C. elegans VAB-8 and UNC-73 regulate the SAX-3receptor to direct cell and growth-cone migrations

Natsuko Watari-Goshima1, Ken-ichi Ogura2, Fred W Wolf1,4, Yoshio Goshima2,3 & Gian Garriga1

During nervous system development, a small number of conserved guidance cues and receptors regulate many axon trajectories.

How could a limited number of cues and receptors regulate such complex projection patterns? One way is to modulate receptor

function. Here we show that the Caenorhabditis elegans kinesin-related protein VAB-8L, which is necessary and sufficient for

posterior cell and growth-cone migrations, directs these migrations by regulating the levels of the guidance receptor SAX-3

(also known as robo). Genetic experiments indicate that VAB-8L and the Rac guanine nucleotide exchange factor activity of

UNC-73 (trio) increase the ability of the SLT-1 (slit) and UNC-6 (netrin) guidance pathways to promote posterior guidance.

The observations of higher SAX-3 receptor abundance in animals with increasing amounts of VAB-8L, and of physical interactions

between UNC-73 and both VAB-8L and the intracellular domain of the SAX-3, support a model whereby VAB-8L directs cell and

growth-cone migrations by promoting localization of guidance receptors to the cell surface.

The migrations of neuronal cell bodies and their growth cones con-tribute to the overall form and pattern of connectivity of nervoussystems. Attractive and repulsive molecules guide these neuronsand growth cones to their final destinations. These moleculesand their receptors are conserved among species as diverse asnematodes and mammals1,2. UNC-6 (netrin), for example, guidesthe ventral extensions of C. elegans, Drosophila melanogaster andvertebrate axons through its receptor UNC-40 (DCC)3–9. UNC-6(netrin) can also repel growth cones, acting through the con-served UNC-5 receptor. In C. elegans, the same UNC-6 gradient thatattracts growth cones ventrally also repels UNC-5–expressing growthcones dorsally10–12.

On reaching the midline, growth cones decide whether to cross it, adecision that is orchestrated by regulating guidance-receptor respon-siveness. As commissural axons approach the midline of the spinalcord, their ability to cross it is enabled by altering their responsivenessto netrin and slit. The formation of heteromers between the netrinreceptor DCC and the slit receptor robo inhibits the sensitivityof commissural growth cones to netrin13, and expression of thereceptor Rig-1 represses their sensitivity to slit, allowing axons tocross14. In Drosophila, a different mechanism allows axons to crossthe midline: expression of commissureless routes robo to the lysosomefor degradation15,16.

Once mouse commissural axons cross the midline, they respond to aWnt4 gradient, turning anteriorly toward higher concentrations of theWnt17. The frizzled receptor Fz3 appears to mediate this attractiveresponse. Several C. elegans Wnts are expressed in the tail and guide

migrating cells and growth cones forward18. The Wnt EGL-20 repelsthese axons via frizzled receptors.

In C. elegans, the gene vab-8 directs most posterior migrations. Thesemigrations require vab-8 (ref. 19), and ectopic expression of vab-8 canreroute anteriorly projected axons toward the posterior20. The vab-8locus encodes a kinesin-related isoform, VAB-8L, that acts in cells topromote the posterior migration of their growth cones20. How VAB-8Lregulates these migrations remains enigmatic.

In the present study, we find that VAB-8L regulates guidancereceptors. Mutations that reduce or eliminate the function of the Rhoand Rac guanine nucleotide exchange factor (GEF) UNC-73 (trio) andthe receptors SAX-3 (robo), UNC-5 (Unc5) and UNC-40 (DCC)suppress rerouting of the ALM process caused by VAB-8L misexpres-sion. We provide genetic evidence that VAB-8L regulates these receptorsvia the Rac GEF activity of UNC-73 and show that VAB-8L controls thelevels of the SAX-3 receptor. Finally, we find that the spectrin repeats ofUNC-73 can physically interact with both VAB-8L and the intracellulardomains of SAX-3, UNC-5 and UNC-40. Based on these findings andthose of Levy-Strumpf and Culotti21, we come to the surprisingconclusion that VAB-8L promotes the posterior migration of cells andgrowth cones by regulating the activity of guidance receptors that alsofunction in dorsal-ventral (DV) guidance. The additional finding thatthese effects require UNC-73 Rac GEF activity is equally surprising asRac GTPases are thought to function downstream of guidance receptorsin signal transduction. Although the DCC and robo receptors wereknown to be negatively regulated at the midline, these results providethe first evidence for a positive regulation of these receptors.

Received 14 November 2006; accepted 19 December 2006; published online 21 January 2007; doi:10.1038/nn1834

1Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, University of California, Berkeley, California 94720-3204, USA. 2Department ofMolecular Pharmacology and Neurobiology, Yokohama City University Graduate School of Medicine, Yokohama, 236-0004, Japan. 3CREST Japan Science and TechnologyCorporation Kawaguchi 332-0012, Japan. 4Current address: Ernest Gallo Clinic & Research Center, 5858 Horton St., Suite 200, Emeryville, California 94608, USA.Correspondence should be addressed to G.G. (garriga@berkeley.edu).

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RESULTS

VAB-8L is sufficient for posterior growth-cone migrations

The ALMs are bilaterally symmetrical mechanosensory neurons thatsense touch to the anterior body22. Each ALM extends a single anteriorprocess to near the tip of the head (Fig. 1) and occasionally a shortposterior process22,23. We refer to long extensions such as the anteriorones in wild-type animals as ‘processes’, and ignore short protrusionssuch as the posterior ones. We previously reported that when VAB-8L isexpressed in the ALMs, its processes are rerouted toward the posterior:VAB-8L is a long isoform that contains an N-terminal kinesin-likemotor domain and that promotes growth-cone and some cell migra-tions20. The mec-7 promoter drives expression in the ALM, AVM, PVMand PLM mechanosensory neurons (Fig. 1a). ALMs of strains thatcarried Pmec-7::vab-8L::gfp transgenes had a single posterior process63–72% of the time (Fig. 1c,e,h) and bipolar processes4–13% of the time (Fig. 1f–h).

