R6 Dispatch

Axon guidance: Netrin receptors are revealedSarah Guthrie

Netrins are molecules that guide growing axons andthat are strikingly similar in sequence and in function inflies, nematodes and vertebrates. Now, members of afamily of netrin receptors have been identified in allthree animal groups and shown to have crucial,conserved roles in axon navigation.

Address: Department of Developmental Neurobiology, UMDS, Guy’sHospital, London SE1 9RT, UK.

Electronic identifier: 0960-9822-007-R0006

Current Biology 1997, 7:R6–R9

© Current Biology Ltd ISSN 0960-9822

By a process of sequential decision-making, axons navigateto their targets in the developing nervous system. Untilrecently, little has been known of the molecular cues thatensure axons grow with such precision to remote destina-tions. These molecules may be involved in short-rangecell-to-cell interactions, or diffuse from more distantregions to elicit chemoattraction or chemorepulsion ofgrowth cones (reviewed in [1]). Prominent among suchdiffusible molecules are the netrins, which manifest bothchemoattractant and chemorepellent properties. Netrinshave been characterized in invertebrates and vertebrates,and a recent flurry of papers reports the discovery of netrinreceptors in flies, nematodes and rodents. Loss-of-functionand gain-of-function studies now highlight the importanceof these receptors in governing axonal responses to netrinat the neural midline.

Midline signallingAmong axon pathfinding decisions, a class that haveproved particularly amenable to experimental study arethose involving the midline — a region with similar fea-tures in vertebrates, nematodes and flies (Fig. 1). Groupsof ectodermal, often glial, cells act as the pathways for the

formation of axonal commissures — axonal connectionsbetween the two sides of the embryonic nervous system.Other axons are deflected away from the midline, or growalongside it. Midline cells are thus implicated in produc-ing the signals that segregate axonal pathways.

The Spanish neurobiologist Santiago Ramón y Cajal firstsaw the central role of the midline in axon guidance. Hesuggested that the extension of commissural axons fromtheir dorsal cell bodies towards the ventral midline floorplate of the spinal cord occurs in response to a diffusiblechemoattractant emanating from this region [2]. This ideawas eventually tested by culturing explants of embryonicrat dorsal spinal cord at a distance from floor-plateexplants in collagen gels. Commissural axons grew fromthe dorsal explants only when floor-plate tissue waspresent, presumably in response to a factor diffusing fromthis source (Fig. 2) [3]. When floor plates were juxtaposeddirectly with dorsal spinal explants, the axons deviatedtowards the floor plate [3].

Netrin and its relativesAttempts to find the floor-plate-derived chemoattractantculminated in the purification of two active proteins fromembryonic chick brain, named netrin-1 and netrin-2, andthe cloning of their genes [4]. Netrin-1 and netrin-2 areexpressed in the floor plate and the ventral neural tube,respectively, as might be expected for such chemoattrac-tants [5]. Aggregates of netrin-secreting cells cause theoutgrowth and orientation of commissural axons in vitro(Fig. 2) [5]. The sequences of the netrins revealed similar-ities with the extracellular matrix protein laminin, and theunc-6 protein of the nematode Caenorhabditis elegans [6,7].

Unc-6 mutant worms show perturbations in mesodermal cellmigration, in the ventral axon extension of dorsal neurons,and in the dorsal axon navigation of ventral neurons [6].

Figure 1

Representation of axon pathways in transversesections through (a) the nematode and (b) theembryonic vertebrate neural tube. Ventrallydirected axons are shown in red, and dorsallydirected axons in blue. Ventrally directed anddorsally directed axon pathways are those thatmay be dependent on the chemoattractant andchemorepellent effects of netrin, respectively.In vertebrates, these are the pathways takenby the axons of commissural and some classesof cranial motor neurons, respectively.

Dorsal(a) (b)

Ventral

Dorsal

Ventral

unc-6 expression

netrin-1netrin-2 expression© 1996 Current Biology

Early unc-6 expression is restricted to the ventral epider-moblasts and ventral cephalic sheaths, upon which neuronsgrow and mesodermal cells migrate [8]. This expressionreveals a neat parallel in the presumed chemoattraction ofventrally directed axons to the midline in both worms andvertebrates. Defects in the axon pathfinding of ventralneurons away from the midline in unc-6 mutants suggestedthat netrin may also mediate the chemorepulsion of ventralneurons in vertebrates. Amazingly, this was borne out inexperiments showing the chemorepulsion by netrin oftrochlear motor neurons, a ventral cell group whose axonsgrow dorsally [9].

