Genetic analysis of growth cone migrations in Caenorhabditis elegans

Genetic Analysis of Growth Cone Migrations inCaenorhabditis elegans

David C. Merz and Joseph G. Culotti

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and Department of Medical andMolecular Genetics, Faculty of Medicine, University of Toronto, Toronto, Canada M5G 1X5

Received 20 April 2000; accepted 25 April 2000

ABSTRACT: Model organisms likeCaenorhabditiselegansallow the study of growth cone motility andguidance in vivo. We are using circumferential axonguidance in C. elegansto study both the mechanisms ofguidance and the interactions between different guid-ance systemsin vivo. A genetic screen has identifiedsuppressors of the specific axon guidance defects causedby ectopic expression of UNC-5, the repulsive receptorfor the UNC-6/netrin guidance cue. These mutationsidentify eight genes whose products are required for thefunction of UNC-5 in these cells. In principle, the func-tions of some of these genes may involveunc-73, which

encodes a multidomain, cytoplasmic protein that is anactivator of the rac and rho GTPases. Loss of UNC-73causes errors in axon guidance, and it is hypothesizedthat UNC-73 acts in multiple signaling pathways used byguidance receptors on the growth cone surface to regu-late the underlying cytoskeleton. Here we summarizeand discuss these recent developments that are advanc-ing our understanding of growth cone signal transduc-tion in vivo. © 2000 John Wiley & Sons, Inc. J Neurobiol 44: 281–288,

2000

Keywords:growth cone; UNC-73; signal transduction;Caenorhabditis elegans

The neuronal growth cone (GC) leads the extendingaxon to its appropriate target, often over long dis-tances involving changes in direction and substrate.Similar to the leading edge of a migrating cell, the GCsends out finger-like projections called filopodia andsheet-like projections called lamellipodia. Thesestructures contain dense and highly polymerized actinmicrofilaments. Filopodial and lamellipodial projec-tions are dynamic and reflect the patterns of actinpolymerization and depolymerization, extending andretracting, attaching and detaching from the substrate.In response to cues that they encounter in the envi-ronment, these projections are stabilized or destabi-lized to varying degrees. Stabilization allows force tobe generated by the cytoskeleton against the substrate,

and this results in movement of the GC in the direc-tion of that particular filopodium or lamellipodium.Conversely, local destabilization favors movement ina different direction. The nature of the local responseswithin the GC depends on the repertoire of signaltransduction molecules and cell surface receptors thatare present within the GC (Hamelin et al., 1993).

CAENORHABDITIS ELEGANS AND THEGROWTH CONE

The nematodeC. eleganshas a relatively simple ner-vous system. The adult hermaphrodite has only 302neurons, and the entire pattern of connectivity isknown from serial electron micrographs (White et al.,1986). Long nerve tracts are positioned along eitherthe longitudinal or the circumferential axes, and mostC. elegansneurons exhibit little or no branching ofaxons. Thus, deviations from a normal nervous struc-ture may be readily detected. The development of

Correspondence to:J. G. Culotti ([email protected]).Contract grant sponsor: MRC (JGC).Contract grant sponsor: NCIC (JGC).Contract grant sponsor: Spinal Cord Research Foundation

(JGC).© 2000 John Wiley & Sons, Inc.

281

green fluorescent protein (GFP) reporter constructshas allowed the visualization of neuronal morpholo-gies and of migrating GCs in living animals (Knobelet al., 1999). In the future, it may be feasible toobserve directly the distributions and interactions offluorophore-tagged proteins within migrating GCsinvivo. There are significant advantages to studyingbiological events as they occur in an intact animalrather than with simplifiedin vitro systems, and it isnow possible to carry out many of the assays normallydonein vitro using the nervous system ofC. elegans.

As a genetic model system, C. elegansoffers thepotential to isolate genetic mutations that disrupt abiological event like axon guidance and thus identifygene products essential for that event. Signaling part-ners within a pathway may be isolated through sub-sequent screens for modifier mutations (enhancers andsuppressors). Many axon guidance mutants have beenidentified through an uncoordinated locomotion phe-notype or by the direct visualization of a disruption inaxonal morphology (Brenner, 1974; Hedgecock et al.,1990; McIntire et al., 1992; Forrester and Garriga,1997; reviewed by Antebi et al., 1997). We are usingC. elegansto address basic issues in motility andguidance of neuronal GCsin vivo. The purpose of thisreview is to summarize some of the results and someof the questions that have arisen from these studies.

