Axon Guidance Mechanisms and Molecules: Lessons from Invertebrates

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AXON GUIDANCE MECHANISMS AND MOLECULES: LESSONS FROM INVERTEBRATES

Sofia J. Araújo and Guy Tear Vertebrates and invertebrates share the formidable task of accurately establishing the elaborate connections that make up their nervous systems. Researchers investigating this process have the challenge of identifying the molecules and mechanisms that underlie this process. Each group of organisms offers their own advantages for piecing together the conserved constituents. Broadly speaking, the invertebrates have allowed the discovery of relevant genes through classical genetic screens for mutations that affect the process of axon guidance, whereas vertebrates provide numerous systems for the elaboration of the functional mechanisms. Here, we focus on the role of invertebrates in characterizing the molecular mechanisms of axon guidance.

During embryonic development, axons have to travel The advantages of invertebrates considerable distances to reach their final targets. Pioneer neurons extend the first axons and navigate in Remarkably, this navigation takes place in a highly an environment rich in cell surface, extracellular ordered and stereotyped manner, and it has become matrix and soluble molecules that act as guidance cues clear that the solution to the shared challenge of to attract or repel the axons. At the tip of each axon is axon guidance in vertebrates and invertebrates has the growth cone that bears the receptors that react to been highly conserved. The rapid advances in our these cues, thereby directing extension and guiding the understanding of how this process takes place in formation of the axon pathway1–3.Invertebrate studies all organisms has been significantly aided by making gave the initial clues to the now universally accepted full use of the advantages that invertebrates bring idea that the same guidance molecule can act both as a to the problem. Combining the information from repellent and attractant on different axons, as is the both systems will help us understand how cells can case for the grasshopper semaphorin I molecule4,or integrate extensive extracellular information to can act on the same neuron at different times of devel- follow an unerring migration pathway and will yield opment. Later-developing neurons can also grow as clues on how to encourage axon regeneration after follower axons and track pathways that have been laid injury or disease. In this review, we will focus on the down by the pioneers to reach their targets. contribution that invertebrates studies have made in During the past decade, our understanding of axon Molecular Neurobiology the characterization of molecular mechanisms guidance mechanisms has advanced a great deal5. Department, Medical of axon guidance, whereas a review next month Invertebrate organisms such as the fruitfly Drosophila Research Council Centre for Developmental discusses how vertebrates have aided this study. melanogaster, the nematode Caenorhabditis elegans Neurobiology, New Hunts Rather than simply listing the molecules identified by and the grasshopper have had a prominent role in the House, Guy’s Campus, invertebrate studies, we have attempted to describe identification of key players and the mechanisms that are King’s College, the contexts within which the molecular functions involved in this process. The advantages of organisms London, SE1 1UL, UK. were first identified to show how these types of stud- include their simple nervous system architecture — Correspondence to G.T. e-mail: [email protected] ies have aided our understanding and can inform for example, each hemisegment of the Drosophila doi:10.1038/nrn1243 vertebrate studies. ventral nerve cord contains about 300 neurons and

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C. elegans has a total of 302 neurons — and the ability guidance, such as regulation of receptor availability32,33, to identify and follow individual neurons through the use of many different isoforms34 and crosstalk development. Through the study of such simple between signalling pathways. systems, we might define a well-conserved model for guidance decisions. In addition, Drosophila and C. elegans CNS midline — crossing choices allow the use of classical genetic analyses. Mutagenesis In invertebrates, as in higher organisms, axons must screens in both organisms have revealed many essential travel large distances to reach their final targets. To com- genes that are necessary for normal axon outgrowth plete this journey accurately, a key strategy is to break the (TABLE 1; for an extended version of this table, see Online distance down into a series of smaller trajectories TABLE 1) and this has paved the way for understanding between so-called guidepost cells or intermediate targets. the molecular pathways. These analyses have involved The initial observation of guidepost cells came from the screening for new genes (loss- and gain-of-function), analysis of axon outgrowth in the grasshopper limb identifying interacting genes (ENHANCER AND SUPPRESSOR bud35.Here, the Ti1 neuron navigates its way to the CNS SCREENS) and testing in vivo the function of candidate through key landmarks that are provided by neuronal genes that have been identified elsewhere or in molecular somata. Ablation of these cells result in the failure of the screens. These gene-discovery tools have allowed neuron to efficiently complete its pathfinding36. the identification of central players in axon guidance Another key intermediate target is the midline of the that are now known to have identical functions in CNS ventral nerve cord (VNC), which has a role in invertebrates and vertebrates. directing axons from one side of the nervous system to The efficacy of a genetic analysis relies on detectable the other. In the fly, the axon tracts of the CNS have phenotypic traits. The advent of anatomical probes an orthogonal organization, with longitudinal tracts to visualize subsets of axon pathways, an approach positioned either side of the specialized midline cells. pioneered by S. Benzer in his screens for monoclonal Within each segment, two commissural tracts (the antibodies against specific neural antigens6,7, has led to anterior and posterior commissures) extend across several large-scale genetic screens to look for mutations the midline to join the two sides. Most axons have a that affect the development of both the central nervous contralateral projection across the midline, whereas the system (CNS) and the peripheral nervous system (PNS) rest extend exclusively on one side of the midline (FIG. 1). (reviewed in REF. 8). Using these antibodies6,7,9–11 or The C. elegans VNC has a similar organization, with two specific cellular markers12,it has been possible to look for axon bundles separated by a midline structure that mutations that affect the development of the CNS and consists of an epidermal ridge topped by a row of motor PNS axonal pathways13–21,the axon trajectories of motor neurons37.In the worm, most VNC axons join the VNC neurons22,23 and adult24 or larval photoreceptor25 path- at its anterior or posterior end, choosing a fascicle on the ways. These screens relied on the histological examina- left or right side. Other axons join the VNC along its tion of axonal projections in homozygous mutant length and can extend ipsilaterally on their own side or animals. However, mutations in many genes that encode extend over the midline to the contralateral side38 key axon guidance molecules might not produce infor- (FIG. 1).This simple decision of whether or not to cross mative phenotypes in homozygous mutant animals. the CNS midline has become a fundamental model, and Many functions that are required for axon guidance in fly and worm genetic screens have identified several the embryo might be provided redundantly by both genes that are necessary for the guidance of axon the maternal and zygotic genomes, or are necessary outgrowth at the midline13,39. for similar processes in other cells. Analysis of later In Drosophila and C. elegans, the same signals act to developmental stages is hampered by the fact that many attract or repel axons at the midline, and these are also mutations can result in early lethality. For these reasons, conserved in vertebrates. An important midline signal is genetic screens analysing systematic gain-of-function the UNC-6 molecule, which was first identified in a phenotypes26,27 or identifying suppressors or enhancers of screen of uncoordinated C. elegans animals39 and was a particular axon guidance phenotype28,29 have been used subsequently found to be conserved from worm to man successfully. More recently, CLONAL MARKERS that allow the (where it is named netrin)40. The unc-6/netrin gene detection of mutant cells within a MOSAIC background30,31 encodes a ligand that is secreted from the ventral epider- ENHANCER AND SUPPRESSOR have been used to screen for molecules that are required moblasts and the midline pioneer neurons AVG and SCREENS during axon guidance at later stages of development, for PVT41,probably forming a gradient with a high point at Systems that are used to identify genes that exacerbate or reduce example, from the adult eye to the brain. These inverte- the midline. UNC-6 can act simultaneously as an attrac- the phenotype caused by brate mutagenesis screens have resulted in the identifica- tant and repellent, as both axons that normally grow to or mutations in other genes. tion of many molecules that are essential for the process away from the midline are affected in the unc-6 mutant. of axon guidance, and have established their role in vivo. The axonal response to the UNC-6 signal depends on the CLONAL MARKER A marker that allows the For many of these genes, other studies that provide nature of the UNC-6 receptor that is expressed by the identification of the progeny insight into many of the molecular pathways involved axons that navigate at the midline. The C. elegans screen derived from a single cell in this process have followed. In this way, the study of also identified mutations in unc-40 that primarily disrupt (a clone). axon guidance in invertebrate organisms has provided axon outgrowth ventrally to the midline, and mutations raw material, in the form of new genes, for vertebrate in unc-5 that affect the axons that normally extend MOSAIC Tissue containing two or more studies. In addition, invertebrate model organisms have dorsally. These genes encode the netrin receptors; genetically distinct cell types. revealed new mechanisms that participate in axonal unc-40 encodes a receptor of the immunoglobulin (Ig)

