Inhibition of Netrin-Mediated Axon Attraction by a Receptor Protein Tyrosine Phosphatase

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Science  02 Jul 2004:
Vol. 305, Issue 5680, pp. 103-106
DOI: 10.1126/science.1096983

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During axon guidance, the ventral guidance of the Caenorhabditis elegans anterior ventral microtubule axon is controlled by two cues, the UNC-6/netrin attractant recognized by the UNC-40/DCC receptor and the SLT-1/slit repellent recognized by the SAX-3/robo receptor. We show here that loss-of-function mutations in clr-1 enhance netrin-dependent attraction, suppressing ventral guidance defects in slt-1 mutants. clr-1 encodes a transmembrane receptor protein tyrosine phosphatase (RPTP) that functions in AVM to inhibit signaling through the DCC family receptor UNC-40 and its effector, UNC-34/enabled. The known effects of other RPTPs in axon guidance could result from modulation of guidance receptors like UNC-40/DCC.

Axons in the developing nervous system respond to attractive and repulsive guidance cues of the netrin, slit, semaphorin, and ephrin families (13). The interpretation of a guidance signal as a repellent or an attractant is context-dependent and influenced by the activities of other signaling pathways (4, 5). Thus, the netrin receptor UNC-40 contributes to both axon attraction (acting on its own) and repulsion (in cooperation with the second netrin receptor, UNC-5, or the slit receptor robo) (610).

Receptor protein tyrosine phosphatases (RPTPs) are implicated in axon growth and guidance (11, 12). In general, the inputs that regulate RPTPs, as well as their potential targets, are unknown. Phosphatases are presumed to affect axon guidance by antagonizing kinases, and many tyrosine kinases have been implicated in axon outgrowth and guidance (1319).

The molecules that guide AVM axons toward the ventral midline in C. elegans are similar to those that direct commissural neurons toward the floor plate in vertebrate spinal cords. The AVM neuron sends its axon ventrally toward the attractant UNC-6/netrin (Fig. 1, A and B) and away from the dorsal repellent SLT-1. Mutations in either of these signaling systems result in a 30 to 40% penetrant defect in AVM ventral guidance, whereas mutations in both systems together result in a near-complete failure of ventral guidance (20). Because there are two sources of guidance information, mutants that potentiate signaling through one guidance pathway might suppress the effects caused by loss of the other pathway. A slt-1(null) strain is therefore a useful genetic background to search for mutations allowing enhanced signaling through the unc-6–unc-40 pathway.

Fig. 1.

clr-1 regulates unc-6/unc-40–dependent guidance. (A) Schematic diagram of wild-type and mutant AVM axons. D indicates dorsal; V, ventral; A, anterior; P, posterior. (B) SAX-3/robo and UNC-40/DCC guidance receptors in AVM (green) guide axons toward ventral UNC-6/netrin (blue) and away from dorsal SLT-1/slit (red). (C) clr-1 mutation suppresses AVM guidance defects of slt-1 and sax-3 mutations but not unc-6 and unc-40 mutations. Because clr-1 is essential for viability, we used a temperature-sensitive partial loss-of-function clr-1 allele, e1745ts. All other mutations were strong loss-of-function alleles. Strains were grown at 20°C except the sax-3 and the clr-1; sax-3 mutants. Asterisks indicate data significantly different from clr-1(+) controls (P < 0.001). AVM axon trajectories (arrow) labeled by zdIs5[mec-4::gfp] in (D) slt-1, (E) clr-1; slt-1, (F) unc-6, and (G) clr-1; unc-6. Arrowhead, ALM cell body. Anterior is to the left; dorsal, up. Scale bar, 20 μm.