MECHANISMS OFMIGRATION/GUIDANCE

GC motility is based on the dynamic and coordinatedregulation of the cytoskeleton and of adhesion to thesubstrate. Signaling pathways that provide positionalor directional input to the GC have many potentialpoints of access to this motility machinery.In vivoandin vitro studies have pointed to the small GTPasesof the Rho family, including rho, rac, and cdc42, askey regulators of local changes in the actin cytoskel-eton during cell or GC migration, cell shape changes,or cytokinesis (Nobes and Hall, 1995). InC. elegans,the novel Rho family member MIG-2 is involved inseveral cell migration events and activated alleles ofmig-2disrupt, in addition to cell migrations, the guid-ance of some GC migrations (Zipkin et al., 1997). Invertebrate systems, many potential downstream effec-tors of the Rho GTPase molecules have been identi-fied biochemically. Genomic sequencing has revealedthat putative homologues of most of these candidateeffectors are present in theC. elegansgenome. How-ever, with some exceptions (Wissman et al., 1997) thein vivo roles of most of these remain unclear. Inaddition, the mechanisms of upstream regulation of

the Rho family GTPases, particularly their proposedactivation/inhibition by cell surface receptors, arelargely unknown.

The unc-73gene was originally identified throughmutations that cause an Unc (uncoordinated) pheno-type. Subsequent analysis showed that mutations inunc-73 can affect a variety of developmental pro-cesses, including cell migration and embryonic hypo-dermal morphogenesis (Garriga and Stern, 1994;Chen et al., 1997; Wissman et al., 1999). The Uncphenotype, however, is due mainly to defects in theguidance of axonal growth cones. In contrast to thedefects associated with the loss of components ofspecific guidance systems, many different types ofaxon guidance errors are observed inunc-73mutants(McIntire et al., 1992). For example, unlike mutationsaffecting the UNC-6/netrin pathway (see below),which affect circumferential but not longitudinal mi-grations,unc-73mutations disrupt GC guidance alongboth axes. This suggests that the protein encoded byunc-73plays a general role in GC guidance or motil-ity, possibly contributing to signaling through multi-ple guidance system pathways. The various cellulardefects observed inunc-73mutants in addition to theGC guidance errors suggest roles in cell motility orpolarity, possibly through regulation of the cytoskel-eton.

Cloning revealed thatunc-73encodes several con-served, cytoplasmic proteins related to the vertebrateTrio (Debant et al., 1996), Kalirin (Alam et al., 1997)and Duet (Kawai et al., 1999) proteins. As shown inFigure 1, the largest isoform of UNC-73 (UNC-73A)contains several types of potential protein–proteininteraction domains, including spectrin repeats, anSH3 domain, and immunoglobulin (Ig)- and fibronec-tin type III (FNIII)-like domains. In addition, there aretwo guanine nucleotide exchange factor (GEF) cata-lytic domains called Dbl-homology (DH) domains(Steven et al., 1998). Each DH domain is situatedimmediately adjacent to a pleckstrin homology (PH)domain. PH domains typically mediate binding tophosphoinositides, which may be involved in mem-brane localization or in regulation of DH domainactivity (Lemmon et al., 1997; Nimnual et al., 1998).One of the DH domains (DH1) has exchange factoractivity specific for activation of the rac GTPase andthe other (DH2) for the rho GTPase (Steven et al.,1998; R. Steven, T. J. Kubiseski, J. G. Culotti, and A.Pawson, unpublished data). As evidence of the im-portance of the GEF activity in UNC-73 functioninvivo, a missense mutation that eliminates the Rac-specific GEF activity of DH1 was identified in thestrong hypomorphicunc-73(rh40)allele (Steven etal., 1998). Consistent with the mutant phenotypes in

282 Merz and Culotti

C. elegansthat suggested a defect in cytoskeletalregulation, expression of a peptide containing theDH1 domain in cultured mammalian cells causedmembrane ruffling, indicative of positive stimulationof the actin cytoskeleton (Steven et al., 1998).

The unc-73gene is expressed by neurons duringaxon extension and is required cell autonomously foraxon guidance functions. Thus, we believe thatUNC-73 is involved in the reception or the transduc-tion of guidance information within the GC, passinginformation from transmembrane receptors to rac andrho, which subsequently direct the actin-mediated ex-tension or retraction of filopodia and lamellipodia.This scenario is also consistent with the other cellpolarity and motility defects observed inunc-73mu-tants.