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Table 1 | Invertebrate axon guidance molecules Molecule Molecular function Guidance function Secreted ligands UNC-6/Netrin A, B Chemoattractant/chemorepellent Midline and longitudinal axon guidance, motor neuron target selection Slit/SLT-1 Chemorepellent Midline and longitudinal axon guidance BeatIa Modulator of CAM function Longitudinal axon fasciculation UNC-129 TGFβ – netrin signalling Motor neuron axon guidance Kuzbanian/ADAM10 Metalloprotease Robo signalling and longitudinal axon guidance Wnt5 Chemorepellent — Drl ligand Midline crossing SemaIIa Chemorepellent Axon fasciculation and motor neuron target selection Cell surface proteins UNC-5 Repellent Netrin receptor Midline and motor neuron axon guidance UNC-40/Frazzled Attractive/repellent Netrin receptor Midline and motor neuron axon guidance Robo/SAX-3 and Robo2 Slit receptor Midline crossing and longitudinal axon positioning Robo3 Slit receptor Longitudinal axon positioning Ptp10A, Ptp69D,Ptp99A and Dlar Orphan receptor PP Midline, motor neuron and photoreceptor axon guidance Ptp52F Orphan receptor PP Midline, motor neuron and photoreceptor axon guidance FasI Adhesion molecule Midline and longitudinal axon guidance FasII Adhesion molecule Longitudinal and motor neuron axon guidance FasIII Adhesion molecule Axon fasciculation and motor neuron target selection FasIV/SemaIa Repellent signal — plexin ligand Motor neuron and longitudinal axon guidance N-cadherin Adhesion molecule Longitudinal and photoreceptor axon guidance Off-track Plexin co-receptor Motor neuron axon guidance Plexin Sema receptor Motor neuron axon guidance α, β-integrin Adhesion molecule Midline and longitudinal axon guidance Derailed Wnt5 receptor Midline crossing Neuroglian Adhesion molecule Motor neuron and longitudinal axon guidance Connectin Adhesion molecule Motor neuron axon guidance Sidestep Adhesion molecule/ligand Motor neuron axon guidance DsCam Adhesion molecule/ligand Photoreceptor axon guidance InR Receptor tyrosine kinase Photoreceptor axon guidance Notch Delta receptor Motor neuron axon guidance Delta Notch ligand Motor neuron axon guidance Flamingo Orphan 7TM receptor/adhesion molecule Photoreceptor axon guidance Intracellular proteins Commissureless Chaperone molecule Midline axon guidance, motor neuron target selection Trio/UNC-73 Guanine exchange factor (GEF) Motor neuron, longitudinal and photoreceptor axon guidance Ena Actin binding UNC-40, Robo, Dlar signalling Abl Actin binding/kinase UNC-40, Robo, Dlar signalling Non-stop Ubiquitin hydrolase Glia function in photoreceptor axon guidance Jab1/Csn5 Proteosome function Glia function in photoreceptor axon guidance Rho/Rac/Cdc42 Regulator of actin cytoskeleton UNC-40, Robo, Dlar signalling GEF64C and Sos GEF Robo signalling MLCK Regulator of myosin activity Robo signalling UNC-115 Actin binding Netrin signalling UNC-44 Binds actin and receptor molecules Netrin signalling Capulet Binds adenylyl cyclase and actin Robo signalling Chickadee Actin polymerization regulator Motor neuron outgrowth Mical Redox state regulator Sema signalling MAX-1 Adaptor protein? Netrin repellent signalling Dock Adaptor protein Photoreceptor axon guidance Pak Kinase Photoreceptor axon guidance CAM, cell adhesion molecule; PP, protein phosphatase; TGF, transforming growth factor. See our website for an extended version of this table.

