AXON GUIDANCE MECHANISMS AND MOLECULES: LESSONS FROM INVERTEBRATES

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1 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. Molecular Neurobiology Department, Medical Research Council Centre for Developmental Neurobiology, New Hunts House, Guy s Campus, King s College, London, SE1 1UL, UK. Correspondence to G.T. guy.tear@kcl.ac.uk doi: /nrn1243 During embryonic development, axons have to travel considerable distances to reach their final targets. Remarkably, this navigation takes place in a highly ordered and stereotyped manner, and it has become clear that the solution to the shared challenge of axon guidance in vertebrates and invertebrates has been highly conserved. The rapid advances in our understanding of how this process takes place in all organisms has been significantly aided by making full use of the advantages that invertebrates bring to the problem. Combining the information from both systems will help us understand how cells can integrate extensive extracellular information to follow an unerring migration pathway and will yield clues on how to encourage axon regeneration after injury or disease. In this review, we will focus on the contribution that invertebrates studies have made in the characterization of molecular mechanisms of axon guidance, whereas a review next month discusses how vertebrates have aided this study. Rather than simply listing the molecules identified by invertebrate studies, we have attempted to describe the contexts within which the molecular functions were first identified to show how these types of studies have aided our understanding and can inform vertebrate studies. The advantages of invertebrates Pioneer neurons extend the first axons and navigate in an environment rich in cell surface, extracellular matrix and soluble molecules that act as guidance cues to attract or repel the axons. At the tip of each axon is the growth cone that bears the receptors that react to these cues, thereby directing extension and guiding the formation of the axon pathway 1 3.Invertebrate studies gave the initial clues to the now universally accepted idea that the same guidance molecule can act both as a repellent and attractant on different axons, as is the case for the grasshopper semaphorin I molecule 4,or can act on the same neuron at different times of development. Later-developing neurons can also grow as follower axons and track pathways that have been laid down by the pioneers to reach their targets. During the past decade, our understanding of axon guidance mechanisms has advanced a great deal 5. Invertebrate organisms such as the fruitfly Drosophila melanogaster, the nematode Caenorhabditis elegans and the grasshopper have had a prominent role in the identification of key players and the mechanisms that are involved in this process. The advantages of organisms include their simple nervous system architecture for example, each hemisegment of the Drosophila ventral nerve cord contains about 300 neurons and 910 NOVEMBER 2003 VOLUME 4

2 ENHANCER AND SUPPRESSOR SCREENS Systems that are used to identify genes that exacerbate or reduce the phenotype caused by mutations in other genes. CLONAL MARKER A marker that allows the identification of the progeny derived from a single cell (a clone). MOSAIC Tissue containing two or more genetically distinct cell types. C. elegans has a total of 302 neurons and the ability to identify and follow individual neurons through development. Through the study of such simple systems, we might define a well-conserved model for guidance decisions. In addition, Drosophila and C. elegans allow the use of classical genetic analyses. Mutagenesis screens in both organisms have revealed many essential genes that are necessary for normal axon outgrowth (TABLE 1; for an extended version of this table, see Online TABLE 1) and this has paved the way for understanding the molecular pathways. These analyses have involved screening for new genes (loss- and gain-of-function), identifying interacting genes (ENHANCER AND SUPPRESSOR SCREENS) and testing in vivo the function of candidate genes that have been identified elsewhere or in molecular screens. These gene-discovery tools have allowed the identification of central players in axon guidance that are now known to have identical functions in invertebrates and vertebrates. The efficacy of a genetic analysis relies on detectable phenotypic traits. The advent of anatomical probes to visualize subsets of axon pathways, an approach pioneered by S. Benzer in his screens for monoclonal antibodies against specific neural antigens 6,7, has led to several large-scale genetic screens to look for mutations that affect the development of both the central nervous system (CNS) and the peripheral nervous system (PNS) (reviewed in REF. 8). Using these antibodies 6,7,9 11 or specific cellular markers 12, it has been possible to look for mutations that affect the development of the CNS and PNS axonal pathways 13 21,the axon trajectories of motor neurons 22,23 and adult 24 or larval photoreceptor 25 pathways. These screens relied on the histological examination of axonal projections in homozygous mutant animals. However, mutations in many genes that encode key axon guidance molecules might not produce informative phenotypes in homozygous mutant animals. Many functions that are required for axon guidance in the embryo might be provided redundantly by both the maternal and zygotic genomes, or are necessary for similar processes in other cells. Analysis of later developmental stages is hampered by the fact that many mutations can result in early lethality. For these reasons, genetic screens analysing systematic gain-of-function phenotypes 26,27 or identifying suppressors or enhancers of a particular axon guidance phenotype 28,29 have been used successfully. More