidomain redox proteins, characterized by a signature amino‑terminal flavoprotein monooxygenase (MO) enzyme domain165,166. In Drosophila melanogaster, Mical is required for the assembly of neuromuscular connectivity downstream of the axon repellent semaphorin-1a (Sema‑1a) and its receptor PlexA167,168. How plexins activate MICALs is unknown, but the dependence of Sema‑1a–PlexA signalling on the Mical MO domain implicates redox reactions in neuronal semaphorin function168. How does the MO domain mediate semaphorin function? Intriguingly, this enzymatic region of the Mical protein can bind filamentous (F)‑actin and, in its activated state, induces the disassembly of actin filaments169. This F‑actin disassembly activity is mediated by the post-translational oxidation of a methionine residue (M44) at the pointed-end of actin filament subunits. This modification induces a severing of actin filaments and a decrease in actin polymerization170. Work on bristle development in D. melanogaster, which is both a Sema‑1a– PlexA-dependent and F‑actin-dependent process, indicates that the oxidation and subsequent disassembly of F‑actin by the Mical MO domain is necessary in vivo for repulsive Sema‑1a–PlexA signalling169,170. In addition to directly modifying F‑actin, MICALs also indirectly regulate the cytoskeleton through collapsin response mediator proteins (CRMPs), a small family of microtubule assembly factors171. Exposure of mouse sensory neurons to SEMA3A leads to the generation of hydrogen peroxide by the MICAL1 MO domain, which subsequently oxidizes CRMP2. Oxidation of CRMP2 induces the formation of a transient complex between CRMP2 and thioredoxin. This interaction is required for the sequential phosphorylation and inactivation of CRMP2 by cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase‑3β (GSK3β), which leads to the inhibition of microtubule assembly and growth cone collapse in vitro172. Thus, although MICALs do not directly modify the microtubule cytoskeleton they seem to control microtubule dynamics through CRMPs. However, as F‑actin is also a substrate of the MICAL MO domain, it will be important to determine whether the modification of F‑actin and CRMP2 by MICAL is a sequential event or is regulated spatiotemporally or by specific cofactors.

does not directly bind Mical but nevertheless requires Mical for certain Sema‑2a‑dependent axon guidance functions. Genetic and biochemical analyses support a model in which PlexB binds PlexA to gain access to and signal through Mical following Sema‑2a stimulation43. A third strategy is bidirectional signalling. The ability of guidance cues to act as both ligands (forward signalling) and receptors (reverse signalling) has been demonstrated for various ligand–receptor pairs, such as ephrins and EPHs, neuregulins and ERBBs, and semaphorins and plexins30,45,46. To date, neuronal receptor functions for transmembrane semaphorins have been best-characterized for Sema‑1a, which can serve as a receptor for PlexA to drive synapse formation and neurite targeting in D. melanogaster 47–49 (FIG. 1). However, the role of SEMA6D as a PLXNA1 receptor during cardiac development 50 and the ability of many membrane-associated semaphorins to interact with cytosolic signalling proteins51–54 suggest that reverse signalling is a general property of transmembrane semaphorins. Furthermore, recent work indicates that in addition to PlexA, the secreted semaphorins Sema‑2a and Sema‑2b may act as ligands for Sema‑1a receptors in D. melanogaster 55 (FIG. 1). Although further work is needed to fully characterize these remarkable interactions, these findings suggest that the functions of

some semaphorins may be mediated by other transmembrane semaphorins acting as receptors. In summary, neurons can expand their semaphorin signalling capacities by regulating the subunit composition of their receptors and by utilizing semaphorins as ligands or receptors. Many questions remain about these mechanisms. For example, to what extent do semaphorin reverse signalling and heterodimeric plexin interactions contribute to neural circuit development? How widespread are their roles in vertebrates? Furthermore, on the basis of work on other families of axon guidance cues, do additional diversification mechanisms such as posttranslational modification, regulated proteolysis or alternative splicing await discovery for semaphorin signalling? Transcriptional and post-transcriptional regulation. The tight control of guidance receptor expression at specific times and locations is essential for generating appropriate responses to guidance cues. Various mechanisms have been described that ensure the correct spatiotemporal expression and presentation of guidance receptors. These range from regulated endocytosis to spatially restricted receptor degradation. Rather than considering all these mechanisms here, several of which have been reviewed recently24,56,57, this section focuses on the role of transcriptional and post-transcriptional regulation in semaphorin receptor expression. Transcriptional control has emerged as a common and potent regulatory strategy in axon guidance58 and, recently, transcriptional mechanisms have also been implicated in neuronal semaphorin receptor regulation. For example, it is becoming clear that neuropilins are a convergent target of multiple transcription factors during neuron migration. Ablation of COUP transcription factor 2 (COUP‑TF2; also known as NR2F2) in mice lowers NRP1 and NRP2 levels in PAX6‑positive cells that migrate from the caudal ganglionic eminence to the amygdala. As a consequence, these cells are misrouted, leading to patterning defects of the amygdala59. Another example is the repression of Nrp1 and/or Nrp2 expression in telencephalic interneurons by the homeobox transcription factor proteins DLX1, DLX2 and NKX2.1. This repression allows these cells to enter the striatum, which expresses the repulsive neuropilin ligands SEMA3A and SEMA3F60,61. Collectively, these results underscore a role for transcription factors in semaphorin receptor regulation in neurons. Still, most of the data available are restricted to the regulation of neuropilins in neuron migration. Thus, an important future goal is to elucidate how widespread these mechanisms apply by assessing the transcriptional regulation of additional (co-)receptors in different aspects of neural circuit development. Another intriguing issue is how the transcription factors that regulate semaphorin receptor expression are controlled themselves. Such regulators are likely to include intrinsic and extrinsic signalling molecules. Insight into intrinsic mechanisms that fine-tune the transcriptional regulation of semaphorin receptors was recently provided by studies in the developing chick retinotectal system62. Embryonic retinal ganglion cell (RGC) axons travel from the eye to the optic tectum, their main target in the brain. En route, they



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RE1‑silencing transcription factor (REST). A member of the Kruppel-type zinc finger transcription factor family that represses gene transcription by binding the neuron-restrictive silencer element in DNA (NRSE or RE1).