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C M Wt comm robo slit fra

HSN

PDE

PVQ LRWt unc-6 unc-40 unc-5 sax-3 Figure 1 | Axon pathways at the CNS midline of Drosophila and Caenorhabditis elegans. a | Simplified schematic of the trajectories taken by commissural (C, green), ipsilateral (I, blue) and motor (M, red) neurons in the Drosophila central nervous system (CNS). In the wild-type (Wt) embryo, most CNS axons extend along a commissural pathway and cross the midline (dashed line) in one of two commissural axon tracts. These axons cross the midline only once. The ipsilaterally projecting axons extend on one side of the CNS only, whereas the motor neurons extend out to the periphery either on their own side of the CNS or after crossing the midline (not shown). The Drosophila CNS is bilaterally symmetrical, with the same organization of neurons on either side of the midline. In commissureless (comm) mutants, the C neurons fail to cross, whereas in the robo mutant, I axons can cross the midline and C axons can recross, resulting in whorls of axons at the midline. In the absence of slit, all CNS axons extend towards the midline region and are unable to leave. When frazzled (fra) (or both netrin genes) is removed some axons fail to cross the midline, and breaks appear in the longitudinal tracts. b | Simplified schematic of the trajectories of some neurons in the C. elegans ventral nerve cord (VNC). The C. elegans VNC is asymmetric, with many more axons running in the right fascicle (R) than in the left fascicle (L). Many axons enter the VNC from the anterior or posterior end of the tract; for example, the AV (brown) or PVQ (blue) neurons. Other axons join the VNC along its length; for example, the HSN (orange) and PDE (green) axons, and they might extend contralaterally (PDEL) or ipsilaterally (PDER) once in the VNC. Motor neurons, such as DA5 and DA6 (red), have their cell bodies at the ventral midline (dashed line), and they extend dorsally away from the VNC. In unc-6 mutants, the D neurons fail to extend away from the VNC, and the PDE and HSN neurons fail to extend to the VNC. Mutations in unc-40 primarily affect the axons extending to the VNC, but they can also disrupt extension away from the ventral midline. In the absence of unc-5 activity, the D motor neurons are unable to extend dorsally. Mutations in sax-3 cause a VNC phenotype, where axons from the AV, HSN or PVQ neurons fail to remain in their fascicle and inappropriately cross the midline.

superfamily that perceives netrin as an attractant, and Other genetic studies in invertebrates have identified unc-5 encodes a molecule with Ig and THROMBOSPONDIN further components that are necessary for netrin sig- domains that also binds netrin but sees it as a repellent. nalling. Clues to the nature of the molecules that are The axons that are repelled by UNC-6 express both downstream of the netrin receptors have come from UNC-5 and UNC-40, indicating that UNC-5 might act screens in C. elegans,looking for mutations that interact to convert UNC-40 signalling from positive to negative. with unc-5 or unc-40 (REFS 22,47,48).These screens identi- Homologues of these molecules have also been identified fied ced-10 (a Rac GTPase), unc-115 (an actin-binding in Drosophila,where mutational studies indicate that protein), unc-34 (a homologue of Drosophila enabled, they have the same role in directing axon guidance42–45. ena) and unc-44 (ankyrin). These molecules point to The frazzled gene encodes the receptor for the attractive pathways from the cell surface that direct changes to the netrin response and is required for midline crossing. In actin cytoskeleton in the growth cone. CED-10 is a mem- addition to this role, Frazzled might act to capture and ber of the Rac/Rho/Cdc42 family of GTPases, which are present netrin to direct growth of longitudinal axons46. key regulators of actin cytoskeleton dynamics in most Drosophila Unc5 can also direct repulsion away from the migratory cells. UNC-115 is a target of several Racs, pos- midline, where it acts independently of Frazzled sibly including ced-10, and like its vertebrate homologue, 43 49 50 THROMBOSPONDIN (Unc40) .Curiously, this activity of Unc5 requires its Ablim , it binds actin filaments . UNC-44 probably acts A homotrimeric glycoprotein cytoplasmic domain, a domain that in vertebrates binds to link cell-surface molecules to actin. Ena is a target found in platelets, and in the to and inhibits the vertebrate Unc40/Frazzled homo- of the Drosophila Abelson tyrosine kinase (Abl)51 that extracellular matrix of logue, Deleted in Colorectal Cancer (DCC). However, acts with profilin to regulate actin polymerization52. endothelial cells and fibroblasts. It is involved in platelet Drosophila Unc5 does function with Frazzled to direct These same pathways act downstream of other axon aggregation. motor neurons away from the CNS to the periphery43. guidance receptors (see later in text). Also identified was