Netrin function has also been analysed using a gene trapvector to generate mice deficient in netrin-1 [10]. A smallamount of wild-type transcript was detected in mutantembryos, indicating an incomplete abolition of gene func-tion. Nevertheless, within the spinal cords of theseanimals, commissural axon outgrowth is impaired:although initially directed ventrally, fewer commissuralaxons extend towards or reach the ventral midline. Whencultured at a distance from dorsal spinal cords, mutantfloor plates elicit reduced or no commissural axon out-growth compared with wild-type floor plates. But whenthe tissues are directly apposed, commissural axons canstill orient towards the mutant floor plate, suggesting thatchemoattractant properties persist (Fig. 2). Retention ofthis orienting capacity is also demonstrated by the abilityof the mutant floor plates to repel trochlear axons in vitro,and axon trajectories of trochlear motor neurons in themutant animal appear normal. In other brain regions thereare dramatic defects involving the absence of the corpuscallosum and the hippocampal and anterior commissures.

In recent studies, two Drosophila netrins have been identi-fied [11,12]. The Drosophila Netrin-A and Netrin-B genesare most similar to netrin-1, and both are expressed byventral midline glia as well as in certain muscles. Flieswith deletions for both Netrin-A and -B genes show vari-able defects, ranging from complete absence of commis-sures in most segments to commissures containing areduced number of axons. In peripheral regions, motoraxons of the intersegmental nerve navigate incorrectly,branching, stalling or projecting past their target [11].Restoration of Netrin-A or -B expression specifically in themidline of these mutants rescues commissure formation.By contrast, flooding the nervous system with ectopicNetrin-A/B yields thinner commissures, reminiscent of loss-of-function mutants, while expression on all muscles leadsto motor-axon pathfinding errors [11,12]. These cleverexperimental strategies emphasize the importance of thelocalized expression of netrin for precise axon guidance.

Receptors for netrinPinning down the precise functions of an axon guidancemolecule depends in large part on the identification of its

receptor (or receptors). In the nematode, all known unc-6functions also require unc-5 and/or unc-40, and mutationsin these genes impair the migration and axon extension ofventral and dorsal cells, respectively [6]. Whereas unc-5 isknown to encode a putative receptor responsible for dor-sally directed migrations [13], the status of unc-40 hasbeen less clear. The recent cloning and characterization ofunc-40 has shown that it encodes a receptor with a trans-membrane domain and an extracellular domain with fourimmunoglobulin modules followed by six fibronectin typeIII repeats [14]. In neurula-stage embryos, unc-40 isexpressed by ventral motor neurons, dorsal neurons, someneuroblasts and mesodermal cells, all of which are knownto respond to unc-6. In an unc-40 mutant background,specifically directing unc-40 expression to dorsal touch

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Figure 2

Experimental strategy for analysing influences on (a) axon outgrowthand (b) chemoattraction in collagen gel co-cultures. In (a), explants ofembryonic rat spinal cord (taken at embryonic day 11; E11) are placedat a distance from floorplate explants or aggregates of netrin-secretingcells. In (b), explants of floor plate or cell aggregates are placedadjacent to dorsal spinal cord explants, to show chemoattraction.Panels below illustrate the results of experiments in which netrin-deficient floor plates are used in these assays, or anti-DCC antibodiesare applied to the co-cultures.

(a) Axon outgrowth assay (b) Chemoattraction assay

Time 0: E11 embryonic rat dorsal spinal cord co-cultured with floor plate or netrin-secreting cells (green shading)

After 40 hours in culture: commissural axons grow out and are attracted by floor-plate tissue or netrin

After 40 hours in culture with a netrin-defient floor plate or in the presence of anti-DCC antibodies: commissural axons fail

to grow out but are still attracted

Floor plate or netrin-secreting cells

© 1996 Current Biology

Cell body

Axon

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neurons that normally project ventrally, but which are dis-rupted in unc-40 mutants, enables these neurons to projectaxons to the ventral midline. This ‘rescue’ experimentdemonstrates that unc-40 acts cell-autonomously, favour-ing a model in which unc-40 is a receptor.

In vertebrates, two previously identified proteins arehomologues of unc-40. These are ‘deleted in colorectalcancer’ (DCC) and neogenin, both members of theimmunoglobulin family of adhesion molecules [15,16].Members of this family have far-reaching roles in devel-opment, and can interact with extracellular matrix pro-teins, such as laminin. DCC is a tumour suppressor gene,which is frequently lost in colorectal cancers [15];neogenin has been implicated in neuronal differentiation[16]. The expression and function of DCC has recentlybeen studied in rat embryos [17]. In the spinal cord, DCCis expressed in the region containing commissuralneurons and in the motor columns, while neogenin isexpressed in the ventricular regions. Immunolocalizationshowed that the DCC antigen is present on commissuralaxons en route to the floor plate, and on axons and growthcones of commissural neurons growing in vitro in responseto netrin-1.