The AVM and PVM neurons each project a single process ventrallyinto the ventral nerve cord, where they turn anteriorly and extend

toward the head22,23. In Pmec-7::vab-8L::gfp transgenic worms, AVMand PVM processes always extended normally to the ventral nerve cord,but occasionally they either branched in the cord, extending bothanterior and posterior processes, or turned posteriorly and extended asingle process to the tail (data not shown). The bilaterally symmetricPLMs each extend a short posterior process and a long anteriorprocess22,23. In Pmec-7::vab-8L::gfp worms, the anterior process wasusually shorter and the posterior process longer than in wild-typeworms (data not shown). Thus, ectopic expression of VAB-8L in all ofthese mechanosensory neurons can promote posterior outgrowth oftheir processes. Because the frequency of the ALM rerouting was easierto quantify than the effects on the A/PVM and PLM processes, wefocused on the ALM in the present study.

VAB-8L–dependent ALM rerouting requires UNC-73

To identify molecules that function with VAB-8L, we crossed differentmutations into the Pmec-7::vab-8L::gfp background and asked whetherthey affected the frequency of VAB-8L–dependent ALM rerouting. Wefocused on genes that were known to function in cell and growth-conemigrations and found that mutations in most of these genes had noeffect on rerouting frequency (Supplementary Table 1 online). Muta-tions in unc-73, by contrast, suppressed ALM rerouting (Fig. 2a).UNC-73 is a Rho guanine nucleotide exchange factor (RhoGEF) that ismost closely related to mammalian trio and kalirin and to Drosophilatrio24–26. UNC-73 and its orthologs possess two distinct RhoGEFdomains. The more N-terminal domain (RhoGEF-1) is specific forRac GTPases, and the second (RhoGEF-2) is specific for RhoGTPases26,27. unc-73 encodes eight protein isoforms with distinctactivities28. Isoforms that contain RhoGEF-1 function in axon gui-dance, whereas those that contain RhoGEF-2 have other functions27–29.Two mutations that are predicted to disrupt RhoGEF-1 isoformssuppressed ALM rerouting (Fig. 2a). The rh40 missense mutationis particularly revealing because it specifically eliminates Rac GEFactivity26, suggesting that one or more of the C. elegans Rac GTPasesare necessary for VAB-8L’s ability to reorient the direction of ALMprocess outgrowth. Suppression of rerouting by the hypomorphic allelee936 was significantly stronger than that by rh40 (Fig. 2a), indicatingthat Rac GEF activity is not the only UNC-73 function required forVAB-8L–dependent rerouting. Because mutations that eliminate thefunction of RhoGEF-2 containing isoforms result in extensive lethality,we were unable to assess whether these mutations resulted in a moresignificant suppression.

ALM rerouting requires guidance cues and their receptors

We were inspired to test the roles of guidance receptors in ALMrerouting by the findings of Levy-Strumpf and Culotti21 thatoverexpression of the DCC homolog UNC-40 could also causeALM rerouting and that VAB-8L–dependent ALM rerouting wassuppressed by unc-40 mutations. These investigators also found thatALM rerouting caused by UNC-40 overexpression was not suppressed

HeadALM

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a Figure 1 VAB-8L expression in the ALM neurons reroutes their processes

posteriorly. (a) Schematic diagram of the mechanosensory touch neurons

ALM, AVM, PVM and PLM. The ALM and PLM neurons are bilaterally

symmetric, and only one of each is drawn in the diagram. ALM has a long

anterior process in wild-type worms. (b–g) Epifluorescence (b,c,f) and

corresponding Nomarski (d,e,g) photomicrographs of zdIs5[Pmec-4::gfp];

gmEx177[Pmec-7::vab-8L::gfp] larvae. Large arrows indicate the positions

of the ALM cell bodies and the small arrowheads point to the ALM processes(b,c,f). The small arrows indicate the positions of PLM cell bodies in c and f.

(h) The frequencies of ALM process rerouting in strains that misexpress VAB-

8L. Scale bar, 20 mm. Error bars represent standard errors of proportions.

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by vab-8 mutations, placing vab-8 genetically upstream of unc-40. Wefound that mutations in unc-40 as well as in other genes encodingguidance cues and their receptors suppressed VAB-8L–dependent ALMrerouting. A mutation in sax-3, for example, suppressed reroutingby 27% (Fig. 2a). SAX-3 is the robo receptor ortholog and mediatesthe effects of the guidance cue SLT-1 (slit)30,31. Loss of SLT-1 caused aweaker suppression than loss of SAX-3 (Fig. 2a). This is notsurprising because the phenotypes of sax-3 mutants can be muchmore severe than those of slt-1 mutants, indicating either thatadditional SAX-3 ligands exist or that SAX-3 possesses ligand-independent functions30.