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unc-129,a TGFβ homologue47,53,max-1 (a multi-domain cyclase-associated, monomeric actin-binding molecule), cytoplasmic protein)22 and three uncharacterized genes implying a similar set of downstream molecules to (seu-1, -2 and -3)47. UNC-129 might form part of those regulated by the netrin molecules. Ena/Unc34 another signalling pathway that intersects with the seems to be required downstream of Robo/Sax3 in pathway downstream of UNC-5. Drosophila and C. elegans71,72. Ena binds directly to A second main signalling system at the midline that Robo, primarily through CC2, but it might also bind was initially identified in invertebrates is the Robo/Slit CC1, thereby providing a link from Robo to the actin pathway. A screen in Drosophila to identify genes that are cytoskeleton. Robo2 does not contain CC2, but it can required for axon guidance in the CNS identified muta- still transduce the Slit signal, and a Robo variant that tions in roundabout (robo)13.In the absence of robo func- lacks CC2 retains some function. tion, axons that normally never cross the midline now do Genetic studies using overexpressed abl identify Abl so, and axons that normally cross the midline only once as a repressor of Robo activity. Abl can bind Robo, both can cross several times. Robo encodes a receptor molecule through its SH3 DOMAIN to CC3 and through its SH2 of the Ig superfamily that is conserved across species and domain to a region outside the conserved stretches. It contains four conserved intracellular motifs54 (TABLE 1).Its catalyses a tyrosine phosphorylation in Robo CC1, ligand is the midline repellent signal Slit55,56.Robo is among other sites, and this might block Robo activity, as required both to prevent ipsilaterally projecting axons mutations that eliminate the phosphorylation sites gener- from crossing the midline and to prevent contralaterally ate a constitutively active receptor73.Whether Robo also projecting axons from recrossing. Before axons cross the brings Abl to Ena to regulate its activity is not clear. By midline, Robo is prevented from reaching the surface of contrast, slightly different experiments indicate that Abl the crossing neurons by Commissureless (Comm)57, can act together with Capt to mediate the activity of all making the axons unresponsive to Slit32,33,58.This sorting the Robo proteins76.Halving abl or capt activity, com- of Robo away from a plasma membrane location requires bined with a reduction in the activity of two Robo family the activity of the Nedd4 ubiquitin ligase32.The comm members, gives rise to a midline-crossing phenotype. So, gene is transiently expressed and its protein accumulates Abl seems to be a central component in midline repel- at the midline, thereby preventing it from continuing to lent signalling, but its precise role is not yet clear. Similar act on Robo once the axons have crossed the midline32,33. genetic interaction studies have also revealed the involve- Robo can then accumulate to render the axons sensitive ment of several molecules downstream of Robo — Rho to the Slit repellent and to prevent re-crossing. GTPases74,myosin light chain kinase (MLCK)74, and the Mutations in the single C. elegans robo homologue guanine exchange factors (GEFs) Sos75 and Gef64C73.It sax-3 also cause axons to misroute across the midline59. is still unclear how all these components function However, genetic studies in the worm have indicated together, but there could be another pathway where that Robo might have other ligands in addition to Slit, as Robo acts through the GEFs to downregulate Rac and the axon-outgrowth phenotypes caused by the absence Cdc42, thereby activating Rho to inhibit axon outgrowth of the only Slit gene slt-1 are not as severe as those seen through the action of MLCK on the cytoskeleton. in sax-3 mutants60.In contrast to the situation in the The Robo/Slit system might not be the only signalling worm, mutations in Drosophila slit cause a more severe pathway that mediates repulsion at the midline, as the phenotype than that seen in robo mutants. This is partly combined loss of the protein phosphatases Ptp10D and explained by the presence of two additional robo genes Ptp69D also causes a midline-crossing phenotype some- — robo2 and robo3 (REFS 61–63). Robo2 cooperates with what similar to that seen in embryos lacking robo77.The Robo to control midline crossing. When both robo and ptp mutations show a genetic interaction with robo and robo2 are removed, the CNS axons behave as in a slit slit,indicating that they might also function within the mutant, where all axons extend towards and remain at Robo pathway or a pathway that intersects with that the midline. This indicates that Robo2 normally pro- of Robo to mediate repulsive signalling. Possibilities vides the activity that is required to drive axons away for Ptp action include the activation of Robo through from the Slit signal at the midline61,62.In addition, the dephosphorylation, or antagonism of Abl activity. different Robo proteins have a role in specifying how far Further molecules that seem to mediate Robo axons extend away from the midline64,65 (see below). signalling are the metalloproteinase Kuzbanian (Kuz)63, They also determine sensory axon target selection in the calmodulin75,the integrins and their ligands78.The pre- CNS66 and dendrite outgrowth in the adult CNS67,in cise role for these molecules is presently unclear, but addition to regulating the migration of other cell types they clearly have important roles in regulating axon such as muscle68,glia69 and tracheal branches70. guidance. For example, the metalloproteinase UNC-71 Robo and Sax3 share four conserved cytoplasmic is also necessary for motor neuron guidance79, and loss domains (CC0–3), which are also found in the verte- of integrins causes widespread guidance defects80.

SH DOMAINS brate Robo molecules, whereas Drosophila Robo2 and 3 Whether these molecules are essential for normal Slit Src-homology domains are lack both CC2 and CC3 (REFS 61,62).This conservation signalling or whether midline repellent signalling is par- involved in interactions with indicates that these domains might transduce the Slit ticularly sensitive to perturbations in other signalling phosphorylated tyrosine signal. Genetic and molecular screens have identified pathways remains to be clarified. residues on other proteins (SH2 molecules that act in this transduction71–76, and these Not only do axons make choices regarding whether domains) or with proline-rich sections of other proteins (SH3 have shown that Robo signals through Ena/Unc34, or not to cross the midline, but in Drosophila they also domains). Abl, the Rho GTPases and Capulet (Capt; an adenylyl choose whether to extend in the anterior or posterior

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a b Midline c Fas II Fas II Fas II

vMP2 dMP2 MP1

Slit Robo Robo3 Robo2 MP1 MP2 N-cadherin Figure 2 | Axon pathways in the Drosophila CNS. a | Fasciclin II labels three main fascicles on either side of the Drosophila midline. Stage 17 embryos, showing the medial, intermediate and lateral FasII fascicles in red and pioneer axons of MP1, dMP2 and vMP2 neurons in green. At this stage, axons of dMP2 and vMP2 run along the first (medial) FasII fascicle, and axons of the MP1 run along the second (intermediate) FasII fascicle. Axons of pCC also run along the medial FasII fascicle (not shown). According to these observations, the medial FasII-positive tract has been named the pCC/MP2 pathway, and the intermediate tract the MP1 pathway89. b | Schematic representation of some axonal pathways joining the various longitudinal fascicles parallel to the midline. Each follower axon can specifically recognize the surface of particular axons that have extended earlier. These axons can select to join pre-existing pathways and bundle together to form the fascicles. The axons switch between these pathways as they extend towards their own individual target. c | The Robo code model of how axons choose their pathway on either side of the midline. The Slit protein is secreted by midline cells and diffuses to create a gradient across the central nervous system. The distance travelled by the axons away from the midline depends on the combination of Robo proteins that they express. Axons that express Robo protein continuously do not cross the midline, but choose a longitudinal fascicle on their own side according to which other Robo proteins they express. Axons that do not initially express the Robo protein cross the midline, where Robo levels now increase, and they choose a FasII-positive pathway depending on the combination of Robo proteins that they now express. Axons expressing Robo, Robo2 and Robo3 are most strongly repelled from the midline and enter the lateral fascicle. Axons expressing only Robo and Robo3 extend into the intermediate zone and axons that only express Robo stay closer to the midline in the medial zone. As the level of Slit signalling through the Robo receptors decreases, the neurons might recognize N-cadherin and remain in their correct zone. The axons then use their specific surface markers (such as FasII or Connectin) to select the appropriate pathway. Reproduced, with permission, from REF.89  Company of Biologists (1997).