It is important to note that, even in putative nulls orin alleles such asunc-73(rh40)in which all rac GEFactivity is eliminated, most of the axon guidancedefects ofunc-73mutants have incomplete penetranceor expressivity. This indicates that GCs do not requireabsolutely the function of UNC-73 for motility or formost guidance functions. This is presumably due tothe presence of parallel signaling pathways, i.e., otheractivators of rac and rho that can partially compensate

for a loss of UNC-73 activity. Alternatively, theremay be rho- and rac-independent pathways for cy-toskeletal regulation. It is probable that UNC-73 func-tions more as part of a cytoplasmic signaling networkthan as part of a simple linear pathway.

A comparison of the phenotypes caused by loss-of-function versus activating mutations in the Rhofamily member encoded bymig-2 suggests that thereis redundancy at the level of the GTPases, becauseactivated alleles cause more severe defects in axonguidance than do null alleles (Zipkin et al., 1997). Thecomplete sequence of theC. elegansgenome hasrevealed the presence of several genes encoding po-tential Rho family GEF proteins that could partiallycompensate for the loss of UNC-73. The low expres-sivity of unc-73axon guidance defects suggests ge-netic strategies for usingunc-73mutants as sensitizedgenetic backgrounds to identify other signaling com-ponents in the growth cone. For example, geneticscreens for mutations that enhance the severity of therelatively weak axon guidance defects ofunc-73might identify genes critical for parallel signalingpathways. Alternatively, genetic screens for suppres-sors ofunc-73may identify genes whose products liewithin or in parallel to theunc-73pathway.

The pleiotropic phenotypes caused by mutations inGC signaling components like UNC-73 or MIG-2underscore the commonality of the mechanisms bywhich cells regulate the actin cytoskeleton. Thus, thesame proteins, and possibly the same cytoplasmicsignaling pathways, are utilized by various tissues atdifferent times in development to regulate cell shape,polarity, and motility as well as GC guidance. Thesemaphorin family of secreted proteins is best knownfor its roles in the directional guidance of GCs. How-ever, the most significant defects caused by loss of thesemaphorin II homologuemab-20 in C. elegansin-volve hypodermal morphogenesis (Roy et al., 2000).

CIRCUMFERENTIAL AXON GUIDANCE

The secreted UNC-6 protein, a member of the lami-nin-related UNC-6/netrin family of guidance cues,plays a key role in guiding circumferential (dorsoven-tral) cell and growth cone migrations along the innersurface of the hypodermis ofC. elegans(Hedgecocket al., 1990; Ishii et al., 1992). There are two subtypesof transmembrane receptors for UNC-6/netrin pro-teins: UNC-40/DCC (Chan et al., 1996; Keino-Masuet al., 1996) and UNC-5 (Leung-Hagesteijn et al.,1992; Leonardo et al., 1997). GCs expressing bothUNC-5 and UNC-40 transmembrane receptors arerepelled away from ventrally expressed UNC-6,

Figure 1 Functional domains of the UNC-73 cytoskeletalregulator. The UNC-73A isoform is predicted to contain atleast six distinct protein domains. Eight spectrin-like repeatsare located at the N-terminus. In addition, there is an SH3-like domain, which may mediate binding to proline-richregions of other proteins. Other putative protein–proteininteraction domains of unknown function include one eachrelated to Ig and FNIII domains. The Dbl-homology do-mains DH1 and DH2 can catalyze GDP/GTP exchange forrac and rho, respectively, thereby regulating the extensionof filopodia and lamellipodia. Each DH domain is adjacentto a pleckstrin homology domain (PH1 and PH2). Thesemay be involved in membrane localization or in regulationof DH domain activity by PH domain-binding phosphoi-nositides (PI).

Growth Cone Genetics 283

whereas those expressing UNC-40 but not UNC-5 areattracted toward UNC-6 or are insensitive to it.

The critical role of UNC-5 in dictating a repulsiveresponse to the UNC-6 cue was shown by ectopicexpression of UNC-5 in neurons that normally ex-press only the UNC-40 receptor subtype and whoseGCs are normally attracted to UNC-6 or do not ex-hibit a response to it. The touch sensory neurons havelaterally positioned cell bodies and extend axonsalong the inner surface of the hypodermis either lon-gitudinally or, as with the AVM neuron shown sche-matically in Figure 2, ventrally (toward UNC-6) andthen longitudinally. The ectopic expression of UNC-5in these neurons caused the dorsalward projection oftheir axons in an UNC-6–dependent manner (Hame-lin et al., 1993). Consistently, touch neuron axonsextended in a dorsalward direction immediately fromthe cell soma and turned in their normal longitudinaldirection upon reaching the dorsal nerve cord.