Mossy fibres In the hippocampus, mossy fibres are unmyelinated axons that project from granule cells in the dentate gyrus to pyramidal neurons in the CA3 region.

Stratum lucidum A thin layer in the CA3 region just inwards of the stratum pyramidale (the layer containing pyramidal neurons) that contains mossy fibre axons and the proximal dendrites of CA3 pyramidal neurons.

encounter various guidance cues, including SEMA3A. However, until RGC axons come across SEMA3A in the distal part of their trajectory, they do not express NRP1 and hence are not sensitive to SEMA3A63. Analogous changes in the SEMA3 responsiveness of axons en route to their targets have been reported for commissural and dopaminergic axons64–66. Such spatiotemporally controlled changes serve to avoid axon stalling or the (premature) entry of axons into specific regions. In chick RGCs, the onset of NRP1 expression coincides with a marked upregulation of miR‑124, a neuron-specific microRNA (miRNA). miRNAs are short, non-coding RNAs that recognize target sequences in specific mRNAs and inhibit protein expression67. miR‑124 knockdown reduces NRP1 expression in RGCs and induces axon pathfinding errors that resemble those that are observed following NRP1 or SEMA3A knockdown62,63. Unexpectedly, miR‑124 does not bind NRP1 mRNA directly but instead targets RE1‑silencing transcription factor co-repressor 1 (CoREST) mRNA to regulate NRP1 levels (CoREST is the primary cofactor of REST and is required for the repressive effects of REST on transcription). CoREST can repress NRP1 expression in a human keratinocyte cell line, thereby controlling SEMA3A responsiveness68. REST-mediated transcriptional regulation of NRP1 expression in neurons remains to be shown, but together these data support the idea that miR‑124 inhibits CoREST in RGCs, thereby releasing REST inhibition of NRP1 expression. It is important to note that, in mice, miR‑124 has 17 validated mRNA targets, including the transcripts for laminin and β1 integrin, and many predicted targets. It is therefore likely that miR‑124 targets transcripts other than Nrp1 mRNA in RGCs, some of which may affect RGC axon guidance. Furthermore, although direct miRNA-mediated control of plexins in neurons remains to be shown, a more general role for miRNAs in the refinement of neuronal semaphorin signalling is suggested by the observations that Nrp2 mRNA is targeted by miR‑188 in hippocampal neurons, SEMA6A mRNA is targeted by miR‑27 in endothelial cells, NRP1 is targeted by miR‑9, miR‑181b and miR‑320a in angiogenesis and colorectal cancer, and Plxnb1 is targeted by miR‑241 in cervical cancer 69–73. Furthermore, several plexin and neuropilin mRNAs contain predicted binding sites for miRNAs that are known to be expressed in neurons (for example, miR‑124 and miR‑134 (REFS 74,75)). Semaphorin–plexin cis interactions. Besides regulation of their abundance or presentation, guidance receptors can be controlled through cis inhibitory interactions with their ligands. This type of receptor regulation has been reported for several ligand–receptor systems, including ephrins and EPHs, Notch and Delta, and, recently, SEMA6s and their PLXNA receptors76 (FIG. 1). Dorsal root ganglion (DRG) and sympathetic neurons both express PLXNA4, but only sympathetic growth cones are repelled by the transmembrane semaphorin SEMA6A expressed on surrounding cells77–79. This differential responsiveness is caused by the co‑expression of PLXNA4 with SEMA6A and SEMA6B in DRG neurons. These SEMA6 molecules

bind to PLXNA4 in cis thereby hindering trans interactions between SEMA6s in the surrounding tissue and PLXNA4 in DRG neurons80. DRG growth cones also use PLXNA4 to respond to SEMA3A in the extracellular environment78. Apparently, SEMA6A–PLXNA4 cis interactions do not prevent cis interactions between PLXNA4 and SEMA3A–NRP1. This finding is puzzling as interactions between PLXNA4 and SEMA3A or SEMA6A (in cis and trans) all rely on the PLXNA4 sema domain17,80 (BOX 1). This apparent discrepancy might be explained by the segregated distribution of PLXNA4–SEMA6A and PLXNA4– NRP1 complexes in the plane of the membrane of the same neuron or by the ability of SEMA3A and NRP1 to disrupt SEMA6A–PLXNA4 cis interactions on the same cell. Cis inhibitory regulation between SEMA6s and PLXNAs has also been reported in hippocampal neurons. However, here, the role of PLXNA2 resembles that of SEMA6A in DRG neurons; that is, PLXNA2 acts as a cis inhibitor. During hippocampal development, mossy fibres expressing PLXNA4 invade the stratum lucidum in which SEMA6A is expressed. SEMA6A is co‑expressed with PLXNA2, and the latter prevents interactions between SEMA6A on pyramidal neurons and PLXNA4 on in-growing mossy fibres, thereby rendering the stratum lucidum permissive for mossy fibres81. Collectively, these data indicate that cis interactions between SEMA6s and PLXNAs may render both the ligand and receptor unavailable for trans interactions. It is evident that further insight is needed into the molecular control of these signalling modes. Furthermore, it will be interesting to learn whether cis interactions also influence reverse signalling by transmembrane semaphorins and whether responses to other classes of semaphorins are regulated through cis inhibition.