commissure. This decision is regulated by the Derailed the ‘labelled pathways’ hypothesis92,which proposed that (Drl) receptor and its ligand Wnt5 (REFS 28,81).Drl is an axon tracts have different molecular labels on their cell atypical receptor tyrosine kinase, similar to vertebrate surface, labels that subsequent axons can differentiate to Ryk82, that is expressed specifically by the commissural allow their own extension93.As many of the follower axons that extend in the anterior commissure (AC)81.In axons have to extend over long distances to new targets, drl mutants, these axons now erroneously cross the mid- they must be able to select between alternative fascicles line in the posterior commissure (PC). Elegant genetic and recognize when to join or leave a particular fascicle as experiments have revealed that the Drl receptor is sensi- they approach their targets or need to switch pathways. tive to a repellent ligand in the PC81 and that signalling Various molecules have been identified that fulfil this through Drl does not require its kinase activity83. labelling role. Predominant among these are Neuroglian Recently, the repellent ligand that is responsible for this (Nrg)11 and the Fasciclins94,which are expressed on signalling has been identified as Wnt5. Mutations in subsets of axon fascicles in Drosophila and grasshopper wnt5 give rise to a similar phenotype as seen in drl,in embryos9,95. Nrg and Fasciclin (Fas) II and III are all which AC axons extend in the PC. Also, Wnt5 is able to members of the Ig receptor superfamily, and they share bind Drl28.The involvement of Wnt5 on axon guidance features with several vertebrate molecules, including L1, indicates that ligands previously described for their neural cell adhesion molecule (NCAM), Tag1 and role in fate specification could have secondary roles in neural–glial related CAM (NrCAM). Nrg and the mediating axon guidance in the invertebrates as has Fasciclins, like their vertebrate counterparts, can act as been shown in vertebrates84–86.For example, TGFβ homophilic cell adhesion molecules, consistent with a (UNC-129) also mediates midline axon guidance47,53. role in promoting fascicle choice through selective adhe- sion96–98.However, in the absence of either FasI or III, CNS longitudinals — axon pathway choices there are no significant defects in axon outgrowth, indi- Studies in invertebrates have shown the important role of cating that redundant or compensatory mechanisms are a pre-existing axons in guiding subsequent axon significant feature for the Drosophila adhesion mole- outgrowth87. Once pioneer neurons have established a cules13,99, as phenotypes only emerge in double mutant scaffold of axon pathways, axons that develop later fre- combinations100.FasII is expressed in three main axon quently choose to selectively bundle or fasciculate with pathways on either side of the midline, and it has a role in individual tracts to reach their target area. If particular longitudinal selective fasciculation (FIG. 2a).In FasII loss- axon pathways are removed, the follower neurons that of-function mutants, a partial defasciculation of these join these pathways stall and fail to extend88–91.This led to pathways occurs, which can be rescued by the specific

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expression of FasII in the axons that join these path- A detailed examination of the timing and sites of ways101.In addition, transgenic constructs that drive axon fasciculation and defasciculation during out- expression in more axons than normal can induce a growth of the longitudinal tract pioneers implies a key gain-of-function phenotype in which pairs of pathways role for glia109.The initial fasciculation of anteriorly and that should remain separate now fasciculate abnormally. posteriorly directed pioneer axons takes place over the Another molecule that has been implicated in surfaces of glial cells, and the later defasciculation of pathway labelling and selection is Drosophila these pioneers to establish individual longitudinal path- N-cadherin102.N-cadherin is an essential cadherin that, ways also takes place around glia. The loss of these glial like other classical cadherins, possesses a cytoplasmic cells by mutation or ablation disrupts this early pattern domain that physically interacts with β-catenin and an of fasciculation decisions109,110.The molecules that are extracellular domain that is involved in homophilic cell involved have yet to be identified. adhesion. Loss-of-function mutations in N-cadherin cause interruptions in FasII-positive fascicles, Motor neuron pathway and target selection sometimes generating axonal bifurcations that are In both insects and vertebrates, motor axons leave the probable signs of excessive defasciculation or defective CNS in common bundles that separate as they enter fasciculation. In C. elegans,mutations in hammerhead-1 the periphery and begin navigating to their individual (HMR-1), a molecule similar to N-cadherin, cause targets23,111.So, regulation of axon defasciculation is of defects in fasciculation that resemble the Drosophila prime importance for the motor axons, and clues to N-cadherin mutant phenotype103.Extracellular-matrix the mechanisms that regulate these events have been components might also have a role in pathway disclosed in Drosophila.Some of these mechanisms are selection; for example, genetic analyses in Drosophila probably retained in the vertebrates. The motor axons and C. elegans indicate that laminin is required for exit the CNS within the intersegmental nerve (ISN) and axonal pathfinding104,105. segmental nerve (SN) before defasciculating into five Recent work has reinforced the idea of labelled pathways that innervate the muscle fibres23.The SNa pathways. The three Robo receptors are expressed in and SNc pathways emerge from the SN root, whereas overlapping domains that define medial, intermediate the ISNb and ISNd pathways arise from the ISN root in and lateral zones within the longitudinal pathways at such a way that individual motor axons eventually increasing distances from the midline61,65.Robo2 is innervate individual target muscles. Failure to defascic- expressed only on those axons that extend within the ulate at the right choice point can cause growth cones to most lateral third of the longitudinal pathways — the continue along the common path, so bypassing their one furthest away from the midline. The expression of correct targets (FIG. 3). Robo3 is restricted to axons that extend within the As for the CNS longitudinal axons, FasII is a key lateral two-thirds of the longitudinal pathways, and mediator of axon fasciculation for motor axons. When Robo is expressed by all axons in the longitudinal path- the level of FasII on motor axons is increased above its ways. The final, overlapping pattern of expression of the normal level, the adhesion it provides cannot be over- three Robo receptors sets up a series of zones within come and the axons fail to defasciculate at their appro- the longitudinal tracts (FIG. 2c).Manipulation of the priate choice points112.This indicates that modulation number of Robo receptors that are expressed by an indi- of the adhesive interactions between axons underlies vidual longitudinal axon results in a discrete displace- fasciculation decisions. In flies, as in vertebrates, it seems ment of the axon to a new zone61,65.It is proposed that that the decision to fasciculate or defasciculate is deter- the combination of Robo receptors that a particular mined by the balance of attractive and repulsive forces axon expresses dictates the axonal sensitivity to midline on the axons relative to their environment. Slit (and so the distance that the axon extends), Several genes that regulate the process of selective although a computational analysis indicates that the fasciculation/defasciculation have been identified number of Robo receptors that an axon expresses is through genetic screens in Drosophila23. Beaten path sufficient to dictate the distance that they extend from (beat) mutants show a bypass phenotype that is similar the midline106. Only when an axon is positioned within to the axonal overexpression of FasII, hinting that this its appropriate zone does it select its preferred fascicle, molecule has a role in the choice to defasciculate. The and in this way the same pathway labels such as FasII can beat gene encodes a secreted protein that can fold to be re-used in each zone. Signalling through N-cadherin mimic an Ig domain113,114.It is expressed by motor neu- might also have a role in the decision as to when to rons and accumulates in the exit junction where these select a fascicle. Robo signalling inhibits cell adhesion axons defasciculate115.Genetic interactions between beat mediated by N-cadherin107.So, while Robo receptors are and two CAMs, fasII and connectin (conn; an adhesion active, the axons do not use the adhesion provided by molecule that is also expressed by motor axons)115 reveal the underlying N-cadherin. As the axons move down that reduced adhesion within distinct axon fascicles the Slit gradient, Robo signalling will be reduced at a compensates for reduced beat function and suppresses certain distance, depending on the number of receptors beat mutant phenotypes. This indicates that Beat acts in they bear. At this point, N-cadherin-mediated adhesion defasciculation as an anti-adhesive molecule to decrease could begin to act to maintain the axons in their the attraction between fasciculated neurons115.Recently, particular zone, from where they go on to choose the a family of 14 beat-like genes has been identified114. appropriate fascicle108. The closest relative to the original beat (now called