That netrin-1 binds to DCC was established by incubat-ing DCC-expressing cells with netrin-1 and then carryingout immunolocalization using antibodies to both proteins.In the presence of heparin to block non-specific binding,netrin is bound only by cells expressing DCC. Neogenin-expressing cells were also found to bind netrin. However,direct binding of netrin to DCC or neogenin in a cell-freesystem was not confirmed, leaving open the possibilitythat additional factors are required. DCC–netrin interac-tions were explored by applying anti-DCC antibodies tocultures in which commissural axons have been elicited bynetrin or by a nearby floor plate. Under both conditions,axon outgrowth is suppressed, suggesting that netrin-dependent outgrowth is induced via DCC. Perhaps sur-prisingly, chemoattraction assays showed that, in thepresence of anti-DCC antibodies, commissural axon traj-ectories still reoriented in proximity to a floor plate oraggregate of netrin-secreting cells (Fig. 2). It is unclearwhether this simply reflects a failure of the antibody topenetrate the tissue explant, or instead indicates that thereorienting activity of netrin-1 is mediated by a distinctreceptor. Analysis of DCC mutant mice could help resolvethis question.

Completing the symmetry of netrins and their receptorsacross animal groups comes the recent identification ofthe Drosophila gene frazzled, which encodes a proteinwith close homology to DCC and neogenin [18].Frazzled protein is localized on commissural and longitu-dinal axons in the central nervous system and on motoraxons in the periphery. In frazzled mutant embryos,

commissures are thinner than normal, echoing the phe-notypes of the netrin mutants; longitudinal tracts are alsosometimes interrupted and motor axon pathfinding isabnormal. As with nematode unc-40 mutants, the forma-tion of commissures can be restored by targeting frazzledexpression to neurons, but not muscles, of frazzledmutant embryos [18]. This cell-autonomous action,coupled with matching expression patterns of netrins andfrazzled on axon pathways and neurons, respectively,strengthens the case for frazzled being a netrin receptor.But in this study [18], as in the study on nematodes,binding of the netrin homologue to the DCC homologuewas not directly demonstrated.

Are there other chemoattractants?Dissecting pathways of DCC–netrin action still leavesunanswered questions. Netrin is capable in vitro of exert-ing both a growth-promoting and an orienting influenceon commissural axons growing in a collagen matrix. Yetabrogating netrin–DCC interactions in vitro, using eitheranti-DCC antibodies or netrin-deficient floor plates,impairs growth into the matrix but not chemoattraction(Fig. 2). Similarly, floor plates from netrin-deficient miceretain chemoattractant (and chemorepellent) properties.

One way of reconciling these data would be for netrin’saction to be exclusively trophic, with chemoattractantfunction depending on another molecule. Alternatively,additional chemoattractants might compensate for the lossof netrin function when it is experimentally abolished. Butin either case, the existence of DCC-independent path-ways is implied. Other evidence also points to additionalchemoattractants and chemorepellents, such as findingsthat axons of the posterior commissure, oculomotor andspinal motor axons respond to chemorepellent influencesof the floor plate but not of netrin ([19] and A. Varela-Echavarría, A. Tucker, AW Püschel and S.G., unpublishedobservations).

More roles for netrinsThe motor neuron phenotypes in Netrin and frazzledmutant flies provide intriguing evidence that, in additionto their established role in axon guidance at the midline,netrins are involved in motor axon navigation to specificmuscles [11]. In the vertebrate embryo, netrin is alsoexpressed in the dermamyotome [5], whereas DCC isexpressed by young motor neurons [17], perhaps implyingchemoattraction. Conflicting with this is the finding thatseveral cranial motor axon subpopulations are repelled bynetrins ([9] and A. Varela-Echavarría, A. Tucker, AWPüschel and S.G., unpublished observations). Thisparadox might be explained if changes in receptor profilescould modulate the responses of motor axons to netrinduring their developmental history, with chemorepulsionwithin the neural tube being followed by chemoattractionof growth cones in their target field.

Still missing is information about vertebrate homologuesof unc-5, whose possible role in chemorepulsion remains tobe unravelled. In nematodes, the responses of unc-5-expressing cells require the cooperation of unc-40, raisingthe question of how the functions of these moleculesmight combine to mediate growth-cone responses. Beyondthis, the expression of netrin and its receptors has not beenmapped in later development and more rostral regions ofthe vertebrate brain. The defects in commissures innetrin-deficient mice suggests that the story of netrins andaxon guidance may be only just beginning.