Novel mechanisms in neural circuit development It is becoming increasingly clear that semaphorins influence a wide variety of cellular processes in the developing nervous system, ranging from neuron migration35,82–85 and axonal pruning 86,87 to synapse formation and function88–96. Many of these effects rely on their ability to act as repulsive or attractive proteins that control the cytoskeleton. However, evidence is emerging that semaphorins can also influence developing neurons through downstream targets other than the cytoskeleton97,98. A plethora of recent studies have identified novel functions for semaphorins in the development of neuronal connections. It is not possible to comprehensively summarize all these data here, so the following section focuses on a few studies that exemplify the diverse roles of semaphorins and illuminate previously unexplored or poorly characterized aspects of neural circuit development, with a particular emphasis on the vertebrate nervous system. The first part will consider how semaphorins help to specify axons and dendrites in young neurons, followed by a discussion on novel semaphorin-dependent mechanisms that subsequently guide these neurites to their targets in a topographical or lamina-specific manner. Once arrived at their targets, axons must identify their



REVIEWS a

b SEMA3A

SEMA3A

Extracellular NRP1

Intracellular sGC

Presumptive dendrites (NRP1+) Presumptive axon (NRP1+)

↑cGMP ↓PKA

↓cAMP

P P ↓STK11 ↑GSK3β

↑PDE4

↑PKG ↑Expression and membrane targeting of Cav2.3

Cytoskeleton

↓ Axon identity

↑Dendrite identity

Figure 2 | Novel role for SEMA3A in dendrite specification. Semaphorin 3A Nature Reviews | Neuroscience (SEMA3A) promotes the formation of dendrites and inhibits axon formation. a | Schematic of a neuropilin 1 (NRP1)-positive young neuron facing a SEMA3A gradient. b | Exposure of young neurons to SEMA3A leads to a local increase in cyclic GMP signalling, possibly through the activation of soluble guanylyl cyclase (sGC) localized asymmetrically at the developing apical dendrite. cGMP activates phosphodiesterase 4 (PDE4), through the activation of protein kinase G (PKG), to lower cyclic AMP levels. In turn, the decrease in cAMP levels lowers protein kinase A (PKA)-dependent phosphorylation of serine/threonine-protein kinase 11 (STK11) and glycogen synthase kinase 3β (GSK3β). Reduced phosphorylation of STK11 and GSK3β inhibits cytoskeletal changes that are required for axon formation. A rise in cGMP levels also leads, through, as yet, unidentified mechanisms, to the increased expression and plasma membrane targeting of voltage-gated calcium Cav2.3 channels on presumptive dendrites. These channels may be activated through PKG-dependent mechanisms and promote dendrite identity. Dashed arrows indicate parts of the signal transduction pathways that need to be studied further.

postsynaptic partners and generate synaptic contacts, often at specific cellular domains of the postsynaptic cell. Recent work suggests that these processes are also controlled by semaphorins. Regulation of neuronal polarity. Neurons are highly polarized cells that are characterized by one long axon and multiple shorter dendrites. How is this remarkable polarity established? Our current knowledge of neuronal polarity establishment mostly concerns the intrinsic cues involved, such as partitioning-defective (PAR) proteins, small GTPases, and various protein kinases and phosphatases99. Although largely unidentified, it is evident that extracellular signals are required to control these intrinsic cues and induce axon–dendrite polarization. For example, work in Caenorhabditis elegans reveals a requirement for UNC‑6 (netrin) and LIN‑44 (WNT) in axon specification and neuronal polarity 100–102. Furthermore, brainderived neurotrophic factor and transforming growth factor-β direct axon formation in vertebrate neurons103,104. So far, most studies investigating the regulation of neuronal polarization have focused on axon specification. How immature neurites turn into dendrites is not entirely understood. The prevailing view has been that