UNC-6 (netrin) is a conserved cue that guides migrating cells andgrowth cones along the dorsal-ventral (DV) axis. The strong loss-of-function unc-6 mutation ev400 suppressed VAB-8L–dependent ALMrerouting (Fig. 2a,b). There are two known UNC-6 receptors: UNC-5and UNC-40 (refs. 3,12). Whereas UNC-5 mediates UNC-6’s repellentactivity, UNC-40’s role is more complex: it is essential for UNC-6attraction, but also plays a role in UNC-6 repulsion11,32. These geneticobservations are consistent with studies demonstrating that vertebrateorthologs of UNC-5 and UNC-40 can form heteromers that mediatenetrin repulsion33. The unc-5 null mutation ju27 suppressed reroutingto the same extent as unc-6(ev400) (Fig. 2a), and this suppression wasconfirmed for two additional alleles of unc-5 (data not shown). We werenot able to generate worms that contained both the unc-40(e1430)mutation and our integrated Pmec-7::vab-8L::gfp transgene gmIs14,presumably because the combination caused lethality. We were able,however, to generate worms that carried unc-40(e1430) and the extra-chromosomal transgenic Pmec-7::vab-8L::gfp array gmEx177. Similar tothe results of Levy-Strumpf and Culotti21, unc-40(e1430) worms showed27% suppression of the ALM rerouting caused by gmEx177 (Fig. 2b).

We next asked whether the genes required for ALM rerouting act inthe same or distinct pathways. The logic behind these experiments isthat if two molecules act in the same pathway as guidance cue andreceptor, worms bearing mutations in both genes should not be moreseverely affected than worms bearing a single mutation. If, by contrast,the molecules act in distinct pathways, the double mutants shouldbe more severely affected. The sax-3 slt-1 double mutants showed the

same level of suppression as the sax-3 single mutant (Fig. 2a), suggest-ing that SLT-1 acts through SAX-3 to promote ALM rerouting.Similarly, the unc-5; unc-6 double mutants were no more suppressedthan the single mutants (Fig. 2a). These observations are consistentwith the canonical view that SAX-3 (robo) receptors mediate the effectsof SLT-1 (slit) cues and UNC-5 receptors mediate the effects of UNC-6(netrin) cues.

UNC-40 and SAX-3 heteromeric receptors have been proposed tomediate specific SLT-1 functions34, raising the possibility that SLT-1acts through an UNC-40 and SAX-3 receptor complex to mediate ALMrerouting. We tested this possibility by observing the effect of combin-ing unc-40 and slt-1 mutations in the Pmec-7::vab-8L::gfp background.Although the slt-1(eh15) mutation did not cause a significant suppres-sion of the rerouting caused by gmEx177, it did enhance the suppres-sion by unc-40(e1430) (Fig. 2b). Taken together with the finding thatSLT-1 functions through SAX-3 for rerouting, this result suggests thatUNC-40 and the SLT-1–SAX-3 pathway function in parallel. Levy-Strumpf and Culotti21 have shown that unc-40 mutations do notenhance the suppression of ALM rerouting caused by unc-6 mutations,indicating that UNC-6 acts through UNC-40 in this process.

High SAX-3 expression can induce ALM rerouting

Both VAB-8L and UNC-73 act cell-autonomously in regard to cellmigration and axon guidance20,26. To test whether SAX-3 functions inthe ALM to promote rerouting, we asked whether expression ofSAX-3 specifically in ALM could rescue the suppression caused by asax-3 mutation. Although the extrachromosomal transgenic Pmec-7::sax-3 array gmEx364 alone had no effect on the direction taken bythe ALM process, it rescued the suppression of VAB-8L–dependentALM rerouting caused by the sax-3 mutation, restoring the level ofrerouting to that seen in gmIs14 (Supplementary Fig. 1 online).

Because excess UNC-40 could induce ALM rerouting21, we testedwhether excess SAX-3 could also cause the ALM to reroute itsprocess posteriorly. We generated a second Pmec-7::sax-3 transgenicarray (gmEx335) by injecting ten times the concentration of thePmec-7::sax-3 construct used to produce gmEx364. The higher levelsof SAX-3 caused the ALMs to extend a single process posteriorly 3% of

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Figure 2 Mutations in genes encoding UNC-73, guidance cues and their receptors suppress VAB-8L–dependent ALM rerouting. (a) Suppression of VAB-8L–

dependent ALM rerouting by unc-73, slt-1, sax-3, unc-5 and unc-6 mutations. Asterisks denote statistically significant differences between gmIs14 worms

and gmIs14 worms carrying mutations in these genes. **P o 0.005, ***P o 0.0005. Levels of suppression by unc-73(e936) and unc-73(rh40), and by

slt-1(eh15) and sax-3(ky123), are significantly different (***P o 0.0005; *P o 0.05). n.s., not significant. (b) Suppression of VAB-8L–dependent ALM

rerouting by an unc-40 mutation. Asterisks denote statistically significant differences between gmEx177 and different gmEx177 strains. ***P o 0.0005; n.s.,

not significant. slt-1 enhances the suppression by unc-40 (*P o 0.05). The suppression by unc-6 is much stronger than that by unc-40 (***P o 0.0005). In

unc-73, slt-1, sax-3, unc-5, unc-6 and unc-40 mutants, the ALMs extend a single anterior process as in the wild type (data not shown). Error bars represent

standard errors of proportions.

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the time (Fig. 3) and produced bipolar processes 5% of the time(Fig. 3e–g). We observed no ALM rerouting in transgenic wormscarrying a Pmec-7::gfp transgene generated by injecting the construct atthe same concentration, suggesting that rerouting caused bygmEx335 was due to high levels of SAX-3 and not to the presence ofthe mec-7 promoter (Fig. 3g). We also observed ALM rerouting inPmec-7::sax-3::gfp transgenic worms (gmEx338) generated using thesame high concentrations of the construct (data not shown), and theintegrated version of this transgene (gmIs28) caused ALM rerouting22% of the time (Fig. 3g). Because of the higher frequency of ALMrerouting produced by gmIs28, we used it in subsequent experiments.

VAB-8L and UNC-73 function upstream of SAX-3

To order vab-8 and sax-3 genetically, we asked whether loss ofvab-8 could suppress SAX-3–dependent ALM rerouting and foundthat it could not (Fig. 3g). The finding that loss of sax-3 can suppressVAB-8L–dependent rerouting (Fig. 2a), but loss of vab-8 cannotsuppress SAX-3–dependent rerouting, suggests that vab-8 acts geneti-cally upstream of sax-3.