A genetic screen for suppressors of the abnormalaxonal trajectories caused by ectopic expression ofUNC-5 identified eight genes whose products arerequired for the function of UNC-5 in this assay (Fig.2; Colavita and Culotti, 1998). These included theknown genesunc-6, unc-40, unc-34, andunc-44, and

four novel mutants:unc-129, seu-1, seu-2,andseu-3.Because this genetic screen involved the reversion ofabnormal axonal projections back to more normalprojections, it would not be expected to identify mu-tants that disrupt GC motility or guidance in a generalfashion. Rather, this type of screen is expected toidentify genes whose products affect more specificallythe function of UNC-5 or other dorsoventral guidancesystem components. The characterization of the mu-tants thus isolated is expected to help elucidate themechanism by which UNC-5 expression causes arepulsive response to the UNC-6 guidance cue.

unc-44encodes a protein related to ankyrin (Ot-suka et al., 1995), which in vertebrates forms specificconnections between transmembrane proteins and theactin cytoskeleton (Bennett, 1992).unc-44mutants inC. eleganshave previously been reported to exhibitaxon guidance defects (McIntire et al., 1992). Thesedefects only partially overlap with those ofunc-5,although they do more closely resemble those causedby unc-40mutations (McIntire et al., 1992). Interest-ingly, in both UNC-44 and the cytoplasmic region ofUNC-5, there is a predicted protein–protein interac-tion domain called a Death Domain (Hofmann andTschopp, 1995; Ponting et al., 1999). The Death Do-mains characterized in proteins of apoptotic signalingpathways can exhibit either homophilic or hetero-philic binding. By analogy, through direct interactionswith UNC-5, UNC-44 could assist in holding theUNC-5 receptor in a functional complex with othersignaling or cytoskeletal components. UNC-5 andUNC-44 could compete for some other Death Domainbinding partner, although this result is more specula-tive. If UNC-44/ankyrin normally acts to stabilizeinteractions between transmembrane adhesion mole-cules and the cytoskeleton, antagonism by UNC-5may provide a mechanism by which the repellantUNC-5 receptor (when bound to the UNC-6 ligand)can destabilize such connections.

unc-129mutants on their own exhibit defects indorsalward axonal projection that resemble thosecaused byunc-6 or unc-5 mutations. However, theunc-129defects are less severe than null mutants inunc-6 or unc-5. Cloning revealed thatunc-129en-codes a member of the transforming growth factor(TGF)-b/bone morphogenic protein (BMP) family ofgrowth factors (Colavita et al., 1998). Reporter con-struct analysis indicates that it is expressed by dorsalbut not ventral body wall muscles and that this dif-ferential expression is critical for its guidance func-tion (Colavita et al., 1998). Ectopic expression ofUNC-129 by ventral muscles causes axon guidancedefects as severe as anunc-129loss-of-function mu-tation, as well as cell migration defects similar to

Figure 2 Guidance of the AVM neuron GC by the UNC-6/netrin guidance cue and the UNC-40 and UNC-5 receptorsubtypes. AVM neurons normally express the UNC-40 butnot the UNC-5 receptor subtype. AVM GCs migrate ven-trally to the ventral nerve cord, then turn anteriorly andmigrate within the ventral nerve cord to the nerve ring. Thenormal ventralward migration phase depends on ventrallyexpressed UNC-6 and other, unknown guidance cues. Ec-topic expression of the UNC-5 receptor in AVM causesGCs to migrate in a dorsalward direction to the dorsal nervecord. The subsequent longitudinal extension is unaffected.Mutations inunc-6, unc-40, unc-34, unc-44, unc-129, seu-1,seu-2, or seu-3suppress the dorsalward migrations, indicat-ing that the products of these genes are required for UNC-5function in this neuron. The presence of UNC-5, even whenunable to cause dorsalward repulsion, is sufficient to inter-fere with the ventralward migration phase.


those caused byunc-5,6,40mutations (Colavita et al.,1998).