once one neurite has turned into an axon, the other neurites all become dendrites by default. Alternatively, both axons and dendrites may be specified by particular molecular signals. Recent work provides support for the latter model by showing that SEMA3A contributes to dendrite specification through the inhibition of axon specification and the promotion of dendrite identity. These polarizing effects of SEMA3A are dependent on NRP1 and on the regulation of cyclic nucleotides105,106 (FIG. 2). These findings are in line with previous studies suggesting that cyclic AMP and cyclic GMP signalling transduces the actions of several extracellular factors on neuronal polarization. Increasing cAMP levels promotes axon differentiation and suppresses dendrite formation, whereas raising cGMP levels has the opposite effect 107. Consistent with the ability of SEMA3A to promote dendrite formation, exposure of young neurons to SEMA3A triggers a decrease in cAMP and an increase in cGMP levels 105,106. Although it is unknown how SEMA3A regulates cyclic nucleotide levels in young neurons, it is interesting to note that sema3a can induce the local production of cGMP by soluble guanylyl cyclase (sgc) in polarized Xenopus laevis neurons and that sgc is asymmetrically localized to the developing apical dendrite of embryonic cortical neurons108,109. This specific subcellular distribution of sgc may enable sema3a to selectively raise cGMP levels in presumptive dendrites. How do the reciprocal changes in cAMP and cGMP levels downstream of SEMA3A determine dendrite identity? In hippocampal neurons, the decrease in cAMP levels triggered by SEMA3A lowers the protein kinase A (PKA)-dependent phosphorylation of the axon determinants serine/threonine kinase 11 (STK11) (also known as LKB1) and glycogen synthase kinase 3β (GSK3β), leading to the inactivation and activation of the STK11 and GSK3β pathways, respectively 105 (FIG. 2). Normally STK11 and GSK3β target the cytoskeleton to promote or inhibit axon formation, respectively 103,110,111. Intriguingly, the decrease in cAMP levels that is observed following SEMA3A exposure may be attributed directly to elevated cGMP levels. cGMP can downregulate cAMP through activation of the cAMP-metabolizing enzyme phosphodiesterase 4 (PDE4), and inhibition of PDE4, or protein kinase G (PKG), relieves the SEMA3A‑induced suppression of cAMP and PKA-dependent STK11 and GSK3β phosphorylation105,107 (FIG. 2). Together, these data suggest that SEMA3A causes an elevation of cGMP levels, which reduces cAMP and PKA activity, leading to the suppression of the PKA-dependent STK11 or GSK3β phosphorylation that is crucial for axon specification. Work in X. laevis indicates the existence of another cGMP-dependent pathway downstream of sema3a in neuronal polarity establishment. In X. laevis spinal commissural interneurons, the increase in cGMP by sema3a induces the expression and targeting of functional voltage-gated calcium Cav2.3 channels in presumptive dendrites, which promotes dendrite specification and inhibits axon identity through unknown mechanisms (FIG. 2). Unexpectedly, pkg is not required for the cGMP-dependent effects on Cav2.3 channel expression,



REVIEWS but is nevertheless essential for the acquisition of dendrite identity. One explanation for this observation is that pkg gates calcium entry through Cav2.3 channels by inducing membrane polarization and thereby helps to specify the dendrite108. In addition, as described above, PKG may antagonize PKA-dependent axon specification pathways105. In all, these data indicate that SEMA3A can determine dendrite identity through the cGMP-mediated, PKG-dependent and -independent regulation of Cav2.3 channels. Collectively, these findings show that SEMA3A facilitates dendrite formation and inhibits axon specification through the activation of different cGMP-dependent signalling pathways. The ability of SEMA3A to control the polarization of cortical neurons in vivo105 elegantly illustrates how semaphorins are reiteratively used throughout development. The same SEMA3A gradients that first attract migrating cortical neurons82 and help to establish their polarity 105 are used later on in development to guide cortical dendrites and axons in opposite directions and to establish the elaboration of dendritic complexity 95,109,112–114. Several fundamental questions remain to be answered about the role of SEMA3A in neuronal polarization. For example, how are the expression and plasma membrane targeting of Cav2.3 channels by cGMP regulated and how do these channels subsequently determine neurite fate? From a more general perspective, are there synergistic or antagonistic interactions between SEMA3A and other extrinsic polarization factors, some of which are known to regulate the same downstream signalling cascades99? Topographical and lamina-specific connectivity. Once neuronal polarity has been established, axons and dendrites begin to elaborate and generate remarkably complex patterns of connectivity. Intriguingly, neurons in one region of the nervous system may connect to a target region while maintaining their neighbour–neighbour relationships in the target field, a process termed topographical map formation. Considerable progress has been made in defining the molecular basis of topographical mapping, particularly in the visual and olfactory systems115,116. Topographical connections are established by several axon guidance cue and receptor pairs that show graded expression in projection neurons or their targets. For example, low to high anterior–posterior (A–P) gradients of ephrinAs in the tectum guide RGC axons to specific A–P positions according to their EPHA receptor levels116. However, axons do not solely rely on molecular signals in the environment to establish connectivity, but also on molecules expressed by fellow axons, including axon guidance cues. For example, repulsive interactions between ephrinAs and EPHAs expressed on sensory and motor afferents, respectively, underlie the segregation and targeting of these axon pathways in the periphery 117. Although the role of axon guidance cues expressed by axons is still poorly defined, recent studies unveil an intriguing contribution of axonally produced semaphorins to olfactory topographical map development. In the main olfactory system of mammals, olfactory sensory neurons (OSNs) in the olfactory epithelium, which is