Similarly, we were able to order unc-73 and sax-3 genetically by askingwhether an unc-73 mutation was able to suppress SAX-3–dependentrerouting. The finding that unc-73 was required for VAB-8L–dependentbut not SAX-3–dependent ALM rerouting indicates that unc-73 actsgenetically downstream of vab-8 and upstream of sax-3 (Fig. 3g).

We tested whether other suppressors of VAB-8L–dependent rerout-ing also suppressed SAX-3–dependent rerouting. As described above,SAX-3 and UNC-40 heteromeric receptors have been proposed tomediate certain SLT-1 responses34. As another approach to testingwhether SAX-3 and UNC-40 act together, we asked whether UNC-40was necessary for SAX-3’s ability to promote ALM rerouting and foundthat an unc-40 null mutation did not affect SAX-3–dependent rerout-ing. This finding is consistent with our previous observation indicatingthat UNC-40 and SAX-3 act independently in this process (Fig. 2b).

Neither unc-5 nor unc-6 mutations suppressed SAX-3–dependentrerouting (Fig. 3g), indicating that SAX-3 does not mediate the effectsof UNC-6. We also found that slt-1(eh15) did not suppress SAX-3–dependent ALM rerouting (Fig. 3g), indicating that SAX-3 does notrequire activation by SLT-1 to cause rerouting. Activation of SAX-3 byother ligands might be sufficient to cause rerouting.

These observations and those of Levy-Strumpf and Culotti21 suggestthat VAB-8L coordinately controls SAX-3, UNC-5 and UNC-40receptor expression or function. SLT-1 and other ligands acting thoughthe SAX-3 receptor, and UNC-6 acting though the UNC-5 andUNC-40 receptors, can cause ALM to extend its process posteriorlywhen VAB-8L is present.

ALM cell migration defects

The studies described above involve phenotypes that are produced byprotein misexpression, raising the concern that these effects do notreflect the normal roles of these molecules. Several of the proteins,however, have been shown to function in early ALM development.Shortly after being generated, the ALM cell bodies migrate a shortdistance toward the posterior (Fig. 4a). Only after completing thismigration do the ALMs extend their anterior processes during embryo-genesis. Loss or reduction of SAX-3, UNC-73 and VAB-8L have all beenshown to disrupt ALM cell migration19,30,35,36. We scored the ALM cellbody positions in early L1 worms bearing mutations in these genes and,as predicted from previous studies, all of the mutants had ALMmigration defects: 95% of ALMs were located between the hypodermalcells V2 and V3 in wild-type worms, whereas the percentages of ALMslocated anterior to the V2 cells were significantly higher in vab-8, unc-73 and sax-3 mutants (Fig. 4b).

We also tested whether unc-5 and unc-6 mutations disruptedALM cell migration and found that more ALMs were shifted anteriorto the V2 cell (Fig. 4b). The effects of the vab-8, unc-5 and unc-6mutations were weak, so we further tested the involvement of thesegenes by scoring the positions of ALM cell bodies in a gmEx369[Pmec-3::gfp; Pmec-7::gfp] transgenic background, which sensitizesthe background to perturbations in ALM migration. We found thatthis background enhanced the ALM migration defects of vab-8, unc-5and unc-6 mutants.

We also found that expression of VAB-8L in the ALMs of gmIs14worms caused the cell bodies of these neurons to migrate too farposteriorly 49% of the time (Fig. 4b). The undermigration and over-migration ALM phenotypes of worms lacking or expressing excessVAB-8L, respectively, indicate that the different levels of VAB-8L canregulate the final positions of migrating cells. The excessive ALMmigration phenotype of gmIs14 worms raised the possibility that thererouting phenotype that we observed was caused not by excess VAB-8L but by the abnormal positions of the ALM cell bodies. To addressthis issue, we scored both the positions of the ALM cell bodies andwhether the ALM processes were rerouted in L1 worms using thetransgene gmEx369. We found that 86% of the ALM processes werererouted when the ALMs were in their normal positions between V2

ALM

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Figure 3 Posterior rerouting of ALM caused by excess SAX-3. (a–f) Anterior

(a), posterior (c) and bipolar (e) processes of ALMs in gmEx335[Pmec-7::sax-

3, Pmec-3::gfp, Pttx-3::gfp]. White arrows point to the ALM cell bodies. The

small arrowheads indicate the ALM processes. b, d and f show DIC images of

a, c and e, respectively. (g) The frequencies of SAX-3–dependent ALM

rerouting in various mutant backgrounds. slt-1 does not suppress the ALM

rerouting in gmEx335[Pmec-7::sax-3]. vab-8, unc-73, unc-40, unc-5, and

unc-6 do not suppress the ALM rerouting of gmIs28[Pmec-7::sax-3::gfp].200 worms were scored for each strain. n.s., not statistically significant.

Scale bar, 20 mm. Error bars represent standard errors of proportions.

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and V3, and 85% were rerouted when the ALMs migrated too far andwere posterior to V3. This observation argues that excess VAB-8Lindependently caused excess ALM migration and process rerouting.

We asked whether loss of unc-73, sax-3, unc-6 or unc-5 couldsuppress the ALM overmigration phenotype caused by misexpressionof VAB-8L in the ALM (Fig. 4b). As with ALM rerouting, mutations inall of these genes suppressed the overmigration phenotype of gmIs14.Thus, the migrations of the ALM cell bodies requires the functions ofthe same molecules as VAB-8L–dependent ALM rerouting, suggestingthat our model for VAB-8L function, which is based on the ALMrerouting results, is relevant to the role played by endogenous VAB-8L.