recently, CLONAL MARKERS that allow the detection of mutant cells within a MOSAIC background 30,31 have been used to screen for molecules that are required during axon guidance at later stages of development, for example, from the adult eye to the brain. These invertebrate mutagenesis screens have resulted in the identification of many molecules that are essential for the process of axon guidance, and have established their role in vivo. For many of these genes, other studies that provide insight into many of the molecular pathways involved in this process have followed. In this way, the study of axon guidance in invertebrate organisms has provided raw material, in the form of new genes, for vertebrate studies. In addition, invertebrate model organisms have revealed new mechanisms that participate in axonal guidance, such as regulation of receptor availability 32,33, the use of many different isoforms 34 and crosstalk between signalling pathways. CNS midline crossing choices In invertebrates, as in higher organisms, axons must travel large distances to reach their final targets. To complete this journey accurately, a key strategy is to break the distance down into a series of smaller trajectories between so-called guidepost cells or intermediate targets. The initial observation of guidepost cells came from the analysis of axon outgrowth in the grasshopper limb bud 35.Here, the Ti1 neuron navigates its way to the CNS through key landmarks that are provided by neuronal somata. Ablation of these cells result in the failure of the neuron to efficiently complete its pathfinding 36. Another key intermediate target is the midline of the CNS ventral nerve cord (VNC), which has a role in directing axons from one side of the nervous system to the other. In the fly, the axon tracts of the CNS have an orthogonal organization, with longitudinal tracts positioned either side of the specialized midline cells. Within each segment, two commissural tracts (the anterior and posterior commissures) extend across the midline to join the two sides. Most axons have a contralateral projection across the midline, whereas the rest extend exclusively on one side of the midline (FIG. 1). The C. elegans VNC has a similar organization, with two axon bundles separated by a midline structure that consists of an epidermal ridge topped by a row of motor neurons 37.In the worm, most VNC axons join the VNC at its anterior or posterior end, choosing a fascicle on the left or right side. Other axons join the VNC along its length and can extend ipsilaterally on their own side or extend over the midline to the contralateral side 38 (FIG. 1).This simple decision of whether or not to cross the CNS midline has become a fundamental model, and fly and worm genetic screens have identified several genes that are necessary for the guidance of axon outgrowth at the midline 13,39. In Drosophila and C. elegans, the same signals act to attract or repel axons at the midline, and these are also conserved in vertebrates. An important midline signal is the UNC-6 molecule, which was first identified in a screen of uncoordinated C. elegans animals 39 and was subsequently found to be conserved from worm to man (where it is named netrin) 40. The unc-6/netrin gene encodes a ligand that is secreted from the ventral epidermoblasts and the midline pioneer neurons AVG and PVT 41,probably forming a gradient with a high point at the midline. UNC-6 can act simultaneously as an attractant and repellent, as both axons that normally grow to or away from the midline are affected in the unc-6 mutant. The axonal response to the UNC-6 signal depends on the nature of the UNC-6 receptor that is expressed by the axons that navigate at the midline. The C. elegans screen also identified mutations in unc-40 that primarily disrupt axon outgrowth ventrally to the midline, and mutations in unc-5 that affect the axons that normally extend dorsally. These genes encode the netrin receptors; unc-40 encodes a receptor of the immunoglobulin (Ig) NATURE REVIEWS NEUROSCIENCE VOLUME 4 NOVEMBER

3 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. 912 NOVEMBER 2003 VOLUME 4

4 a I C M Wt comm robo slit fra b HSN PDE L Wt R PVQ unc-6 unc-40 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. unc-5 sax-3 THROMBOSPONDIN A homotrimeric glycoprotein found in platelets, and in the extracellular matrix of endothelial cells and fibroblasts. It is involved in platelet aggregation. superfamily that perceives netrin as an attractant, and unc-5 encodes a molecule with Ig and THROMBOSPONDIN domains that also binds netrin but sees it as a repellent. The axons that are repelled by UNC-6 express both UNC-5 and UNC-40, indicating that UNC-5 might act to convert UNC-40 signalling from positive to negative. Homologues of these molecules have also been identified in Drosophila,where mutational studies indicate that they have the same role in directing axon guidance The frazzled gene encodes the receptor for the attractive netrin response and is required for midline crossing. In addition to this role, Frazzled might act to capture and present netrin to direct growth of longitudinal axons 46. Drosophila Unc5 can also direct repulsion away from the midline, where it acts independently of Frazzled (Unc40) 43.Curiously, this activity of Unc5 requires its cytoplasmic domain, a domain that in vertebrates binds to and inhibits the vertebrate Unc40/Frazzled homologue, Deleted in Colorectal Cancer (DCC). However, Drosophila Unc5 does function with Frazzled to direct motor neurons away from the CNS to the periphery 43. Other genetic studies in invertebrates have identified further components that are necessary for netrin signalling. Clues to the nature of the molecules that are downstream of the netrin receptors have come from screens in C. elegans,looking for mutations that interact with