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aa Wild type Dlar (PlexA)118. PlexA loss-of-function phenotypes resemble those of SemaIa in that the SNa does not defasciculate at ISN its choice point. The defasciculation phenotype observed 12 13 in SemaI and PlexA mutants can be suppressed by the ISNb 6 * removal of one copy of FasII or by reducing Conn 7 levels118,119. So, decreasing axon–axon attraction can com- pensate for a reduction in axon–axon repulsion. Similarly, bcSemaIIa acts as a repellent secreted by the epithelium to Wild type side drive fasciculation during the outgrowth of the Ti1 Dorsal neuron in the grasshopper limb120.It seems that there is a

ISN switch in the balance of attractive and repellent forces to SNa allow axon separation at defasciculation choice points. ISNb Immunoprecipitation and genetic-interaction experi- Muscles ments have shown that PlexA associates with the SNc transmembrane protein Off-track (Otk)121.Otk and PlexA associate as components of a receptor complex that is involved in repulsive signalling in response to 121 ISNd semaphorins .More recently, PlexA has been shown to interact with Mical (molecule interacting with CasL), and this interaction seems to be required for Sema1a–PlexA-mediated repulsive axon guidance122. CNS Drosophila Mical is a large multidomain cytosolic Figure 3 | Motor neuron axon pathways in Drosophila. axonal protein that belongs to a family of FLAVOPROTEIN a | Abdominal motor axon projections in the wild type and in a MONOOXYGENASES.Homozygous Mical mutants show Dlar mutant. Wild-type and Dlar motor neuron branches as seen motor axon phenotypes that are similar to those of PlexA in filleted stage 17 embryos. The intersegmental nerve b (ISNb) or Sema1a mutants122.Mical contains domains that are defasciculates from the ISN and innervates ventral longitudinal muscles 6, 7, 12 and 13. In Dlar mutants, the ISNb fails to important for interacting with actin, intermediate fila- defasciculate from the ISN (arrowhead) and does not innervate ments and cytoskeletal-associated adaptor proteins. For the ventral longitudinal muscles126. The asterisk marks the these reasons, Mical is an excellent candidate for directly position where ISN and ISNb can be distinguished as separate mediating the cytoskeletal alterations that are characteris- fascicles. ISNb, intersegmental nerve b. Reproduced, with tic of semaphorin signalling. The characteristics of Mical  permission, from REF.123 Elsevier Sciences (1996). strongly indicate that redox signalling is important for b | Schematic diagram of wild-type motor axon projections. Each main nerve branch is shown in a different colour as it semaphorin-mediated axonal repulsion. emerges from the exit junction outside the central nervous Overexpression of Drosophila Plexin B (PlexB) on system (CNS). In wild-type motor axons, the ISN (red) and SN neurons disrupts ISNb motor axon guidance, making (orange/green) defasciculate into five pathways that innervate the the axon stall and fail to innervate some of its ventral muscle fibres. The SNa (green) and SNc (orange) pathways muscle targets123. PlexB binds to the active form of Rac emerge from the SN root and the ISN (red), ISNb (blue) and ISNd through its cytoplasmic domains (and to RhoA through (brown) pathways arise from the ISN root. c | side mutants display an extreme bypass phenotype. In side mutants, as in a different region), but this interaction is not required beat and Dlar mutants, axons from the main motor axon for Rho activation. Instead, the in vivo function of these pathways fail to defasciculate at choice points and do not PlexB–Rac–RhoA interactions seems to be to suppress innervate most of the ventral musculature. Rac activity by sequestering it from its downstream effector, the serine-threonine kinase Pak, and to enhance RhoA signalling123. beatIa114) is beatIc,which seems to function in a pro- The involvement of cell signalling in the regulation of adhesive fashion, revealing complementary functions in fasciculation has also been disclosed by the discovery that the Beat family of genes. Mutations in three other mem- Dlar,a homologue of the human receptor protein tyro- bers of this family lead to much more subtle phenotypes sine phosphatase (RPTP) LAR (leukocyte antigen-related than those observed in beatIa114. like)124,controls motor neuron pathway choice. Dlar is The axonal attraction driven by FasII has another expressed exclusively by neurons, and it functions in counterpoint in the activity of the Semaphorin (Sema) motor axon guidance125,126.Mutations in Dlar cause the family. The Sema proteins are a large family of secreted failure of some motor axons to leave their common and transmembrane axon guidance molecules with both motor pathway at choice points and enter their appropri- attractive and repulsive characteristics, which signal ate muscle territories. Instead, these motor axons show a through multimeric receptor complexes (reviewed in bypass phenotype, failing to defasciculate from the main REF. 116). The transmembrane SemaIa is expressed by nerve bundle126 (FIG. 3a).In addition to Dlar, Drosophila FLAVOPROTEIN Drosophila motor axons where it acts as a repellent to has four more Rptp genes, and genetic interaction MONOOXYGENASES mediate axonal defasciculation; in the absence of SemaIa, studies127–129 show that Dlar, Ptp10D, Ptp69D, Ptp99A A subclass of proteins that are motor axons remain fasciculated at their defasciculation and Ptp52F all participate in the regulation of extension involved in the catalysis of redox 117 reactions and use flavin-adenine choice points .Genetic interaction experiments have of the ISN and the bifurcation of the SNa, cooperating or dinucleotide as a coenzyme. shown that the SemaIa functional receptor is Plexin A competing, depending on the context126–128.