VAB-8L regulates SAX-3 levels

The genetic experiments described above indicate that VAB-8L regulatesthe SAX-3 pathway. We reasoned that if VAB-8L regulates SAX-3, wemight see a change in the distribution or abundance of the SAX-3protein in ALM neurons that produce different levels of VAB-8L. To testthis hypothesis, we used gmIs28, the integrated Pmec-7::sax-3::gfp trans-gene. We began to detect SAX-3::GFP at the 1.5-fold stage of embryo-genesis in the ALMs and their sister cells, the BDU neurons; by thetwofold stage we detected SAX-3::GFP in these cells in 54% of embryos(Fig. 5). By contrast, we rarely detected a SAX-3::GFP signal in vab-8null mutant embryos at the 1.5-fold stage, and only 27% of twofold stageembryos had a detectable signal in ALM and BDU (Fig. 5d–f,m).

The requirement of VAB-8L for efficient expression of SAX-3::GFPpredicts that expression of vab-8L from the mec-7 promoter willincrease the levels of SAX-3::GFP in the ALM. In a strain carryingboth gmIs28[Pmec-7::sax-3::gfp] and gmIs14[Pmec-7::vab-8L::gfp], wefound that the percentage of embryos with a detectable green fluor-escent protein (GFP) signal in ALM and BDU was much higher at bothstages as compared to strains carrying gmIs28 alone (Fig. 5g–i,m). Webelieve that the GFP expression can be attributed to SAX-3::GFP inthese embryos for three reasons. First, GFP appears to be at the cellmembrane, the expected localization for the SAX-3 receptor. Second,we detect no VAB-8L::GFP in gmIs14 embryos in the ALM and BDUneurons at these stages of embryogenesis (Fig. 5j–m). Finally, weexpressed an integrated, untagged version of vab-8L driven from themec-3 promoter. This transgene caused the ALMs to have posterior andbipolar processes 41% of the time and increased the frequency of ALMsexpressing SAX-3::GFP in twofold embryos by 16% (P ¼ 0.02). Thus,increasing the dose of VAB-8L increases the levels of SAX-3::GFP.

UNC-73 can bind VAB-8L, SAX-3, UNC-5 and UNC-40

Our pathway for VAB-8L function could reflect direct or indirectinteractions among the molecules. To test the possibility that theseinteractions are direct, we asked whether the proteins could interactphysically in a yeast two-hybrid assay. UNC-73B is an isoform thatcontains the Rac GEF but not the Rho GEF domain26. We foundthat the full-length UNC-73B interacted with a VAB-8L fragment(Fig. 6 and Supplementary Fig. 2 online). The VAB-8L–interactingdomain of UNC-73B was narrowed down to the smaller fragment thatcontains spectrin repeats (Fig. 6 and Supplementary Fig. 2). Thecytoplasmic domains of SAX-3, UNC-5 and UNC-40 also bound boththe full-length and the same smaller fragment of UNC-73B (Fig. 6 andSupplementary Fig. 2). UNC-73B was able to bind itself, suggestingthat UNC-73 might function as a multimer. The spectrin repeats didnot bind several additional C. elegans proteins that we tested, indicatingthat the interactions that we observed were specific (data not shown;see Supplementary Fig. 2 for negative controls). We propose that theincrease in SAX-3 levels produced by VAB-8L is mediated by proteininteractions between UNC-73 and both VAB-8L and SAX-3.

DISCUSSION

Guidance receptors mediate VAB-8L–dependent ALM rerouting

On the basis of our observations and those of Levy-Strumpf andCulotti21, we propose that VAB-8L increases the activity of receptors

Presumptivegonad

Tail

L1 larva

Percentage ALM cell bodiesV1

P1/2

Wild type

vab-8(e1017)*unc-73(e936)**

n

unc-73(rh40)***

wild typeS

vab-8S†

*,†, # P < 0.05 **,†† P < 0.001 ***, ### P < 0.0001

unc-6S†

unc-5S††

unc-6(ev400)*

mec-7::vab-8L::gfp (gmls14)***unc-73(e936); gmls14###

unc-73(rh40); gmls14###

gmls14; sax-3 ###

gmls14; unc-6 #

gmls14; unc-5 #

unc-5(e53)

sax-3(ky123)***slt-1(eh15)

P3/4 P5/6

2 39 57

83 60 29

176 41 36

15102 45 28

1582 34 41

37 31 52 8

165 31 42 5 2

731 29 47 10 3

13 22 47 25

6 17 46 31

1

51 45 4

17315 35 12

128 50 30

31 49 45

72 68 23

135 52 29

11 67 22

2 23 73

2

2

1

61

65

66

60

61

65

63

60

73

64

63

61

82

75

62

71

72

70

V2 V3 V4

Head

ALMa

b

Figure 4 Loss or reduction of VAB-8, UNC-73, SAX-3, UNC-5 and UNC-6

perturb ALM cell migration. (a) Schematic diagram of a 1.5-fold-stage

embryo, showing the direction of ALM migration. (b) Distribution of ALM cell

bodies in early L1 worms. ALM cell bodies were detected by Nomarski optics.

The gmEx369[Pmec-7::gfp, Pmec-3::gfp] transgene provides a sensitized (S)

genetic background that enhances the ALM defects of migration mutants.

Because gmEx369 is an extrachromosomal array, only ALMs that express

GFP were scored. ALM cell bodies were scored relative to the statichypodermal cells, V1, P1/2, V2, P3/4, V3 and P5/6, shown in the diagram.