Not all of the genes identified as required forUNC-5 function in the touch neurons are so stronglyrequired for UNC-5 function in other cells. UNC-34,UNC-44, and UNC-129 are all, like UNC-6 andUNC-5, involved in the formation of commissuralaxonal projections. However, loss of function muta-tions in the genes encoding these proteins do notproduce commissural axon defects as severe as mu-tations inunc-6or unc-5 (McIntire et al., 1992; Co-lavita et al., 1998) and do not disrupt cell migrationsin the same manner asunc-5mutations (Hedgecock etal., 1987). Other suppressors such asseu-1, seu-2,andseu-3exhibit only minor axon guidance defects ontheir own. Yet they are strongly required for theaxonal misrouting caused by ectopic expression ofUNC-5 in the touch neurons. There are at least twopossible reasons for this discrepancy. First, the touchneurons are responsive to cues other than UNC-6,which provide the directional information needed forthe normal longitudinal and ventralward projectionsof these neurons. Ectopically expressed UNC-5 mustcompete with these other endogenous guidance sys-tems to cause dorsalward projections. This acts tosensitize the systems to subtle weakenings of theefficacy of the UNC-5 signaling pathway. Thus, if amutation inunc-34enfeebles the UNC-5 signal trans-duction mechanism, the competing pathways maynow override or out-compete whatever UNC-5 sig-naling remains, thereby suppressing the UNC-5–in-duced dorsalward axonal projections. In support ofthis explanation, mutations inunc-6, which are nor-mally fully recessive for axon guidance defects, canact as dominant suppressors of the function of UNC-5ectopically expressed in the touch neurons (Colavitaand Culotti, 1998). By the same reasoning, the prod-ucts of these suppressor genes need not act directly inan UNC-5 signaling pathway, but could be part of aparallel pathway that contributes to dorsalward guid-ance.

A second explanation for the identification of thesesuppressors of the function of UNC-5 in the touchneurons is that the GCs of the touch neurons may notexpress, or may not express in a functionally appro-priate form, all of the signaling components necessaryfor a complete UNC-5–mediated response. In supportof the latter idea is the finding that UNC-40 is re-quired for the UNC-5–mediated response in the touchneurons. In contrast, cells and growth cones that nor-mally express and respond to UNC-5 do not abso-lutely require UNC-40 for that response (Hedgecocket al., 1990). Thus, we propose that the touch neuronslack some component(s) that would normally contrib-

ute (e.g., in commissural motorneurons) to UNC-5–mediated repulsion from UNC-6 and that this resultsin a heightened dependence on UNC-40 and UNC-34,for example, in these cells.

An interesting observation from the study of thisectopic expression system is that UNC-5, even whenunable to drive dorsalward GC migrations (i.e., in thepresence of anunc-34 mutation), still almost com-pletely blocked the normal ventralward projections ofneurons such as the AVM touch neuron (Fig. 2;Colavita and Culotti, 1998). Furthermore, the pen-etrance of this effect was much greater than thatcaused by mutations inunc-6or unc-40, so the pres-ence of UNC-5 is doing more in these circumstancesthan simply sequestering UNC-6 or UNC-40 andthereby disabling the UNC-6–mediated attractiveguidance system. Rather, UNC-5 (at presumed highlevels of expression in the touch neurons), even in theabsence of UNC-6, interferes with other guidancesystems that normally direct the AVM GC toward theventral nerve cord. However, longitudinal axonal ex-tensions are never disrupted in these animals, indicat-ing that this interference occurs only between circum-ferential guidance systems and spares longitudinalsystems.

NAVIGATION PROGRAMS

The expression of genes encoding guidance receptorsor possibly other signaling components will largelydetermine the nature of the responses of the GC to thedirectional cues that it encounters. Thus, the nucleusof the neuron provides the GC with a “navigationprogram” (i.e., it instructs the GC to exhibit particularresponses to guidance cues). The actual multistagepathway followed during migration and the timing ofchanges in GC behavior are dictated by the navigationprogram and by local signaling within the GC, possi-bly by the dynamic activation and inhibition of guid-ance system components.

All of the ventral cord motorneurons inC. elegansthat are repelled dorsally by the UNC-6 guidance cueexpress both the UNC-5 and UNC-40 transmembranereceptors (Chan et al., 1996; Su et al., 2000). Incontrast, the motorneurons whose axons remainwithin the ventral cord express UNC-40 but notUNC-5 (Chan et al., 1996). Thus, expression ofUNC-5 by a neuron is sufficient to cause dorsalwardaxonal guidance. But how many genes must be ex-pressed to ensure that a GC migrates dorsally and notventrally? Are there groups of genes that are typicallyexpressed together to cause migration in a specificdirection, or is one gene sufficient? Does each guid-


ance system require a distinct navigation program?We have recently cloned theseu-1gene, whose prod-uct is required for the function of ectopically ex-pressed UNC-5 within the touch neurons, and thepredicted product of this gene is a novel nuclear-localized protein that acts within the touch neurons(H. Zheng, J. G.Culotti, and D. C. Merz, unpublishedresults). From the nucleus, SEU-1 appears to controlthe sensitivity of the neuronal growth cone to UNC-6.Further characterization of this gene should revealsome of the nature of the touch neuron navigationprogram.