unc-5 or unc-40 (REFS 22,47,48).These screens identified ced-10 (a Rac GTPase), unc-115 (an actin-binding protein), unc-34 (a homologue of Drosophila enabled, ena) and unc-44 (ankyrin). These molecules point to pathways from the cell surface that direct changes to the actin cytoskeleton in the growth cone. CED-10 is a member of the Rac/Rho/Cdc42 family of GTPases, which are key regulators of actin cytoskeleton dynamics in most migratory cells. UNC-115 is a target of several Racs, possibly including ced-10, and like its vertebrate homologue, Ablim 49, it binds actin filaments 50. UNC-44 probably acts to link cell-surface molecules to actin. Ena is a target of the Drosophila Abelson tyrosine kinase (Abl) 51 that acts with profilin to regulate actin polymerization 52. These same pathways act downstream of other axon guidance receptors (see later in text). Also identified was NATURE REVIEWS NEUROSCIENCE VOLUME 4 NOVEMBER

5 SH DOMAINS Src-homology domains are involved in interactions with phosphorylated tyrosine residues on other proteins (SH2 domains) or with proline-rich sections of other proteins (SH3 domains). unc-129,a TGFβ homologue 47,53, max-1 (a multi-domain cytoplasmic protein) 22 and three uncharacterized genes (seu-1, -2 and -3) 47. UNC-129 might form part of another signalling pathway that intersects with the pathway downstream of UNC-5. A second main signalling system at the midline that was initially identified in invertebrates is the Robo/Slit pathway. A screen in Drosophila to identify genes that are required for axon guidance in the CNS identified mutations in roundabout (robo) 13.In the absence of robo function, axons that normally never cross the midline now do so, and axons that normally cross the midline only once can cross several times. Robo encodes a receptor molecule of the Ig superfamily that is conserved across species and contains four conserved intracellular motifs 54 (TABLE 1).Its ligand is the midline repellent signal Slit 55,56.Robo is required both to prevent ipsilaterally projecting axons from crossing the midline and to prevent contralaterally projecting axons from recrossing. Before axons cross the midline, Robo is prevented from reaching the surface of the crossing neurons by Commissureless (Comm) 57, making the axons unresponsive to Slit 32,33,58.This sorting of Robo away from a plasma membrane location requires the activity of the Nedd4 ubiquitin ligase 32.The comm gene is transiently expressed and its protein accumulates at the midline, thereby preventing it from continuing to act on Robo once the axons have crossed the midline 32,33. Robo can then accumulate to render the axons sensitive to the Slit repellent and to prevent re-crossing. Mutations in the single C. elegans robo homologue sax-3 also cause axons to misroute across the midline 59. However, genetic studies in the worm have indicated that Robo might have other ligands in addition to Slit, as the axon-outgrowth phenotypes caused by the absence of the only Slit gene slt-1 are not as severe as those seen in sax-3 mutants 60.In contrast to the situation in the worm, mutations in Drosophila slit cause a more severe phenotype than that seen in robo mutants. This is partly explained by the presence of two additional robo genes robo2 and robo3 (REFS 61 63). Robo2 cooperates with Robo to control midline crossing. When both robo and robo2 are removed, the CNS axons behave as in a slit mutant, where all axons extend towards and remain at the midline. This indicates that Robo2 normally provides the activity that is required to drive axons away from the Slit signal at the midline 61,62.In addition, the different Robo proteins have a role in specifying how far axons extend away from the midline 64,65 (see below). They also determine sensory axon target selection in the CNS 66 and dendrite outgrowth in the adult CNS 67,in addition to regulating the migration of other cell types such as muscle 68,glia 69 and tracheal branches 70. Robo and Sax3 share four conserved cytoplasmic domains (CC0 3), which are also found in the vertebrate Robo molecules, whereas Drosophila Robo2 and 3 lack both CC2 and CC3 (REFS 61,62).This conservation indicates that these domains might transduce the Slit signal. Genetic and molecular screens have identified molecules that act in this transduction 71 76, and these have shown that Robo signals through Ena/Unc34, Abl, the Rho GTPases and Capulet (Capt; an adenylyl cyclase-associated, monomeric actin-binding molecule), implying a similar set of downstream molecules to those regulated by the netrin molecules. Ena/Unc34 seems to be required downstream of Robo/Sax3 in Drosophila and C. elegans 71,72. Ena binds directly to Robo, primarily through CC2, but it might also bind CC1, thereby providing a link from Robo to the actin cytoskeleton. Robo2 does not contain CC2, but it can still transduce the Slit signal, and a Robo variant that lacks CC2 retains some function. Genetic studies using overexpressed abl identify Abl as a repressor of Robo activity. Abl can bind Robo, both through its SH3 DOMAIN to CC3 and through its SH2 domain to a region outside the conserved stretches. It catalyses a tyrosine phosphorylation in Robo CC1, among other sites, and this might block Robo activity, as mutations that eliminate the phosphorylation sites generate a constitutively active receptor 73.Whether Robo also brings Abl to Ena to regulate its activity is not clear. By contrast, slightly different experiments indicate that Abl can act together with Capt to mediate the activity of all the Robo proteins 76.Halving abl or capt activity, combined with a reduction in the activity of two Robo family members, gives rise to a midline-crossing phenotype. So, Abl seems to be a central component in midline repellent signalling, but its precise role is not yet clear. Similar genetic interaction studies have