Black vertical lines show that most ALMs in wild-type worms are located

between V2 and V3. The proportions of ALMs located anterior to V2 in

vab-8, unc-73, sax-3, unc-5 and unc-6 mutants compared to those of

wild-type worms are statistically higher. Asterisks represent the P values for

comparisons of the frequencies of anterior ALMs between wild-type and

mutant strains, and crosses P values for comparisons of the wild typeS and

mutantsS carrying gmEx369. For overmigration in gmIs14 [Pmec-7::vab-

8L::gfp], the proportion of ALMs posterior to V3 is compared with that in

wild-type worms (P o 0.0001). unc-73, sax-3, unc-6 and unc-5 mutations

suppress the overmigration seen in gmIs14 statistically significantly.

# represents the P values obtained when comparing the wild type and

mutants carrying gmIs14.

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involved in DV guidance, enhancing their ability to respond to theirligands (Supplementary Fig. 3 online). A role for SLT-1 in posteriorlydirected guidance has already been demonstrated: cells in the head ofthe embryo express SLT-1 when the ALMs migrate and extend theirprocesses, and SLT-1 and SAX-3 are required for the migrations of theALM and CAN cell bodies30,31,37. Both types of neurons migrateposteriorly, and ectopic expression of SLT-1 suggests that its expressionin the head normally repels the CANs posteriorly30. UNC-6 expressionis dynamic, and UNC-6 is expressed in the inner labial and ventral

cephalic sheaths cells in the head during embryogenesis4. We proposethat this source of UNC-6 causes the anterior-posterior (AP) guidanceeffects that we observe. Although the proposal that UNC-6 and UNC-5regulate AP guidance is new, UNC-40 has been shown to regulate theinitial polarization of the Q neuroblasts along the AP axis38.

VAB-8L acts through UNC-73 to regulate guidance receptors

The requirement for UNC-73 in VAB-8L–dependent but not SAX-3–dependent ALM process rerouting suggests that UNC-73 regulatesSAX-3 receptor signaling and possibly signaling by the UNC-5and UNC-40 receptors. Suppression of VAB-8L rerouting by theunc-73(rh40) mutation, which eliminates UNC-73’s Rac GEF activity,is particularly revealing and indicates that Rac signaling regulatesguidance-receptor function, a hypothesis that is supported by thealtered localization of UNC-40 in worms containing activated MIG-2(ref. 21). This is a heterodox view, as Racs are thought to act down-stream of guidance receptors. Racs have specifically been proposed tomediate robo’s repulsive role at the Drosophila midline and UNC-40’sattractive role for C. elegans sensory axons39–42. A model proposing thatRacs can regulate receptors, however, is not unprecedented. Activationof Rac1 in HeLa and MDCK cells can block receptor-mediatedendocytosis43,44, and activation of Racs in leukocytes clusters integrinreceptors and stimulates cell spreading45.

Our yeast two-hybrid analysis suggests that direct interactionsbetween the spectrin repeats of UNC-73B and both VAB-8L and thecytoplasmic domain of SAX-3 are important for this regulation.Because the same region of UNC-73B can also interact with theintracellular tails of UNC-5 and UNC-40, we propose that VAB-8Land UNC-73B might also regulate the levels of these receptors. Physical

Pm

ec-7

::sax

-3::g

fpva

b-8(

e101

7);

Pm

ec-7

::sax

-3::g

fpgm

Is14

;P

mec

-7::s

ax-3

::gfp

gmIs

14

n =

76

n =

54

n =

85

n =

56

n =

94

n =

68

n =

74

n =

59

Pmec

-7::s

ax-3

::gfp

vab-

8(e1

017)

;

Pmec

-7::s

ax-3

::gfp

gmIs1

4;

Pmec

-7::s

ax-3

::gfp

Pmec

-7::v

ab-8

L:gf

p

(gm

Is14)

100

80

60

40

20

0

Per

cent

age

embr

yos

with

GF

P in

ALM

and

BD

U

1.5-fold stage 2-fold stage

a b c

d e f

g h i

j

m

k l

***

*

*

***

Figure 5 VAB-8L level affects the SAX-3::GFP level in ALMs and BDUs of

early embryos. (a–l) Left column shows examples of GFP signal seen at

twofold-stage embryos in Pmec-7::sax-3::gfp(gmIs28) (a), vab-8(e1017);

Pmec-7::sax-3::gfp(gmIs28) (d), Pmec-7::vab-8L::gfp(gmIs14); Pmec-7::

sax-3::gfp(gmIs28) (g) and Pmec-7::vab-8L::gfp(gmIs14) (j). Arrows indicate

ALM and BDU in a, d and g. GFP expressed from ttx-3 promoter (injection

marker) and ectopic expressions of GFP are seen in the strain carrying Pmec-

7::sax-3::gfp(gmIs28) (brackets in a,d,g). In the strains carrying Pmec-7::vab-8L::gfp(gmIs14) alone, the GFP signal in ALM and BDU is not detected

(j,l). In middle column, b, e, h and k are DIC pictures corresponding to a, d, g

and j, respectively. In right column, c, f, i and l are enlarged images of the

areas marked by black open boxes in b, e, h, and k, respectively. Scale bars,

10 mm. (m) Frequencies of embryos with GFP in ALM and BDU in 1.5- and

twofold-stage embryos. The percentages of embryos with a GFP signal in

vab-8(e1017); Pmec-7::sax-3::gfp or Pmec-7::vab-8L::gfp(gmIs14); Pmec-

7::sax-3::gfp-were compared at each stage with Pmec-7::sax-3::gfp(gmIs28)