CONCLUSIONS

Multiple guidance cues provide directional informa-tion to GCs, and these are likely to be at least partiallyredundant to one another. In circumferential guidancein C. elegans, the most important guidance cue ap-pears to be UNC-6, which is produced by epidermo-blasts and neurons primarily in the ventral side of theworm (Wadsworth et al., 1996). However, null muta-tions inunc-6do not completely eliminate migrationstoward or away from the ventral midline. This indi-cates that other, UNC-6–independent systems mustalso be providing directional information to GCs. Tounderstand the interactions between guidance systemsin vivo, it will be necessary to identify all of thesedirectional inputs.

It is not known how the TGF-b–related UNC-129functions in growth cone guidance or whether itsactions are partially or completely independent of theUNC-6 guidance system. Identifying downstream sig-naling components such as receptors for UNC-129will help to clarify its role. It is notable that, incontrast tounc-129, mutations in genes encodingother TGF-b signaling pathway components, includ-ing type I and type II receptors, do not cause axonaldefects (Colavita et al., 1998). UNC-129 may actthrough some atypical signaling mechanism. To iden-tify novel signaling partners, we are carrying outgenetic screens using ectopic UNC-129–expressinglines. Because these lines are more severely defectivefor some phenotypes than forunc-129null alleles, itshould be possible to isolate mutations that partiallysuppress the phenotype of the ectopic-expressinglines. These mutations may identify genes that encodeproteins used to transduce directional informationfrom UNC-129.

Another circumferential guidance system inC. el-egans is that of SAX-3/Robo, an immunoglobulinsuperfamily member that, in flies and vertebrates, is areceptor for the secreted Slit family of ligands (Brose

et al., 1999; Kidd et al., 1999; Li et al., 1999). LikeUNC-6 and UNC-40, SAX-3 inC. elegansis involvedin the guidance of axonal projections toward the ven-tral nerve cord in regulating crossing of the ventralmidline (Zallen et al., 1998). Effects ofsax-3mutantsof the touch neurons have not yet been described. Inaddition, the effects of mutations in aC. elegansSlithomologue(s) have not been reported. However, thisguidance system may also contribute, in parallel to theUNC-6 system, to the circumferential guidance oftouch neuron growth cones.

New technical developments often necessitate therevisiting of old ideas. A recent study used confocalmicroscopy to observe GCs of one class of commis-sural neurons as they extended from the ventral to thedorsal nerve cord (Knobel et al., 1999). This studyrevealed a surprising degree of complexity in GCbehavior, including stalling and collapsing, as theGCs carried out this seemingly simple migration.Rather than being limited to dealing with GC behav-iour as simply attraction or repulsion, we will now beable to assign more specific roles to proteins (such asUNC-73) involved in GC guidance or motility byidentifying more precisely the migration event that isdefective in mutant backgrounds. For example, two ofthose sites at which commissural GCs pause in theirmigrations and spread into anvil-shaped structuresbefore continuing dorsally (Knobel et al., 1999) cor-respond to locations of cells that expressunc-129reporter genes: lateral hypodermal and dorsal musclecells (Colavita et al., 1998). It must now be deter-mined whether UNC-129, which is involved in theseventral-to-dorsal GC migrations, is involved in any ofthese specific GC behaviors. Future studies of axonguidance errors inC. eleganswill have to considereach GC migration as a series of events, a defect inany one of which could result in an axon guidanceerror.

There is a considerable quantity of informationabout the biochemical interactions between signalingcomponents likely to play important roles in GC mo-tility and guidance. A critical problem in the futurewill be to understand the function of these proteinsinvivo in the developing nervous system. For example,where in the cell do guidance proteins carry out theirfunctions, and how can these functions be attributedto spatially localized protein–protein interactions?Genetic model organisms, includingC. elegansandDrosophila, are uniquely suited to this task. A syn-thesis ofin vitro biochemical andin vivo genetic datawill allow a complete picture of GC function.

The authors thank Drs. N. Levy-Strumpf, R. Steven, andT. J. Kubiseski for comments of the manuscript.