located in the nasal cavity, project axons to the olfactory bulb. Each OSN expresses a single type of receptor to detect odorants, and OSNs expressing the same receptor are distributed widely in the olfactory epithelium but converge their axons onto a single glomerulus at a specific dorsal–ventral (D–V) and A–P position in the olfactory bulb115. How do axons from OSNs that are randomly distributed along the A–P axis of the olfactory epithelium find each other and converge into a glomerus at a specific A–P position? One mechanism underlying this intriguing axonal convergence relies on the ability of different types of odorant receptors to induce different expression levels of semaphorins and their receptors (FIG. 3). In response to odorants, odorant receptors activate adenylyl cyclase type 3 (ADCY3) to produce cAMP and induce neuronal depolarization through the regulation of cyclic nucleotide-gated channels115. However, different classes of odorant receptors also produce different cAMP levels in the absence of ligand, leading to differential PKA-mediated and cAMP responsive element-binding protein (CREB)mediated gene expression. Two genes targeted through this transcriptional pathway are Nrp1 and Sema3a 118. Increases in cAMP levels induce higher Nrp1 and reduced Sema3a expression, respectively. Thus, OSNs expressing a particular odorant receptor are characterized by unique cAMP levels and thereby unique and complementary levels of NRP1 and SEMA3A (FIG. 3). Interestingly, in motor axons, SEMA3A can also post-transcriptionally regulate NRP1 expression in an autocrine manner 119, but it remains to be determined whether this regulatory mechanism helps to further fine-tune NRP1 levels in OSNs. The observation that genetic alteration of Nrp1 or Sema3a perturbs the A–P topography of the glomerular map indicates that SEMA3A–NRP1 signalling is required for olfactory topographical mapping 120–123. But how do SEMA3A and NRP1 contribute to the development of topographical OSN projections? Interestingly, rather than serving as a classical target-derived cue, SEMA3A is generated by axons to regulate the topographical organization of NRP1‑expressing OSN axons. As OSN axons project to the olfactory bulb, they are gradually sorted into subbundles before reaching the olfactory bulb according to the amounts of SEMA3A and NRP1 they express120 (FIG. 3). Selective ablation of Sema3a from OSN axons perturbs not only pre-target sorting, but also the final projection sites of these axons in the olfactory bulb, thereby indicating that pre-target sorting is required for topographical map development120. Although it is conceivable that this sorting mechanism predominantly relies on repulsive SEMA3A–NRP1 signalling, it is possible that homophilic (NRP1–NRP1) and heterophilic (for example, NRP1–L1) NRP1 interactions also have a role32,124. An issue that remains to be addressed is how the sub-bundles are oriented along the correct axis before projecting onto the topographical map on the olfactory bulb. This probably requires (intermediate) targetderived positional cues. A possible candidate is SEMA3A, which is expressed by ensheathing glia surrounding the OSN axons120,121, but glia-specific ablation of SEMA3A is required to test this model. This approach would also



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Figure 3 | Axon-derived SEMA3s in olfactory topographical map formation. a, b | Schematic representation of a Nature Reviews | Neuroscience mouse head (a) and of a sagittal cut through a mouse head (b). The red box in (b) indicates position schematics in (c) and (d). c | Semaphorin 3A (SEMA3A) and neuropilin 1 (NRP1) regulate the pre-target sorting of the axons of olfactory sensory neurons (OSNs), which are located in the olfactory epithelium (OE). Each class of OSN expresses a unique odorant receptor (OR) and each OR induces a unique level of cyclic AMP. As cAMP regulates NRP1 and SEMA3A expression in a complementary manner (upregulation and downregulation, respectively), each class of OSN is characterized by unique amounts of NRP1 and SEMA3A. As OSN axons progress to the olfactory bulb (OB), they are sorted according to the levels of NRP1 and SEMA3A that they express. This pre-target axon sorting is necessary but not sufficient for the establishment of anterior–posterior (A–P) topographical connections between the OE and the OB. d | The sequential arrival of OSNs expressing SEMA3F or its receptors, NRP2 and plexin A3 (PLXNA3), regulates the dorsal–ventral (D–V) patterning of olfactory axons. Early OSN axon projections are guided to the OB by SLIT1 and ROBO2 (REFS 126,127). These early axons express and deposit SEMA3F in the embryonic OB (future anterior–dorsal part). In the OE, SEMA3F and NRP2–PLXNA3 are expressed in a complementary and graded manner. As innervation of the OB by OSN axons progresses along the D–V axis, SEMA3F released by early arriving axons repels later-arriving NRP2–PLXNA3‑positive axons to more ventral regions. DM, dorsal–medial; E14, embryonic day 14; NC, nasal cavity; P0, postnatal day 0; VL, ventral–lateral. Part b is modified, with permission, from REF. 174 © (2000) Elsevier Science and from REF. 175 © (2010) John Wiley & Sons Inc.

establish whether glia-derived SEMA3A enforces a waiting period for pioneer mouse OSN axons before they enter the olfactory bulb, as was previously suggested for chick OSN axons125. Taken together, these results indicate a striking role for SEMA3A–NRP1‑mediated pretarget axon sorting in the formation of A–P topography between the olfactory epithelium and bulb. Other recent work illuminates an additional role for OSN axon-derived SEMA3s in preserving topographical relations along the D–V axis. In striking contrast to the A–P organization of the olfactory system, the D–V arrangement of glomeruli in the olfactory bulb is directly related to the D–V position of OSNs in the olfactory epithelium115. How is this positional information of OSNs in the olfactory epithelium conveyed to their target site? Previous studies show that graded expression of ROBO2 and its SLIT1 ligand along the D–V axes of the olfactory epithelium and bulb, respectively, guides OSN axons to specific D–V positions in the olfactory bulb126,127. It has been suggested that ROBO2 and SLIT1 function to guide early pioneer axons to the anterior–dorsal olfactory bulb, whereas olfactory axons that arrive later are

instructed by other cues. This later event may be mediated by NRP2, which is expressed in a low to high D–V gradient in the olfactory epithelium and OSN axon terminals128,129 (FIG. 3). Curiously, the NRP2 ligand SEMA3F is expressed in a pattern complementary to NRP2 in the olfactory epithelium, but is virtually absent from the bulb at early stages94,130. Still, mice deficient in either NRP2 or SEMA3F mice show mistargeting of NRP2‑positive axons along the D–V axis94. On the basis of these data, it is tempting to speculate that SEMA3F and NRP2 order OSNs axons along the D–V axis in the olfactory nerve, as is described above for SEMA3A and NRP2 in A–P patterning. This scenario, however, can not be true as dorsal and ventral OSN axons are organized in separate bundles. Rather, Takeuchi et al.94 report that SEMA3F is secreted in the outer olfactory nerve layer of the anterior–dorsal olfactory bulb by OSN axons. These observations, together with the fact that SEMA3F‑positive OSNs in the dorsal zone mature earlier, and therefore project axons earlier, than the NRP2‑positive neurons in the ventral zone of the olfactory epithelium131, support a model in which



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Maxillary palps A pair of olfactory organs in Drosophila melanogaster, and other insects, that arise from the proboscis and contain a small number of olfactory sensory neurons.