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a Eye disk Sidestep mutants (side) show a loss-of-function phenotype similar to that of beat, SemaIa, Dlar or PlexA mutants, indicating that Side is also involved in Optic stalk promoting defasciculation. However, Side acts through an alternative mechanism. Instead of decreasing axon–axon attractiveness, Side triggers specific defasciculation by increasing the attractiveness of an alternative substrate, the muscles134.Side is a molecule of the Lamina Optic R1–R6 Ig superfamily that is normally expressed on muscle lobe surfaces, and in its absence motor axons fail to defasciculate, do not enter their muscle target regions and R8 instead continue to extend along the motor fascicles. Medulla Being a strong attractant for motor axons, Side proba- R7 bly functions as a ligand for an unknown receptor in motor axon growth cones134. bcd Motor neuron target selection has also been studied R1–R6 through Drosophila genetics. Manipulation of the levels R1–R6 R7 of Netrins A (NetA) and B (NetB), SemaIIa and FasII R8 on muscle surfaces have shown that the differential activities of multiple attractive and repulsive forces are necessary for target selection135.SemaIIa and FasII are R7 R8 R1–R6 R1–R6 R7 R7 expressed by all muscles where they inhibit or promote R8 R8 synaptogenesis respectively135,136.Small relative increases of surface FasII or secreted SemaIIa of neighbouring Dlar/N-cadherin brakeless dock/pak muscles can bias the attraction between the FasII bear- Figure 4 | Projections of R-cell axons to targets in the ing motor neurons and their target cells. The Netrin optic lobe. a | A single ommatidium containing eight R-cell neurons is shown. R-cell axons project through the optic stalk molecules are examples of individual muscle specific into the optic lobe, where they contact their targets. R1–R6 recognition molecules that allow proper targeting. For axons (blue) stop at their target layer in the lamina. The R7 and example, NetB is expressed in two particular muscles R8 axons (red) continue into the underlying medulla, where (6 and 7), where it acts as a short-range attractant to they stop in two distinct layers. b–d | Schematic overcome the SemaIIa repellent signal for the specific representations of various R-cell mutant phenotypes. b |In motor neuron (RP3) that innervates these muscles. Dlar and N-cadherin mutants R7 and R8 terminate in the R8 Interestingly, misexpression of NetB shows that it can region. c | In the absence of Brakeless, R1–R8 all terminate in the medulla. d | In dock and pak mutants, uneven axon also act as a repellent for other motor neurons, indicat- bundles exit the optic stalk towards the lamina. Most fibres ing that it might normally prevent these neurons from follow abnormal paths, with R1–R6 failing to terminate in the inappropriately innervating muscles 6 and 7. lamina and innervating the medulla. As a result, some regions Motor axon guidance and fasciculation/defascicula- are hyperinnervated, whereas others remain uninnervated. tion choices might depend not only on axon–axon and axon–muscle interactions, but also on the interaction of axons with the trachea. The ISN grows in close apposi- Drosophila PTPs have also shed some light on the tion to the transversal branch of the embryonic trachea. intracellular signalling processes that are responsible for This tracheal contact point is clearly important for axon motor axon guidance. The effects of mutations in guidance, as can be observed by using mutants like Drosophila Rac1 resemble the Dlar mutant phenotype, trachealess (trh) or breathless (btl) that lack fully devel- indicating that both proteins cooperate in ISNb guid- oped tracheal structures. Loss-of-function mutations in ance130.Mutations in Drosophila trio (a guanidine trh and btl indicate that tracheal cells could be supplying exchange factor) have been shown to aggravate the Dlar signals for pathfinding and elongation137–139.The use of phenotype in SNb nerves, revealing that Trio and the trachea as an intermediate guide implies that axons Dlar are involved in the same or parallel signalling have to make fasciculation/defasciculation-like deci- pathways in motor axons131.The tyrosine kinase Abl, on sions of when to join or leave this substrate23.Indeed, the other hand, is an antagonist of the Dlar phenotype. axons of the ISN have been proposed to interact with a Ena and Abl associate with Dlar and act as its substrates tracheal branch through a NOTCH–DELTA molecular inter- in vitro, implying that these three molecules form a action in what also seems to use an Abl-dependent NOTCH–DELTA phosphotyrosine-dependent switch that controls intracellular pathway138,139. Two neurogenic genes originally 132 described in Drosophila, the growth cone behaviour at choice points . Ena is products of which interact thought to regulate the actin cytoskeleton by acting on Photoreceptor axon guidance directly. Notch and Delta are Drosophila profilin (Chickadee)52. Drosophila RPTPs The Drosophila adult visual system, which consists of now known to have several most probably signal downstream through Trio, Abl the compound eye and the optic ganglia, is an excellent functions, but were first and Ena to the intracellular effectors of axonal move- system for the study of cellular and molecular mecha- identified as being necessary to prevent ectodermal cells from ment. The Trio homologue UNC-73 is also required for nisms of axon guidance and somatotopy. The com- becoming neuroblasts. axon guidance in C. elegans133. pound eye is a crystal-like array of some 800 identical