also revealed the involvement of several molecules downstream of Robo Rho GTPases 74,myosin light chain kinase (MLCK) 74, and the guanine exchange factors (GEFs) Sos 75 and Gef64C 73.It is still unclear how all these components function together, but there could be another pathway where Robo acts through the GEFs to downregulate Rac and Cdc42, thereby activating Rho to inhibit axon outgrowth through the action of MLCK on the cytoskeleton. The Robo/Slit system might not be the only signalling pathway that mediates repulsion at the midline, as the combined loss of the protein phosphatases Ptp10D and Ptp69D also causes a midline-crossing phenotype somewhat similar to that seen in embryos lacking robo 77.The ptp mutations show a genetic interaction with robo and slit,indicating that they might also function within the Robo pathway or a pathway that intersects with that of Robo to mediate repulsive signalling. Possibilities for Ptp action include the activation of Robo through dephosphorylation, or antagonism of Abl activity. Further molecules that seem to mediate Robo signalling are the metalloproteinase Kuzbanian (Kuz) 63, calmodulin 75,the integrins and their ligands 78.The precise role for these molecules is presently unclear, but they clearly have important roles in regulating axon guidance. For example, the metalloproteinase UNC-71 is also necessary for motor neuron guidance 79, and loss of integrins causes widespread guidance defects 80. Whether these molecules are essential for normal Slit signalling or whether midline repellent signalling is particularly sensitive to perturbations in other signalling pathways remains to be clarified. Not only do axons make choices regarding whether or not to cross the midline, but in Drosophila they also choose whether to extend in the anterior or posterior 914 NOVEMBER 2003 VOLUME 4

6 a b Midline c Fas II Fas II Fas II MP1 vmp2 dmp2 MP1 MP2 Slit Robo Robo3 Robo2 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 pathway 89. 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 (Drl) receptor and its ligand Wnt5 (REFS 28,81).Drl is an atypical receptor tyrosine kinase, similar to vertebrate Ryk 82, that is expressed specifically by the commissural axons that extend in the anterior commissure (AC) 81.In drl mutants, these axons now erroneously cross the midline in the posterior commissure (PC). Elegant genetic experiments have revealed that the Drl receptor is sensitive to a repellent ligand in the PC 81 and that signalling through Drl does not require its kinase activity 83. Recently, the repellent ligand that is responsible for this signalling has been identified as Wnt5. Mutations in wnt5 give rise to a similar phenotype as seen in drl,in which AC axons extend in the PC. Also, Wnt5 is able to bind Drl 28.The involvement of Wnt5 on axon guidance indicates that ligands previously described for their role in fate specification could have secondary roles in mediating axon guidance in the invertebrates as has been shown in vertebrates For example, TGFβ (UNC-129) also mediates midline axon guidance 47,53. CNS longitudinals axon pathway choices Studies in invertebrates have shown the important role of pre-existing axons in guiding subsequent axon outgrowth 87. Once pioneer neurons have established a scaffold of axon pathways, axons that develop later frequently choose to selectively bundle or fasciculate with individual tracts to reach their target area. If particular axon pathways are removed, the follower neurons that join these pathways stall and fail to extend This led to the labelled pathways hypothesis 92,which proposed that axon tracts have different molecular labels on their cell surface, labels that subsequent axons can differentiate to allow their own extension 93.As many of the follower axons have to extend over long distances to new targets, they must be able to select between alternative fascicles and recognize when to join or leave a particular fascicle as they approach their targets or need to switch pathways. Various molecules have been identified that fulfil this labelling role. Predominant among these are Neuroglian (Nrg) 11 and the Fasciclins 94,which are expressed on subsets of axon fascicles in Drosophila and grasshopper embryos 9,95. Nrg and Fasciclin (Fas) II and III are all members of the Ig receptor superfamily, and they share features with several vertebrate molecules, including L1, neural cell adhesion molecule (NCAM), Tag1 and neural glial related CAM (NrCAM). Nrg and the Fasciclins, like their vertebrate counterparts, can act as homophilic cell adhesion molecules, consistent with a role in promoting fascicle choice through selective adhesion However, in the absence of either FasI or III, there are no significant defects in axon outgrowth, indicating that redundant or compensatory mechanisms are a significant feature for the Drosophila adhesion molecules 13,99, as phenotypes only emerge in double mutant combinations 100.FasII is expressed in three main axon pathways on either side of the midline, and it has a role in longitudinal selective fasciculation (FIG. 2a). In FasII lossof-function mutants, a partial defasciculation of these pathways occurs, which can be rescued by the specific NATURE REVIEWS NEUROSCIENCE VOLUME 4 NOVEMBER