Antennal lobe An olfactory structure in Drosophila melanogaster, and other insects, that is composed of glomeruli in which the axons of olfactory sensory neurons synapse with their postsynaptic target neurons.

SEMA3F that is produced by early arriving OSNs repels later-arriving NRP2‑positive and PLXNA3‑positive OSN axons towards more ventral regions of the olfactory bulb94 (FIG. 3). A similar model has been reported in the D. melanogaster olfactory system, where early arriving Sema‑1a‑positive OSN axons from the antenna repel late-arriving PlexA-positive axons from the maxillary palps, leading to segregation of these axons at the antennal lobe132. Collectively, these data indicate an important, evolutionary conserved role for semaphorins in olfactory map formation (FIG. 3). Achieving wiring specificity at a scale that is observed in the olfactory system clearly requires a multistep process, and the semaphorin-dependent mechanisms discussed here serve (together with targetderived molecular cues such as SLITs) to mediate global axon targeting and preserve topographical relationships. Subsequent steps such as the formation of discrete glomeruli require additional, activity-dependent cues, such as ephrinAs and Kirrels115. Whether this refinement process involves semaphorins remains to be determined. Given the widespread neuronal expression of SEMA3s and neuropilins133–135, it will be of great interest to determine whether the axonal mechanisms described for the olfactory system apply more generally to the (topographical) mapping of other axon pathways. Of note, the organization and targeting of mouse sensorimotor pathways are dependent on axon–axon interactions that are partly mediated by NRP1 (REF. 136). Long-range guidance and topographical mapping mechanisms bring axons into close spatial proximity of their target cells, following which short-range cues take over to establish synaptic contacts that are often concentrated in spherical glomeruli or specific planar layers. This stereotypical anatomical arrangement of synapses a

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is a prominent feature of the visual system as well as of many other areas of the CNS137. How such laminaspecific patterns of synaptic connectivity are achieved is the subject of intense study. In the retina, synaptic connections are primarily established in two layers: the outer plexiform layer (OPL) and the inner plexiform layer (IPL). The IPL is located between two cell body layers, the inner nuclear layer (INL) and the ganglion cell layer (GCL), and harbours synapses between bipolar cells, amacrine cells and RGCs. Synapses between photoreceptors, horizontal cells and bipolar cells form in the OPL138. Between 50 and 120 morphologically distinguishable retinal cell types have been reported in vertebrates, each of which displays a specific sublaminar connection pattern in the IPL and/ or the OPL. Thus, to establish synaptic connectivity in the retina, neurites must first be guided into the IPL and OPL, then be targeted to specific sublaminae and subsequently be able to recognize their synaptic partners. Recent studies support key roles for SEMA5A, SEMA5B and SEMA6A in the first two processes in the retina as well as in other regions of the CNS81,139–141. During early postnatal retinal development, SEMA5A and SEMA5B are expressed in the outer neuroblastic layer (ONBL) of the retina (FIG. 4) and loss of SEMA5A and SEMA5B causes the neurites of many subtypes of amacrine cells, bipolar cells and RGCs that express SEMA5 receptors (that is, PLXNA1 and PLXNA3) to aberrantly innervate the INL, OPL and/or ONL139. These findings indicate that SEMA5s constrain the neurites of many retinal neuron subtypes to the IPL. Interestingly, once these neurites are confined to the IPL, some of them are guided to their specific sublaminae by SEMA6A. The IPL can be subdivided into five parallel sublaminae (S1 to S5, with S5 being closest to

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Figure 4 | Transmembrane semaphorins control laminar neurite stratification in the retina. a | Schematic of the embryonic eye. Boxed region is shown in (b) and (c). b | Expression of the repulsive semaphorins SEMA5A and SEMA5B in Nature Reviews | Neuroscience the outer neuroblastic layer (ONBL) during early postnatal development of the retina functions to restrict the neurites of many subtypes of amacrine cells (ACs; for example, vesicular glutamate transporter type 3 (vGlut3)-positive or tyrosine hydroxylase (TH)-positive cells), bipolar cells (BCs; for example, neurokinin 3 receptor (NK3R)-positive cells) and retinal ganglion cells (RGCs) to the inner plexiform layer (IPL) where they form synaptic connections. These effects are mediated by the SEMA5 receptors plexin A1 (PLXNA1) and PLXNA3. c | Within the IPL, various sublaminae can be discerned (S1–S5). SEMA6A is expressed on neurites in sublaminae S4/S5. These neurites lie adjacent to neurites from the S1–S3 sublaminae that express the SEMA6A receptor PLXNA4. During postnatal development, repulsion of PLXNA4‑positive TH‑expressing AC neurites by SEMA6A is necessary for their targeting to sublamina S1. PLXNA4‑negative, M1‑type melanopsin intrinsically photosensitive (ip) RGCs co‑stratify and synapse with TH-positive AC neurites in S1. GCL, retinal ganglion cell layer; INBL, inner neuroblastic layer; INL, inner nuclear layer; NFL, nerve fibre layer.