by a two-tailed z test. Asterisks denote the statistic significance: *P o 0.05,

***P o 0.0005. Error bars represent standard errors of proportions.

402 874

VAB-8L UNC-73B Interaction

196

1

1,070

1,638AD BD

BD

BD

BD

BD

BD

BD

BD

AD

AD

AD

196 1,070

1,638

196 1,070

1,638

196 1,070

1,638

1

1

1

+

+/–*

+

+

+

+*

+

+

897 1,273

363 919

1,108 1,415

UNC-40

UNC-5

SAX-3

Figure 6 Interactions of UNC-73B with VAB-8L, SAX-3, UNC-5 and UNC-40

in the yeast two-hybrid analysis. The fragments of VAB-8L, SAX-3, UNC-5,

UNC-40 and UNC-73 used in the yeast two-hybrid assays are shown with thin

gray boxes under the diagrams of molecular structures. Number of amino

acid at each end of the fragments is shown below the gray boxes. AD and

BD stand for the activation domain and DNA binding domain of GAL4,

respectively, which were fused at the N terminus of each fragment. The

hatched area and black region in VAB-8L protein diagram show a kinesin-like

motor domain and a coiled-coil domain, respectively. The shaded areas in

SAX-3, UNC-5, and UNC-40 diagrams are cytoplasmic domains. The light

gray area in UNC-73B has eight spectrin repeats, and the dark gray domains

are DH and PH domains, which have a GEF activity. Interactions of VAB-8L,SAX-3, UNC-5 and UNC-40 fragments with the full length or the partial

fragment of UNC-73B are indicated at right: +, blue colonies; +/–, many

small colonies. * indicates that only two colonies out of three that we

examined grew on selection plates.

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interactions between the Drosophila UNC-40 homolog frazzled and triohave been described46.

How does VAB-8L regulate receptor function?

Increasing the abundance of VAB-8L in the ALMs results in an increaseof SAX-3. How does VAB-8L achieve this? Two general mechanismscould account for this regulation. VAB-8L could promote eithertranscription of genes encoding guidance receptors or translation oftheir mRNAs. Transcription regulation of the unc-5 gene, for example, isrequired for dorsal turning of the distal tip cell47. Alternatively, VAB-8Lcould regulate receptor transport. VAB-8L could promote the transportof SAX-3 to the cell surface or inhibit its removal by endocytosis. InDrosophila, commissureless is thought to prevent transport of robo tothe growth cone by routing it to the lysosome for degradation15,16.

We favor the model that VAB-8L regulates receptor trafficking forseveral reasons. First, the ability of UNC-73 to interact physically withVAB-8L and the cytoplasmic domain of SAX-3, UNC-5 and UNC-40suggests that these protein interactions mediate VAB-8L’s regulation ofthese receptors. Second, activated MIG-2, a Rac-like GTPase, alters thedistribution of the UNC-40, causing it to accumulate in thread-liketracts below the cell membrane21. Finally, VAB-8 interacts physicallywith UNC-51, which belongs to a family of kinases that regulatemembrane trafficking48,49.

Receptor regulation in axon guidance

If most guidance cues and receptors that regulate growth-cone migra-tions have been identified, then how do these molecules regulateformation of the myriad of axon trajectories that are present in theadult nervous system? One general mechanism that is used at themidline is to regulate the ability of a receptor to respond. Decreasingthe sensitivity of DCC to netrin through the formation of DCC and roboheteromers and decreasing the sensitivity of robo to slit by expression ofthe robo-related receptor Rig-1 allow commissural axons to cross thevertebrate midline13,14. At the Drosophila midline, commissurelessdecreases robo levels, allowing growth cones to cross slit-expressingmidline cells15,50. In each of these cases, robo is inhibited. VAB-8L, bycontrast, promotes receptor function. It seems likely that regulatingreceptor function, both positively and negatively, will be an importantway of using a small set of conserved molecules to specify a largerepertoire of axon trajectories.

METHODSStrains. The wild-type strain of C. elegans used was Bristol N2. Standard

methods of culturing and handling worms were used. All strains were cultured

at 25 1C for scoring. Strains shown in Supplementary Table 1 were scored at

20 1C. Mutations and transgenes used in this study were (i) LGI: unc-73(e936),

unc-73(rh40), unc-40 (e1430), zdIs5 (Pmec-4::gfp); (ii) LGIV: unc-5(e53),

unc-5(ju27), unc-5(e589); (iii) LGV: vab-8(e1017); and (iv) LGX: slt-1(eh15),

sax-3(ky123), unc-6 (ev400).

Molecular biology and strain constructions. Pmec-7::vab-8L::gfp was con-

structed in the same manner as Pmec-3::vab-8L::gfp20. The mec-7 promoter was

amplified from pPD96.41 by PCR. Pmec-7::vab-8L::gfp was co-injected with

pRF4, a plasmid encoding the dominant allele rol-6(su1006), at 150 ng ml–1 and

50 ng ml–1, respectively, to generate gmEx177. gmEx177 was integrated into the

genome by gamma-ray mutagenesis, resulting in the stably transmitting strain

gmIs14. Pmec-3::vab-8L was constructed by removing BstEII fragment from

Pmec-3::vab-8L::gfp20. Pmec-3::vab-8L was co-injected with pRF4 at 150 ng ml–1

and 50 ng ml–1, respectively, to get an extrachromosomal array, and that was

integrated by UV irradiation to obtain gmIs37. The Pmec-7::sax-3 plasmid31

was co-injected at 1 ng ml–1 with Pmec-3::gfp at 10 ng ml–1 and P ttx-3::gfp at

30 ng ml–1 into wild-type worms to produce the extrachromosomal array

gmEx364. For the SAX-3 overexpression experiment, a solution containing

10 ng ml–1 Pmec-7::sax-3, 40 ng ml–1 Pmec-3::gfp, and 50 ng ml–1 P ttx-3::gfp was

injected into the wild type to generate the extrachromosomal array gmEx335.