7 expression of FasII in the axons that join these pathways 101.In addition, transgenic constructs that drive expression in more axons than normal can induce a gain-of-function phenotype in which pairs of pathways that should remain separate now fasciculate abnormally. Another molecule that has been implicated in pathway labelling and selection is Drosophila N-cadherin 102.N-cadherin is an essential cadherin that, like other classical cadherins, possesses a cytoplasmic domain that physically interacts with β-catenin and an extracellular domain that is involved in homophilic cell adhesion. Loss-of-function mutations in N-cadherin cause interruptions in FasII-positive fascicles, sometimes generating axonal bifurcations that are probable signs of excessive defasciculation or defective fasciculation. In C. elegans, mutations in hammerhead-1 (HMR-1), a molecule similar to N-cadherin, cause defects in fasciculation that resemble the Drosophila N-cadherin mutant phenotype 103.Extracellular-matrix components might also have a role in pathway selection; for example, genetic analyses in Drosophila and C. elegans indicate that laminin is required for axonal pathfinding 104,105. Recent work has reinforced the idea of labelled pathways. The three Robo receptors are expressed in overlapping domains that define medial, intermediate and lateral zones within the longitudinal pathways at increasing distances from the midline 61,65.Robo2 is expressed only on those axons that extend within the most lateral third of the longitudinal pathways the one furthest away from the midline. The expression of Robo3 is restricted to axons that extend within the lateral two-thirds of the longitudinal pathways, and Robo is expressed by all axons in the longitudinal pathways. The final, overlapping pattern of expression of the three Robo receptors sets up a series of zones within the longitudinal tracts (FIG. 2c).Manipulation of the number of Robo receptors that are expressed by an individual longitudinal axon results in a discrete displacement of the axon to a new zone 61,65.It is proposed that the combination of Robo receptors that a particular axon expresses dictates the axonal sensitivity to midline Slit (and so the distance that the axon extends), although a computational analysis indicates that the number of Robo receptors that an axon expresses is sufficient to dictate the distance that they extend from the midline 106. Only when an axon is positioned within its appropriate zone does it select its preferred fascicle, and in this way the same pathway labels such as FasII can be re-used in each zone. Signalling through N-cadherin might also have a role in the decision as to when to select a fascicle. Robo signalling inhibits cell adhesion mediated by N-cadherin 107.So, while Robo receptors are active, the axons do not use the adhesion provided by the underlying N-cadherin. As the axons move down the Slit gradient, Robo signalling will be reduced at a certain distance, depending on the number of receptors they bear. At this point, N-cadherin-mediated adhesion could begin to act to maintain the axons in their particular zone, from where they go on to choose the appropriate fascicle 108. A detailed examination of the timing and sites of axon fasciculation and defasciculation during outgrowth of the longitudinal tract pioneers implies a key role for glia 109.The initial fasciculation of anteriorly and posteriorly directed pioneer axons takes place over the surfaces of glial cells, and the later defasciculation of these pioneers to establish individual longitudinal pathways also takes place around glia. The loss of these glial cells by mutation or ablation disrupts this early pattern of fasciculation decisions 109,110.The molecules that are involved have yet to be identified. Motor neuron pathway and target selection In both insects and vertebrates, motor axons leave the CNS in common bundles that separate as they enter the periphery and begin navigating to their individual targets 23,111.So, regulation of axon defasciculation is of prime importance for the motor axons, and clues to the mechanisms that regulate these events have been disclosed in Drosophila.Some of these mechanisms are probably retained in the vertebrates. The motor axons exit the CNS within the intersegmental nerve (ISN) and segmental nerve (SN) before defasciculating into five pathways that innervate the muscle fibres 23.The SNa and SNc pathways emerge from the SN root, whereas the ISNb and ISNd pathways arise from the ISN root in such a way that individual motor axons eventually innervate individual target muscles. Failure to defasciculate at the right choice point can cause growth cones to continue along the common path, so bypassing their correct targets (FIG. 3). As for the CNS longitudinal axons, FasII is a key mediator of axon fasciculation for motor axons. When the level of FasII on motor axons is increased above its normal level, the adhesion it provides cannot be overcome and the axons fail to defasciculate at their appropriate choice points 112.This indicates that modulation of the adhesive interactions between axons underlies fasciculation decisions. In flies, as in vertebrates, it seems that the decision to fasciculate or defasciculate is determined by the balance of attractive and repulsive forces on the axons relative to their environment. Several genes that regulate the process of selective fasciculation/defasciculation have been identified through genetic screens in Drosophila 23. Beaten path (beat) mutants show a bypass phenotype that is similar to the axonal overexpression of FasII, hinting that this molecule has a role in the choice to defasciculate. The beat gene encodes a secreted protein that can fold to mimic an Ig domain 113,114.It is expressed by motor neurons and accumulates in the exit junction where these axons defasciculate 115.Genetic interactions between beat and two CAMs, fasii and connectin (conn; an adhesion molecule that is also expressed by motor axons) 115 reveal that reduced adhesion within distinct axon fascicles compensates for reduced beat function and suppresses beat mutant phenotypes. This indicates that Beat acts in defasciculation as an anti-adhesive molecule to decrease the attraction between fasciculated neurons 115.Recently, a family of 14 beat-like genes has been identified 114. The closest relative to the original beat (now called 916 NOVEMBER 2003 VOLUME 4