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Figure 5 | SEMA3s determine synaptic specificity at the cellular and subcellular Nature Reviews | Neuroscience levels. a | Schematic of direct pathway and indirect pathway striatal medium spiny neurons (MSNs) receiving synaptic inputs from the parafascicular and centromedian intralaminar thalamic nuclei. Direct but not indirect MSNs express plexin D1 (PLXND1). Thalamic afferents express the PLXND1 ligand semaphorin 3E (SEMA3E). b | Genetic ablation of SEMA3E (Sema3e–/–) or PLXND1 (Plxnd1–/–) leads to an increase of thalamic– striatal synapses on the dendrites and soma of direct but not indirect MSNs. Thus, SEMA3E–PLXND1 signalling selectively restricts thalamic–striatal synapse formation on direct MSNs. To control synapse formation, thalamic axon-derived SEMA3E may change the receptivity of direct MSNs to thalamic axons by influencing local signalling in the dendrite or by inducing a retrograde signal in MSNs that repels these axons. c | Schematic of a cortex layer V pyramidal neuron. Boxed region is shown in (d) and (e). d | In wild-type cortical neurons, neuropilin 2 (NRP2) is expressed on the primary apical dendrite but not on second- or higher-order branches of apical dendrites or on basal dendrites. Note that normally, relatively few spines are present on the proximal part of the apical dendrite close to the soma. e | Genetic ablation of NRP2 (Nrp2–/–), its repulsive ligand SEMA3F (Sema3f–/–) or co‑receptor PLXN3A (Plxn3a–/–) alters the distribution of dendritic spines. An increased number of spines can be found in the proximal part of the apical dendrite. SEMA3F may be secreted by cells or neurons in the cortex or by cortical afferent projections.

Triceps muscle A large muscle on the back of the upper limb or forelimb of many vertebrates. It is responsible for extension of the elbow joint (and hence straightening of the limb).

Cutaneous maximus muscle A thin subcutaneous muscle that covers the trunk and flexes the skin.

the GCL)142. SEMA6A is expressed on neurites in S4–S5 and its receptor PLXNA4 is expressed in S1–S3 (FIG. 4). In wild-type mice, PLXNA4‑expressing tyrosine hydroxylase (TH)-positive amacrine cells predominantly stratify their neurites in S1. However, in mice deficient in either SEMA6A or PLXN4A, these cells have aberrant neuronal processes in S4–S5 (REF. 140). Interestingly, the dendrites of PLXNA4‑negative, M1‑type melanopsin intrinsically photosensitive RGCs normally co‑stratify and synapse with TH-positive amacrine neurites in S1 (REF. 143,144). Loss of both SEMA6A and PLXNA4 causes M1‑type melanopsin intrinsically photosensitive RGC dendrites to mistarget to S4–S5 and colocalize with TH-positive neurites. These findings suggest that synaptic connectivity

may be preserved in the absence of SEMA6A and that the mistargeting of M1‑type melanopsin intrinsically photosensitive RGC processes is secondary to defects in amacrine neurite stratification. Thus, SEMA6A and PLXNA4 may not be essential for synaptic target selection, but act to direct synaptic partners to regions where short-range adhesive cues can mediate synapse formation (FIG. 4). In chicks, these adhesive cues include sidekicks and DSCAMs, but in mammalian species they remain largely unidentified137,145,146. It is also important to note that SEMA6A only controls the lamina-specific organization of a small subset of retinal cells, thereby indicating that additional stratification cues must exist. Interesting candidates for these other cues are SEMA4s and their PLXNB receptors, which are prominently expressed in the retina. Although mice deficient in PLXNBs do not show overt retinal stratification defects140, PLXNBs are known to have redundant effects in many neuronal systems147. Thus, further studies assessing unexplored retinal neuron subtypes or using models that have two or more genes simultaneously knocked out are warranted. Synaptic specificity. Upon reaching a target region, axons must choose with which target cells to form connections. Typically, many types of cells are present within a given target region, and axons will form synapses with only a subset of them, often even on specific domains of the target cell. The molecular mechanisms that underlie this specificity are only beginning to be understood148, but recent advances implicate SEMA3s in the control of synaptic specificity at both the cellular and subcellular levels. The first evidence to support such a role for SEMA3s comes from elegant work on the development of spinal reflex circuitry. Spinal reflexes are mediated by synaptic connections between sensory afferents and motor neurons that supply the same muscle. These synaptic contacts can either be direct or indirect. For example, motor neurons and sensory afferents that supply the triceps muscle make direct synaptic contacts, whereas connections between motor neurons and sensory afferents supplying the cutaneous maximus muscle are indirect and require spinal interneurons. How is this wiring specificity achieved? Work by Pecho-Vrieseling et al.149 shows that the formation of direct synapses between sensory afferents and motor neurons that supply the cutaneous maximus muscle is prevented by repulsive interactions between SEMA3E, which is expressed on cutaneous maximus motor neurons, and PLXND1, which is expressed on corresponding sensory afferents. By contrast, direct synaptic contacts between triceps sensory axons and motor neuron dendrites can form, as these motor neurons do not express SEMA3E. Interestingly, SEMA3E and PLXND1 also regulate synaptic specificity in the mammalian striatum. Here, SEMA3E is expressed by thalamic afferents that form glutamatergic synapses with both classes of projection neurons in the striatum, the direct and indirect medium spiny neurons (MSNs). By contrast, PLXND1 is selectively expressed in direct MSNs150 (FIG. 5). Genetic ablation of SEMA3E or PLXND1 causes an increase in