As the control strain for gmEx335, gmEx339 was obtained by injecting

10 ng ml–1 Pmec-7::gfp instead of 10 ng ml–1 Pmec-7::sax-3. We made the

Pmec-3::gfp construct by ligating the PstI-HincII fragment from pPD57.56

(mec-3 promoter) into the PstI-SmaI–cut pPD95.77 (gfp plasmid). To

generate the Pmec-7::gfp construct, the pPD96.41 (mec-7 promoter)

fragment cut at HindIII and XmaI sites was inserted into pPD95.77 cut at

the same sites.

SAX-3::GFP constructs and strains. To construct the Pmec-7::sax-3::gfp

translational fusion, a DNA fragment including a PshAI site and the down-

stream sequence of SAX-3 was amplified by PCR using the 3¢ primer with the

stop codon of SAX-3 changed to a leucine codon and AgeI and StuI sites just

downstream of the altered codon. The PCR product was digested with PshAI

and StuI and then inserted in the Pmec-7::sax-3 construct cut with PshAI. The

AgeI-SpeI fragment of this plasmid was replaced with the AgeI-SpeI fragment

from pPD95.77 (GFP vector) that contains the sequence encoding GFP and

the 3¢ untranslated region. Pmec-7::sax-3::gfp was injected at 10 ng ml–1 with

50 ng ml–1 P ttx-3::gfp to generate the extrachromosomal array gmEx338. UV

irradiation of the worms carrying gmEx338 resulted in the integrated

strain gmIs28.

Scoring of ALM processes. To score ALM processes, we immobilized adult

hermaphrodites in 30 mM sodium azide and viewed them under the �63

objective on a Zeiss Axioskop by GFP fluorescence to determine axonal

morphologies. In wild-type worms, ALMs often had a short posterior process.

To distinguish between this short process and the much longer posterior

processes observed in VAB-8L or SAX-3 misexpression strains, only processes

that were ten times longer than the ALM cell body width were scored as an

‘anterior process’ or ‘posterior process’, depending on the direction of extension.

ALMs with both an anterior process and a posterior process were scored as

having a ‘bipolar process’. We used a two-tailed z test to determine whether the

percentages of wild-type processes were statistically different between two strains.

Scoring of ALM migration. Locations of ALM cell bodies were scored relative

to the static hypodermal cells in early L1 worms before the V cells divided. The

cell bodies of ALM were scored with Nomarski optics. gmEx369 was used to

visualize the cell body position and process orientation of the ALMs. To

generate this transgene a solution containing 1 ng ml–1 Pmec-7::gfp and 10 ng

ml–1 Pmec-3::gfp was injected into wild-type hermaphrodites. Equal numbers of

ALMs on the left and right side were scored for each strain. The statistical

significance of differences in ALM cell body positions between strains was

determined by comparing the proportions of ALMs that migrated outside their

normal range between V2 and V3. The misplaced cells failed to migrate fully

and were anterior to V2, or they migrated too far and were posterior to V3. As

with process rerouting, we used a two-tailed z test to determine if ALM cell

body positions were statistically different between two strains.

Yeast two-hybrid analysis. Yeast two-hybrid analysis was performed essentially

as described by the manufacturer (Takara). For the expression of the GAL4

DNA binding domain (BD) fusion proteins, pGBKT7 was used. For the

expression of the GAL4 activation domain (AD) fusion proteins, pGADT7

or pACT were used. Yeast strain AH109 was co-transformed with the AD and

BD fusion protein expression plasmids.

pB-u73S (DB::UNC-73B (aa 1–1638, full length)), pB-F19 (DB::UNC-73B

(aa 196–1070)), pGBKT7-53 (DB::murine p53), pA-u5C (AD::UNC-5 (aa 363–

919, cytoplasmic region)), pA-u40C (AD::UNC-40 (aa 1108–1415, cytoplasmic

region)), pA-s3C (AD::SAX-3(aa 897–1273, cytoplasmic region)), pF19

(AD::UNC-73B (aa 196–1070)), pF169 (AD::VAB-8L (aa 402–874)) and

pGADT7-T (AD::SV 40 large T-antigen) were used for the analysis. Open

reading frames were amplified by PCR from expressed sequence tag (EST)

clones or RT-PCR products. The EST clones used for the analysis were yk481e9

(unc-5) and yk286b9 (unc-40). The EST clones were kindly provided by

Y. Kohara. pF19 and pF169 were isolated as positive clones that interacted

with UNC-51 by yeast two-hybrid library screening (K.O., unpublished data).

To analyze their interactions, transformants were plated on a selection

medium (SD –Adenine –His –Leu –Trp +X-a-Gal). If the hybrid proteins

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bind each other, GAL4-mediated transcription activation of the HIS3, ADE2

and MEL1 reporter gene occurs, which is detected by the growth and blue color

of the transformants on the selection medium.

Note: Supplementary information is available on the Nature Neuroscience website.

ACKNOWLEDGMENTSWe thank N. Levy-Strumpf and J. Culotti for critical discussions, sharing ofunpublished results and comments on the manuscript. This work was madepossible by their observations on vab-8 and unc-40 interactions, and weare greatly indebted to them for sharing this information. We also thankC. Bargmann and R. Steven for helpful discussions and reagents; T. Lai for thePmec-3::gfp and Pmec-7::gfp constructs; and the C. elegans Genetics Center forsome of the strains used in this study; Y. Kohara for C. elegans EST clones; andP. Vanderzalm for comments on the manuscript. We are grateful to membersof Garriga and Goshima labs for helpful suggestions. This work was supportedby Core Research for Evolutional Science and Technology, Japan Science andTechnology Agency, Grants-in-Aid for Scientific Research in a Priority Areafrom the Ministry of Education, Sports and Culture to K.O. and Y.G., by theYokohama Foundation for Advancement of Medical Science to K.O., and bythe US National Institutes of Health grant NS32057 to G.G.

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureneuroscience

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions

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