8 FLAVOPROTEIN MONOOXYGENASES A subclass of proteins that are involved in the catalysis of redox reactions and use flavin-adenine dinucleotide as a coenzyme. aa b Wild type ISNd ISN Wild type ISNb ISN * ISNb SNa Dorsal Muscles SNc CNS c side Dlar Figure 3 Motor neuron axon pathways in Drosophila. a Abdominal motor axon projections in the wild type and in a Dlar mutant. Wild-type and Dlar motor neuron branches as seen in filleted stage 17 embryos. The intersegmental nerve b (ISNb) defasciculates from the ISN and innervates ventral longitudinal muscles 6, 7, 12 and 13. In Dlar mutants, the ISNb fails to defasciculate from the ISN (arrowhead) and does not innervate the ventral longitudinal muscles 126. The asterisk marks the position where ISN and ISNb can be distinguished as separate fascicles. ISNb, intersegmental nerve b. Reproduced, with permission, from REF. 123 Elsevier Sciences (1996). b Schematic diagram of wild-type motor axon projections. Each main nerve branch is shown in a different colour as it emerges from the exit junction outside the central nervous system (CNS). In wild-type motor axons, the ISN (red) and SN (orange/green) defasciculate into five pathways that innervate the muscle fibres. The SNa (green) and SNc (orange) pathways emerge from the SN root and the ISN (red), ISNb (blue) and ISNd (brown) pathways arise from the ISN root. c side mutants display an extreme bypass phenotype. In side mutants, as in beat and Dlar mutants, axons from the main motor axon pathways fail to defasciculate at choice points and do not innervate most of the ventral musculature. beatia 114 ) is beatic,which seems to function in a proadhesive fashion, revealing complementary functions in the Beat family of genes. Mutations in three other members of this family lead to much more subtle phenotypes than those observed in beatia 114. The axonal attraction driven by FasII has another counterpoint in the activity of the Semaphorin (Sema) family. The Sema proteins are a large family of secreted and transmembrane axon guidance molecules with both attractive and repulsive characteristics, which signal through multimeric receptor complexes (reviewed in REF. 116). The transmembrane SemaIa is expressed by Drosophila motor axons where it acts as a repellent to mediate axonal defasciculation; in the absence of SemaIa, motor axons remain fasciculated at their defasciculation choice points 117.Genetic interaction experiments have shown that the SemaIa functional receptor is Plexin A (PlexA) 118. PlexA loss-of-function phenotypes resemble those of SemaIa in that the SNa does not defasciculate at its choice point. The defasciculation phenotype observed in SemaI and PlexA mutants can be suppressed by the removal of one copy of FasII or by reducing Conn levels 118,119. So, decreasing axon axon attraction can compensate for a reduction in axon axon repulsion. Similarly, SemaIIa acts as a repellent secreted by the epithelium to drive fasciculation during the outgrowth of the Ti1 neuron in the grasshopper limb 120.It seems that there is a switch in the balance of attractive and repellent forces to allow axon separation at defasciculation choice points. Immunoprecipitation and genetic-interaction experiments have shown that PlexA associates with the 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 semaphorins 121.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 guidance 122. Drosophila Mical is a large multidomain cytosolic axonal protein that belongs to a family of FLAVOPROTEIN MONOOXYGENASES. Homozygous Mical mutants show motor axon phenotypes that are similar to those of PlexA or Sema1a mutants 122.Mical contains domains that are important for interacting with actin, intermediate filaments and cytoskeletal-associated adaptor proteins. For these reasons, Mical is an excellent candidate for directly mediating the cytoskeletal alterations that are characteristic of semaphorin signalling. The characteristics of Mical strongly indicate that redox signalling is important for semaphorin-mediated axonal repulsion. Overexpression of Drosophila Plexin B (PlexB) on neurons disrupts ISNb motor axon guidance, making the axon stall and fail to innervate some of its ventral muscle targets 123. PlexB binds to the active form of Rac through its cytoplasmic domains (and to RhoA through a different region), but this interaction is not required for Rho activation. Instead, the in vivo function of these PlexB Rac RhoA interactions seems to be to suppress Rac activity by sequestering it from its downstream effector, the serine-threonine kinase Pak, and to enhance RhoA signalling 123. The involvement of cell signalling in the regulation of fasciculation has also been disclosed by the discovery that Dlar,a homologue of the human receptor protein tyrosine phosphatase (RPTP) LAR (leukocyte antigen-related like) 124,controls motor neuron pathway choice. Dlar is expressed exclusively by neurons, and it functions in motor axon guidance 125,126.Mutations in Dlar cause the failure of some motor axons to leave their common motor pathway at choice points and enter their appropriate muscle territories. Instead, these motor axons show a bypass phenotype, failing to defasciculate from the main nerve bundle 126 (FIG. 3a).In addition to Dlar, Drosophila has four more Rptp genes, and genetic interaction studies show that Dlar, Ptp10D, Ptp69D, Ptp99A and Ptp52F all participate in the regulation of extension of the ISN and the bifurcation of the SNa, cooperating or competing, depending on the context NATURE REVIEWS NEUROSCIENCE VOLUME 4 NOVEMBER