AcknowledgementsMy thanks to A. Hacker, A. Lumsden and A. Varela-Echavarría for extremelyhelpful discussions on the manuscript.

References1. Keynes R, Cook GMW: Axon guidance molecules. Cell 1995,

83:161–169.2. Cajal SR: Histologie du système nerveux de l’homme et des

vertébres. 2 vols, L. Azoulay (trans). Madrid: Instituto Ramon y Cajaldel CSIC;1952–1955.

3. Tessier-Lavigne M, Placzek M, Lumsden AGS, Dodd J, Jessell TM:Chemotropic guidance of developing axons in the mammaliancentral nervous system. Nature 1988, 336:775–778.

4. Serafini T, Kennedy TE, Galko MJ, Mirzayan C, Jessell TM, Tessier-Lavigne M: The netrins define a family of axon outgrowth-promoting proteins homologous to C. elegans UNC-6. Cell 1994,78:409–424.

5. Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M: Netrins arediffusible chemotropic factors for commissural axons in theembryonic spinal cord. Cell 1994, 78:425–435.

6. Hedgecock EM, Culotti JG, Hall DH: The unc-5, unc-6, and unc-40genes guide circumferential migrations of pioneer axons andmesodermal cells of the epidermis in C. elegans. Neuron 1990,2:61–85.

7. Ishii N, Wadsworth WG, Stern BD, Culotti JG, Hedgecock EM: UNC-6, a laminin-related protein,guides cell and pioneer axonmigrations in C. elegans. Neuron 1992, 9:873–881.

8. Wadsworth WG, Bhatt H, Hedgecock EM: Neuroglia and pioneerneurons express UNC-6 to provide global and local netrin cuesfor guiding migrations in C. elegans. Neuron 1996, 16:35–46.

9. Colamarino SA, Tessier-Lavigne M: The axonal chemoattractantnetrin-1 is also a chemorepellent for trochlear motor axons. Cell1995, 81:621–629.

10. Serafini T, Colamarino SA, Leonardo ED, Wang H, Beddington R,Skarnes WC, Tessier-Lavigne M: Netrin-1 is required forcommissural axon guidance in the developing vertebrate nervoussystem. Cell 1996, 87:1001–1014.

11. Mitchell KJ, Doyle JL, Serafini T, Kennedy TE, Tessier-Lavigne M,Goodman CS, Dickson BJ: Genetic analysis of Netrin genes inDrosophila: Netrins guide CNS commissural axons and peripheralmotor axons. Neuron 1996, 17:203–215.

12. Harris R, Sabatelli LM, Seeger MA: Guidance cues at theDrosophila CNS midline: identification and characterization of twoDrosophila netrin/UNC-6 homologs. Neuron 1996, 17:217–228.

13. Leung-Hagesteijn C, Spence AM, Stern BD, Zhou Y, Su M-W,Hedgecock EM, Culotti JG: UNC-5, a transmembrane protein withimmunoglobulin and thrombospondin type 1 domains, guides celland pioneer axon migrations in C. elegans. Cell 1992,71:289–299.

14. Chan SS-Y, Zheng H, Su M-W, Wilk R, Killeen MT, Hedgecock EM,Culotti JG: UNC-40, a C. elegans homolog of DCC (Deleted inColorectal Cancer) is required in motile cells responding to UNC-6 netrin cues. Cell 1996, 87:187–196.

15. Fearon ER, Cho KR, Nigro JM, Kern SE, Simons JW, Ruppert JM,Hamilton SR, Preisinger AC, Thomas G, Kinzler KW, Vogelstein B:Identification of a chromosome 18q gene that is altered incolorectal cancers. Science 1990, 247:49–56.

16. Vielmetter J, Kayyem JF, Roman JM, Dreyer WJ: Neogenin, an aviancell surface protein expressed during terminal neuronaldifferentiation, is closely related to the human tumor suppressormolecule Deleted in Colorectal Cancer. J Cell Biol 1994,127:2009–2020.

17. Keino-Masu K, Masu M, Hinck L, Leonardo ED, Chan SS-Y, CulottiJG, Tessier-Lavigne M: Deleted in Colorectal Cancer encodes anetrin receptor. Cell 1996, 87:175–186.

18. Kolodziej PA, Timpe LC, Mitchell KJ, Fried SR, Goodman CS, Jan LY,Jan YN: frazzled encodes a Drosophila member of the DCCimmunoglobulin subfamily and is required for CNS and motoraxon guidance. Cell 1996, 87:197–204.

19. Shirasaki R, Mirzayan C, Tessier-Lavigne M, Murakami F: Guidance ofcircumferentially-growing axons by netrin-dependent andindependent floor plate chemotropism in the vertebrate brain.Neuron 1997, in press.

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