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ommatidia, each containing eight uniquely identifiable types, and it has been suggested to regulate the photoreceptor neurons (R1–R8 cells) that project retino- transcription factor Runt and enforce target specificity topically to their targets in the optic ganglia (FIG. 4a).The by regulating gene expression150.A similar phenotype to R1–R6 cells project to the first optic ganglion, the lamina, brakeless is shown by non-stop and jab1/csn5 mutants, whereas R7 and R8 project through the lamina to termi- indicating a role for glia in the targeting of R cells to nate in the second optic ganglion, the medulla. As R-cell different layers in the visual system. When lamina glia axons enter the lamina, they encounter both neurons and are missing or their numbers reduced in non-stop glial cells, and the targeting of R-cell axons to different lay- and jsn/csn5 mutants, R1–R6 axons project through the ers indicates that there are molecular signals in the targets lamina into the medulla, implying a role for glia in that allow these areas to be distinguished. The larval visual providing the initial stop signal for R1–R6 axons to system of Drosophila is a simple model that offers the terminate at the lamina151,152.Another hint that some of opportunity to study the projection of a small group of the signals responsible for target selection can arise from photoreceptor neurons, both with respect to pathfinding the target area itself comes from the analysis of the and their fasciculation into a nerve — Bolwig’s nerve25. effects of Netrins and their receptor Frazzled in retinal These are powerful systems to identify essential genes axon projections. These molecules are all expressed in that might not be revealed in embryonic screens, as vision the lamina, but interestingly only Frazzled is required is not essential for viability, but it is possible to create for targeting. When frazzled null clones were created in genetic mosaics in which only the eye tissue is mutant. the eye of heterozygous animals, wild-type retinal fibres Screens using the Drosophila visual system have allowed were incapable of innervating target regions that lacked the identification of genes that encode proteins directly frazzled function153. involved in connectivity, their ligands and the intracellu- Mutations that affect Drosophila N-cadherin were lar pathways triggered by them24,30,140.R-cell genotypes identified in screens for R1–R6 and R7 target selection141. have been manipulated through both gain- and loss-of- When most photoreceptors of cell types R1–R8 are made function studies in wild-type animals30,141.In one of these homozygous for an N-cadherin loss-of-function muta- screens30,mutant R-cell axons were visualized projecting tion, the axonal projections of these cells are affected, and into wild-type optic ganglia and identified, amongst they fail to extend to their appropriate target. This others, trio142 and further alleles of dreadlocks (dock)140 implies a role for N-cadherin in photoreceptor target and p21-activated kinase (Pak)143.Mutations in these selection, in addition to its involvement in axon fascicle genes result in similar phenotypes with highly disorga- formation in the embryo102,141.The phenotypes of Dlar nized projections into the optic ganglia, and they code for mutant R cells are similar to those seen in N-cadherin members of a conserved signal transduction pathway in mutants, hinting at a regulatory or parallel mechanism R-cell growth cones. Dock encodes an SH2/SH3 adaptor between these two molecules154,155.Dosage-sensitive protein, whereas Pak encodes a kinase that binds to Dock genetic interactions in the eye also indicate that Dlar and regulates the actin cytoskeleton downstream from interacts with Trio and Ena155.Parallels between Dlar and the activated Rho GTPases Cdc42 and Rac. Trio encodes a Ptp69D function, both in the embryo and in the visual Rho family guanine exchange factor that activates Rac. system, raise the possibility that the two RPTPs share Genetic and biochemical experiments indicate that Dock common ligands or substrates and activate a common and Trio act in parallel to regulate Pak activity in R-cell signal transduction pathway during photoreceptor target growth cones142.These molecules seem to form a funda- selection30,155,156. mental intracellular signalling pathway, as they are also Genetic screens for photoreceptor guidance also required for axon guidance in the larval visual system34, identified flamingo (fmi) as being required for R1–R6 the embryonic CNS144, motor axons131, the adult brain145 axons to select appropriate targets in the lamina157,158. and the olfactory system146.Receptor molecules that feed Fmi, a cadherin-related cell surface protein, had previ- into this cascade include Dlar131, Dscam (Down syn- ously been shown to regulate planar cell polarity159,160 drome cell adhesion molecule)34 and the Drosophila and dendritic patterning161. R1–R6 axons in fmi mutants insulin receptor (InR)147.Dscam acts through Dock and extend to inappropriate targets in the lamina157, and R8 Pak to promote interactions between the growth cone axons are highly disorganized and often terminate at and an intermediate target during Bolwig’s nerve guid- superficial levels in fmi mosaics158, indicating that Fmi ance34.Genomic analyses have revealed that ALTERNATIVE mediates axon–axon and axon–target interactions and is SPLICING can generate more than 38,000 Dscam isoforms, involved in target selection. This target selection is done and this molecular diversity might contribute to the at a different level from N-cadherin or Dlar target specificity of neuronal connectivity. Drosophila InR binds selection, as there is no overlap between these and the Dock through its cytoplasmic domain, and axon projec- fmi phenotype157. tions of InR mutant photoreceptor cells resemble those of ALTERNATIVE SPLICING 147 During splicing, introns are dock mutants , indicating that InR might activate the Conclusions excised from RNA after Dock–Pak pathway during retinal axon pathfinding. The molecular, genetic and cellular analysis of axon transcription and the cut ends Other molecules are also directly involved in the guidance in several invertebrate systems has converged are rejoined to form a R-cell pathway choice on entering the brain. In the on a set of molecular mechanisms that are conserved continuous message. Alternative absence of Brakeless function, nearly all R1–R6 axons through to the vertebrates. These include the secreted splicing allows the production of different messages from the proceed into the medulla like R7 and R8 (REFS 148,149). ligands Unc6/Netrin and Slit, the receptors Unc40/ same DNA molecule. Brakeless is a nuclear protein that is present in all R-cell Frazzled, Unc5, the Robo molecules and RPTPs, and cell

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adhesion molecules that promote and inhibit axon choices are made. Also, much is to be learnt about how outgrowth. These molecules direct both pioneer and individual axons integrate the simultaneous cues that follower axons to their targets, often easing the problem they receive to generate a single reliable response, and of migrating over large distances by breaking these how they adapt their responses as they move into new down into smaller trajectories. It has become clear that environments along their pathway. Some clues to this the extracellular cues are transduced into the cell to have already come from studies in invertebrates. direct changes to the growth cone cytoskeleton through For example, Robo signalling can silence cadherin a smaller number of intracellular pathways, such as signalling107 and the integrins might stimulate the Abl/Ena and the Rho GTPases. growth cone response to Slit78.Also, molecules such as Despite the success in identifying a large number of Comm can regulate receptor levels32,33, and extrinsic the molecular mechanisms that underlie axon guidance signals might modulate gene expression162.Further in invertebrates and vertebrates, it is probable that many answers to these questions will be forthcoming from the more molecules will be discovered. For example, little is combined use of the experimental advantages of both known about how fasciculation and defasciculation invertebrate and vertebrate systems.

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