9 NOTCH DELTA Two neurogenic genes originally described in Drosophila, the products of which interact directly. Notch and Delta are now known to have several functions, but were first identified as being necessary to prevent ectodermal cells from becoming neuroblasts. a Optic lobe b c d R7 Eye disk R1 R6 R8 Dlar/N-cadherin Lamina Medulla brakeless Optic stalk R1 R6 R7 R8 R1 R6 Drosophila PTPs have also shed some light on the intracellular signalling processes that are responsible for motor axon guidance. The effects of mutations in Drosophila Rac1 resemble the Dlar mutant phenotype, indicating that both proteins cooperate in ISNb guidance 130.Mutations in Drosophila trio (a guanidine exchange factor) have been shown to aggravate the Dlar phenotype in SNb nerves, revealing that Trio and Dlar are involved in the same or parallel signalling pathways in motor axons 131.The tyrosine kinase Abl, on the other hand, is an antagonist of the Dlar phenotype. Ena and Abl associate with Dlar and act as its substrates in vitro, implying that these three molecules form a phosphotyrosine-dependent switch that controls growth cone behaviour at choice points 132. Ena is thought to regulate the actin cytoskeleton by acting on Drosophila profilin (Chickadee) 52. Drosophila RPTPs most probably signal downstream through Trio, Abl and Ena to the intracellular effectors of axonal movement. The Trio homologue UNC-73 is also required for axon guidance in C. elegans 133. R8 R7 dock/pak R1 R6 R7 R8 R1 R6 R7 R8 Figure 4 Projections of R-cell axons to targets in the optic lobe. a A single ommatidium containing eight R-cell neurons is shown. R-cell axons project through the optic stalk into the optic lobe, where they contact their targets. R1 R6 axons (blue) stop at their target layer in the lamina. The R7 and R8 axons (red) continue into the underlying medulla, where they stop in two distinct layers. b d Schematic representations of various R-cell mutant phenotypes. b In Dlar and N-cadherin mutants R7 and R8 terminate in the R8 region. c In the absence of Brakeless, R1 R8 all terminate in the medulla. d In dock and pak mutants, uneven axon bundles exit the optic stalk towards the lamina. Most fibres follow abnormal paths, with R1 R6 failing to terminate in the lamina and innervating the medulla. As a result, some regions are hyperinnervated, whereas others remain uninnervated. 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 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 muscles 134.Side is a molecule of the Ig superfamily that is normally expressed on muscle surfaces, and in its absence motor axons fail to defasciculate, do not enter their muscle target regions and instead continue to extend along the motor fascicles. Being a strong attractant for motor axons, Side probably functions as a ligand for an unknown receptor in motor axon growth cones 134. Motor neuron target selection has also been studied through Drosophila genetics. Manipulation of the levels of Netrins A (NetA) and B (NetB), SemaIIa and FasII on muscle surfaces have shown that the differential activities of multiple attractive and repulsive forces are necessary for target selection 135.SemaIIa and FasII are expressed by all muscles where they inhibit or promote synaptogenesis respectively 135,136.Small relative increases of surface FasII or secreted SemaIIa of neighbouring muscles can bias the attraction between the FasII bearing motor neurons and their target cells. The Netrin molecules are examples of individual muscle specific recognition molecules that allow proper targeting. For example, NetB is expressed in two particular muscles (6 and 7), where it acts as a short-range attractant to overcome the SemaIIa repellent signal for the specific motor neuron (RP3) that innervates these muscles. Interestingly, misexpression of NetB shows that it can also act as a repellent for other motor neurons, indicating that it might normally prevent these neurons from inappropriately innervating muscles 6 and 7. Motor axon guidance and fasciculation/defasciculation 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 apposition to the transversal branch of the embryonic trachea. This tracheal contact point is clearly important for axon guidance, as can be observed by using mutants like trachealess (trh) or breathless (btl) that lack fully developed tracheal structures. Loss-of-function mutations in trh and btl indicate that tracheal cells could be supplying signals for pathfinding and elongation The use of the trachea as an intermediate guide implies that axons have to make fasciculation/defasciculation-like decisions of when to join or leave this substrate 23.Indeed, axons of the ISN have been proposed to interact with a tracheal branch through a NOTCH DELTA molecular interaction in what also seems to use an Abl-dependent intracellular pathway 138,139. Photoreceptor axon guidance The Drosophila adult visual system, which consists of the compound eye and the optic ganglia, is an excellent system for the study of cellular and molecular mechanisms of axon guidance and somatotopy. The compound eye is a crystal-like array of some 800 identical 918 NOVEMBER 2003 VOLUME 4

10 ALTERNATIVE SPLICING During splicing, introns are excised from RNA after transcription and the cut ends are rejoined to form a continuous message. Alternative splicing allows the production of different messages from the same DNA molecule. ommatidia, each containing eight uniquely identifiable photoreceptor neurons (R1 R8 cells) that project retinotopically to their targets in the optic ganglia (FIG. 4a). The R1 R6 cells project to the first optic ganglion, the lamina, whereas R7 and R8 project through the lamina to terminate in the second optic ganglion, the medulla. As R-cell axons enter the lamina, they encounter both neurons and glial cells, and the targeting of R-cell axons to different layers indicates that there are molecular signals in the targets that allow these areas to be distinguished. The larval visual system of Drosophila is a simple model that offers the opportunity to study the projection of a small group of photoreceptor neurons, both with respect to pathfinding