REVIEWS thalamic–striatal synapses on the dendrites and soma of direct MSNs without affecting synapse formation on indirect MSNs150. This finding suggests that SEMA3E and PLXND1 negatively regulate, but not prevent, thalamic–striatal synapse formation on direct MSN neurons150 (FIG. 5). Collectively, these results suggest that PLXND1 and its ligand SEMA3E form a molecular recognition system that determines synaptic specificity in multiple neural systems through different mechanisms. On spinal motor neurons, SEMA3E acts as a classical, target-derived axon repellent, whereas thalamic axons secrete SEMA3E to influence their own synaptic inputs on direct MSNs through an, as yet, uncharacterized mechanism. It seems unlikely, however, that SEMA3E–PLXND1 signalling directly affects the targeting of the thalamic axons. Rather, SEMA3E may control the receptivity of direct MSNs to thalamic axons; for example, by regulating local calcium signalling 151. Alternatively, SEMA3E may elicit the production of a retrograde signal in MSNs that restricts thalamic–striatal synapse development 152. It is evident that additional mechanisms are required to establish wiring specificity in spinal and striatal circuits. For example, even in the absence of PLXND1, sensory afferents innervating the triceps muscle ignore motor neurons connected to the cutaneous maximus muscle. Similarly, in mice deficient in SEMA3E or PLXND1, sensory afferents from the cutaneous maximus muscle make direct contacts with cutaneous maximus but not triceps motor neurons. Notably, other semaphorins and plexins are expressed in the spinal cord and striatum60,61,153–157 and may serve as additional recognition systems for generating sensorimotor and striatal connectivity, most likely in conjunction with the celladhesion based mechanisms that have recently been identified in other systems and species148. A role for SEMA3F in determining subcellular synaptic specificity was recently identified in the cortex and hippocampus. Normally, few spines are found on primary apical dendrites of cortex layer V pyramidal neurons and dentate gyrus granule cells that are immediately proximal of the cell soma95. However, compared with wild-type mice, mice deficient in SEMA3F, NRP2 or PLXNA3 exhibited an increased number of spines on the proximal part of the primary apical dendrites of cortical neurons and granule cells95. Strikingly, no changes were found on second- or higher-order branches of the apical dendrite or on basal dendrites in these genetically modified mice. Moreover, addition of SEMA3F to NRP2‑positive-dissociated cortical neurons led to decreased spine numbers on apical but not basal dendrites. How is this remarkable specificity achieved? Immunohistochemistry and alkaline phosphatase-binding experiments show that NRP2 is selectively expressed on the primary apical dendrite of cortical neurons, both in vitro and in vivo, which allows SEMA3F to regulate spine distribution in a subcellular-specific manner 95. This work raises at least two questions. First, how is SEMA3F provided to the apical dendrite? NRP2– Fc fragment-binding experiments suggest that NRP2

ligands are present in deep pyramidal neuron cortical layers. It remains to be determined whether these ligands are derived from afferents axon projections or released by cortical pyramidal neurons in an autocrine or paracrine manner 95. Second, how is NRP2 expression restricted to the apical dendrite? Many receptors have a compartmentalized distribution in neurons. For example, PLXNA1 is highly expressed on the postcrossing segment of mouse spinal commissural axons, whereas PLXNA2 is enriched in the proximal segment of CA3 pyramidal neurons64,81. A plethora of cellular processes, including regulated exocytosis, endocytosis, trafficking or proteolysis, can control the subcellular distribution of proteins and future studies are needed to unveil the mechanism that is responsible for apical dendrite specific expression of NRP2.

Conclusions and perspectives Work over the past few years has led to the identification of several new and unconventional mechanisms in semaphorin receptor regulation and has implicated this family of molecular cues in previously unexplored or poorly understood aspects of neural circuit development. The ability of transmembrane semaphorins to function as receptors for plexins or, even more surprisingly, semaphorins themselves, and the heteromultimerization of different plexin receptors are all examples of recent insights into the exquisite complexity of semaphorin ligand–receptor signalling. Many intriguing questions with respect to semaphorin–plexin signalling and function remain to be answered. For example, how are the different binding modes between plexins and semaphorins (that is, forward signalling, reverse signalling and cis inhibition) regulated? Furthermore, do mechanisms identified for select semaphorins (for example, receptor functions for transmembrane semaphorins or the roles of axon-derived semaphorins in pre-target sorting and cell type-specific innervation) apply more generally, both for other semaphorins and unrelated guidance cues? One important future challenge is to determine precisely how semaphorin ligand binding translates into plexin activation and, subsequently, the recruitment and activation of downstream signalling cues in vivo. Emerging evidence suggests that uncontrolled semaphorin expression and function has an important role in nervous system-related diseases and regeneration failure. For example, aberrant function and expression of semaphorins and their downstream effectors have been proposed to trigger some of the neuronal structural changes that are observed during neurodegenerative diseases, and to mediate more subtle alterations of neuronal structures that underlie neurodevelopmental and psychiatric disorders (for reviews see REFS 5,6,158,159). In addition, semaphorins contribute to the regenerative failure of severed CNS axons by enhancing the axon growth inhibitory environment near and at the site of injury 160,161. Thus, understanding the mechanistic details of semaphorin signalling and function will be of the utmost importance in the design of strategies to modulate neural injury and disease.



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Acknowledgements

The author thanks A. L. Kolodkin, A. Kumanogoh and C. Siebold for valuable discussions and their critical comments on the manuscript. He also apologizes to all the investigators whose research could not be appropriately cited in the manuscript owing to space limitations.

Competing interests statement

The author declares no competing financial interests.

FURTHER INFORMATION R. Jeroen Pasterkamp’s homepage: http://www.jeroenpasterkamplab.com ALL LINKS ARE ACTIVE IN THE ONLINE PDF


Getting neural circuits into shape with semaphorins - Nature

Getting neural circuits into shape with semaphorins - Nature

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