REFERENCES

Alam MR, Johnson RC, Darlington DN, Hand TA, MainsRE, Eipper BA. 1997. Kalirin, a cytosolic protein withspectrin-like and GDP/GTP exchange factor-like do-mains that interacts with peptidylglycine alpha-amidatingmonooxygenase, an integral membrane peptide-process-ing enzyme. J Biol Chem 272:12667–12675.

Antebi A, Norris CR, Hedgecock EM. 1997. Cell andgrowth cone migrations inC. elegans.II. Cold SpringHarbor Laboratory Press. p 583–609.

Bennett V. 1992. Ankyrins: adaptors between diverseplasma membrane proteins and cytoplasm. J Biol Chem267:8703–8706.

Brenner S. 1974. The genetics ofCaenorhabditis elegans.Genetics 77:71–94.

Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Good-man CS, Tessier-Lavigne M, Kidd T. 1999. Slit proteinsbind Robo receptors and have an evolutionarily con-served role in repulsive axon guidance. Cell 96:795–806.

Chan SS-Y, Zheng H, Su M-W, Wilk R, Killeen MT,Hedgecock EM, Culotti JG. 1996. UNC-40, aC. eleganshomolog of DCC (Deleted in Colorectal Cancer), is re-quired in motile cells responding to UNC-6 netrin cues.Cell 87:187–195.

Chen EB, Branda CS, Stern MJ. 1997. Genetic enhancers ofsem-5define components of the gonad-independent guid-ance mechanism controlling sex myoblast migration inCaenorhabditis eleganshermaphrodites. Dev Biol 182:88–100.

Colavita A, Culotti JG. 1998. Suppressors of ectopic UNC-5growth cone steering identify eight genes involved inaxon guidancein Caenorhabditis elegans. Dev Biol 194:72–85.

Colavita A, Krishna S, Zheng H, Padgett R, Culotti JG.1998. Pioneer axon guidance by UNC-129, aC. elegansTGF-beta. Science 281:706–709.

Debant A, Serra-Pages C, Seipel K, O’Brien S, Tang M,Park S-H, Streuli M. 1996. The multidomain protein Triobinds the LAR transmembrane protein-tyrosine phospha-tase contains a protein kinase domain, and has separaterac-specific and rho-specific guanine nucleotide exchangefactor domains. Proc Natl Acad Sci USA 93:5466–5471.

Forrester WC, Garriga G. 1997. Genes necessary forC.eleganscell and growth cone migrations.Development124:1831-1843.

Garriga G, Stern M.J. 1994. Hams and Egls: genetic anal-ysis of cell migration in Caenorhabditis elegans. Curr.Opin. Genet. Dev. 4:575–580.

Hamelin M, Zhou Y, Su M-W, Scott IM, Culotti JG. 1993.Expression of the UNC-5 guidance receptor in the touchneurons ofC. eleganssteers their axons dorsally. Nature364:327–330.

Hedgecock EM, Culotti JG, Hall DH. 1990. Theunc-5,unc-6, andunc-40genes guide circumferential migrationsof pioneer axons and mesodermal cells on the epidermisof C. elegans. Neuron 4:61–85.

Hedgecock EM, Culotti JG, Hall DH, Stern BD. 1987.

Genetics of cell and axon migrations inCaenorhabditiselegans. Development 100:365–382.

Hofmann K, Tschopp L. 1995. The death domain motiffound in Fas (Apo-1) and TNF receptor is present inproteins involved in apoptosis and axonal guidance.FEBS Lett 371:321–323.

Ishii N, Wadsworth, WG, Stern BD, Culotti JG, HedgecockEM. 1992. UNC-6, a laminin-related protein, guides celland pioneer axon migrations inC. elegans. Neuron9:873–881.

Kawai T, Sanjo H, Akira S. 1999. Duet is a novel serine/threonine kinase with Dbl-Homology (DH) and Pleck-strin-Homology (PH) domains. Gene 227:249–255

Keino-Masu K, Masu M, Hinck L, Leonardo ED, ChanSS-Y, Culotti JG, Tessier-Lavigne M. 1996. Deleted inColorectal Cancer (DCC) encodes a netrin receptor. Cell87:175–185.

Kidd T, Bland KS, Goodman CS. 1999. Slit is the midlinerepellent for the robo receptor inDrosophila. Cell 96:785–794.

Knobel K, Jorgensen EM, Bastiani M. 1999. Growth conesstall and collapse during axon outgrowth inCaenorhab-ditis elegans. Development 126:4489–4498.

Lemmon MA, Falasca M, Ferguson KM, Schlessinger J.1997. Regulatory recruitment of signalling molecules tothe cell membrane by pleckstrin-homolgy domains.Trends Cell Biol 7:237–242.