and their fasciculation into a nerve Bolwig s nerve 25. These are powerful systems to identify essential genes that might not be revealed in embryonic screens, as vision is not essential for viability, but it is possible to create genetic mosaics in which only the eye tissue is mutant. Screens using the Drosophila visual system have allowed the identification of genes that encode proteins directly involved in connectivity, their ligands and the intracellular pathways triggered by them 24,30,140.R-cell genotypes have been manipulated through both gain- and loss-offunction studies in wild-type animals 30,141.In one of these screens 30,mutant R-cell axons were visualized projecting into wild-type optic ganglia and identified, amongst others, trio 142 and further alleles of dreadlocks (dock) 140 and p21-activated kinase (Pak) 143.Mutations in these genes result in similar phenotypes with highly disorganized projections into the optic ganglia, and they code for members of a conserved signal transduction pathway in R-cell growth cones. Dock encodes an SH2/SH3 adaptor protein, whereas Pak encodes a kinase that binds to Dock and regulates the actin cytoskeleton downstream from the activated Rho GTPases Cdc42 and Rac. Trio encodes a Rho family guanine exchange factor that activates Rac. Genetic and biochemical experiments indicate that Dock and Trio act in parallel to regulate Pak activity in R-cell growth cones 142.These molecules seem to form a fundamental intracellular signalling pathway, as they are also required for axon guidance in the larval visual system 34, the embryonic CNS 144, motor axons 131, the adult brain 145 and the olfactory system 146.Receptor molecules that feed into this cascade include Dlar 131, Dscam (Down syndrome cell adhesion molecule) 34 and the Drosophila insulin receptor (InR) 147.Dscam acts through Dock and Pak to promote interactions between the growth cone and an intermediate target during Bolwig s nerve guidance 34.Genomic analyses have revealed that ALTERNATIVE SPLICING can generate more than 38,000 Dscam isoforms, and this molecular diversity might contribute to the specificity of neuronal connectivity. Drosophila InR binds Dock through its cytoplasmic domain, and axon projections of InR mutant photoreceptor cells resemble those of dock mutants 147, indicating that InR might activate the Dock Pak pathway during retinal axon pathfinding. Other molecules are also directly involved in the R-cell pathway choice on entering the brain. In the absence of Brakeless function, nearly all R1 R6 axons proceed into the medulla like R7 and R8 (REFS 148,149). Brakeless is a nuclear protein that is present in all R-cell types, and it has been suggested to regulate the transcription factor Runt and enforce target specificity by regulating gene expression 150.A similar phenotype to brakeless is shown by non-stop and jab1/csn5 mutants, indicating a role for glia in the targeting of R cells to different layers in the visual system. When lamina glia are missing or their numbers reduced in non-stop and jsn/csn5 mutants, R1 R6 axons project through the lamina into the medulla, implying a role for glia in providing the initial stop signal for R1 R6 axons to terminate at the lamina 151,152.Another hint that some of the signals responsible for target selection can arise from the target area itself comes from the analysis of the effects of Netrins and their receptor Frazzled in retinal axon projections. These molecules are all expressed in the lamina, but interestingly only Frazzled is required for targeting. When frazzled null clones were created in the eye of heterozygous animals, wild-type retinal fibres were incapable of innervating target regions that lacked frazzled function 153. Mutations that affect Drosophila N-cadherin were identified in screens for R1 R6 and R7 target selection 141. When most photoreceptors of cell types R1 R8 are made homozygous for an N-cadherin loss-of-function mutation, the axonal projections of these cells are affected, and they fail to extend to their appropriate target. This implies a role for N-cadherin in photoreceptor target selection, in addition to its involvement in axon fascicle formation in the embryo 102,141.The phenotypes of Dlar mutant R cells are similar to those seen in N-cadherin mutants, hinting at a regulatory or parallel mechanism between these two molecules 154,155.Dosage-sensitive genetic interactions in the eye also indicate that Dlar interacts with Trio and Ena 155.Parallels between Dlar and Ptp69D function, both in the embryo and in the visual system, raise the possibility that the two RPTPs share common ligands or substrates and activate a common signal transduction pathway during photoreceptor target selection 30,155,156. Genetic screens for photoreceptor guidance also identified flamingo (fmi) as being required for R1 R6 axons to select appropriate targets in the lamina 157,158. Fmi, a cadherin-related cell surface protein, had previously been shown to regulate planar cell polarity 159,160 and dendritic patterning 161. R1 R6 axons in fmi mutants extend to inappropriate targets in the lamina 157, and R8 axons are highly disorganized and often terminate at superficial levels in fmi mosaics 158, indicating that Fmi mediates axon axon and axon target interactions and is involved in target selection. This target selection is done at a different level from N-cadherin or Dlar target selection, as there is no overlap between these and the fmi phenotype 157. Conclusions The molecular, genetic and cellular analysis of axon guidance in several invertebrate systems has converged on a set of molecular mechanisms that are conserved through to the vertebrates. These include the secreted ligands Unc6/Netrin and Slit, the receptors Unc40/ Frazzled, Unc5, the Robo molecules and RPTPs, and cell NATURE REVIEWS NEUROSCIENCE VOLUME 4 NOVEMBER