Leonardo ED, Hinck L, Masu M, Keino-Masu K, AckermanSL, Tessier-Lavigne M. 1997. Vertebrate homologues ofC. elegansUNC-5 are candidate netrin receptors. Nature386:833–838.

Leung-Hagesteijn C, Spence AM, Stern BD, Zhou Y, SuM-W, Hedgecock EM, Culotti JG. 1992. Unc-5, a trans-membrane protein with immunoglobulin and throm-bospondin type 1 domains, guides cell and pioneer axonmigrations inC. elegans. Cell 71:289–299.

Li HS, Chen JH, Wu W, Fagaly T, Zhou L, Yuan W, DupuisS, Jiang ZH, Nash W, Gick C, Ornitz DM, Wu JY, RaoY. 1999. Vertebrate slit, a secreted ligand for the trans-membrane protein roundabout, is a repellent for olfactorybulb axons. Cell 96:807–818.

McIntire SL, Garriga G, White J, Jacobson D, Horvitz HR.1992. Genes necessary for directed axonal elongation orfasciculation inC. elegans. Neuron 8:307–322.

Nimnual AS, Yatsula BA, Bar-Sagi D. 1998. Coupling ofRas and Rac guanosine triphophatases through the Rasexchanger Sos. Science 279:560–563.

Nobes CD, Hall A. 1995. Rho, rac, and cdc42 GTPasesregulate the assembly of multimolecular focal complexesassociated with actin stress fibers, lamellipodia, and filop-odia. Cell 8:53–62.

Otsuka AJ, Franco R, Yang B, Shim K-H, Tang LZ, ZhangYY, Boontrakulpoontawee P, Jeyaprakash A, HedgecockEM, Wheaton VI, Sobery A. 1995. An ankyrin-relatedgene (unc-44) is necessary for proper axonal guidance inCaenorhabditis elegans. J Cell Biol 129:1081–1092.

Ponting CP, Schultz J, Milpetz F, Bork P. 1999. SMART:identification and annotation of domains from signalling


and extracellular protein sequences. Nucleic Acids Res27:229–232.

Roy PJ, Zheng H, Warren CE, Culotti JG. 2000.mab-20encodes Semaphorin-2A and is required to prevent ec-topic cell contacts during epidermal morphogenesis inC.elegans. Development. 127:755–767.

Steven R, Kubiseski TJ, Zheng H, Kulkarni S, Mancillas J,Ruiz A, Hogue CWV, Pawson A, Culotti JG. 1998.UNC-73 activates the Rac GTPase and is required for celland growth cone migrations inC. elegans. Cell 92:785–795.

Su M-W, Merz DC, Killeen MT, Zhou Y, Zheng H, KramerJ, Hedgecock EM, Culotti JG. 2000. Regulation of theUNC-5 netrin receptor initiates the first re-orientation ofmigrating distal tip cells inC. elegans. Development.127:585–594.

Wadsworth WG, Bhatt H, Hedgecock EM. 1996. Neurogliaand pioneer neurons express UNC-6 to provide globaland local netrin cues for guiding migrations inC. elegans.Neuron 16:35–46.

White JG, Southgate E, Thomson JN, Brenner S. 1986. The

structure of the nervous system of the nematodeCaeno-rhabditis elegans. Philos Trans R Soc Lond B Biol Sci314:1–340.

Wissmann A, Ingles J, McGhee JD, Mains P. 1997.Cae-norhabditis elegansLET-502 is related to Rho-bindingkinases and human myotonic dystrophy kinase and inter-acts genetically with a homolog of the regulatory subunitof smooth muscle myosin phosphatase to affect cellshape. Genes Dev 11:409–422.

Wissman A, Ingles J, Mains PE. 1999. TheCaenorhabditiselegans mel-11myosin phosphatase regulatory subunitaffects tissue contraction in the somatic gonad and theembryonic epidermis and genetically interacts with therac signaling pathway. Dev Biol 209:111–127.

Zallen JA, Yi BA, Bargmann CI. 1998. The conservedimmunoglobulin superfamily member SAX-3/Robo di-rects multiple aspects of axon guidance inC. elegans.Cell 92:217–227.

Zipkin ID, Kindt RM, Kenyon CJ. 1997. Role of a new Rhofamily member in cell migration and axon guidance inC.elegans. Cell 90:883–894.


Documents

Genetic analysis of growth cone migrations in Caenorhabditis elegans