We undertook two parallel approaches to the suppressor screen. First, we generated double mutants between slt-1 and 30 genes implicated in axon guidance or cell migration and examined the resulting AVM phenotypes. Second, we clonally screened a mutagenized slt-1 null mutant (∼1000 genomes), selecting mutants that decreased the penetrance of its AVM guidance defect. Both approaches identified a single strong suppressor, in each case a mutation in the clr-1 gene. The temperature-sensitive mutation clr-1(e1745), tested in the candidate screen, resulted in an almost-complete suppression of the AVM ventral guidance defect of slt-1 null mutants (Fig. 1, C to E). A single mutation from the genetic screen, cy14, yielded a similar suppression of the ventral guidance defect and mapped to chromosome II near the clr-1 locus. Complementation tests showed that cy14 fails to complement the clr-1(e1745) mutant phenotype.

Sequencing the clr-1 open reading frame in the cy14 mutant revealed a molecular lesion for cy14 (21), confirming that cy14 represents a previously unknown clr-1 allele. cy14 is a G-to-A transition in the splice acceptor of intron 5 of clr-1 that leads to the use of a cryptic splice acceptor and consequently to an 18-base-pair deletion in exon 6 (fig. S1, A and B). The mutant allele lacks part of the single immunoglobulin (Ig) domain in the extracellular region of CLR-1 (fig. S1C). CLR-1 encodes a transmembrane protein tyrosine phosphatase that antagonizes fibroblast growth factor receptor (FGFR) signaling (22). An alignment of vertebrate RPTP and CLR-1 catalytic domains assigns CLR-1 to the R5 subdivision of RPTP family members (23) (fig. S1D). However, the extracellular region of CLR-1 is more like the leukocyte common antigen-related protein subfamily of vertebrate RPTPs, because it contains an Ig domain and FN III (fibronectin type III) repeats (fig. S1D). These sequence features indicate that CLR-1 can be classified as an RPTP but not as the clear ortholog of a particular vertebrate RPTP.

Several models could explain the suppression of the slt-1 mutant phenotype by the clr-1 mutation. To distinguish between various possibilities, we examined the effects of the clr-1 mutation in mutant backgrounds that disable elements of different guidance pathways. The clr-1 null phenotype is lethal, and clr-1(cy14) is subviable, so these genetic studies were performed with the use of the temperature-sensitive clr-1 allele e1745ts.

The clr-1 mutation suppressed the AVM defects of a sax-3 mutant (Fig. 1C), arguing that clr-1 function is sax-3–independent. By contrast, the clr-1 mutation failed to significantly suppress AVM defects of unc-6, unc-40, unc-6 slt-1, or unc-40 slt-1 mutants (Fig. 1, C, F, and G). Because AVM guidance defects in unc-6, unc-40, slt-1, or sax-3 mutants are comparable in severity, suppression of slt-1 and sax-3 appears to be pathway-specific. These results suggest that CLR-1 requires UNC-6 and UNC-40 to affect ventral guidance, consistent with the model that CLR-1 acts on UNC-6/UNC-40 signaling.

One way in which CLR-1 could antagonize netrin signaling would be to limit the production or distribution of UNC-6/netrin. To address this possibility, we examined an alternative response to UNC-6. The DA/DB motor axons use UNC-6 as a guidance cue, but they are repelled rather than attracted by ventral UNC-6 (Fig. 2A). Repulsion of DA/DB axons uses both UNC-40 and UNC-5 guidance receptors (24). unc-5 mutants have a severe defect in DA/DB dorsal guidance, whereas unc-40 defects in DA/DB guidance are milder (Fig. 2B). If CLR-1 normally acts to decrease netrin availability, a clr-1 mutation might suppress the mild dorsal guidance defects of an unc-40 mutant by making more UNC-6/netrin available for detection by UNC-5. Contrary to this prediction, the clr-1 mutation resulted in a significant enhancement of unc-40 mutant phenotypes in DA and DB axons (Fig. 2, B to D). This finding suggests a positive role for CLR-1 in netrin repulsion, in contrast with its inhibitory role in netrin attraction. CLR-1 also promotes UNC-6–dependent dorsal mesodermal cell migrations (25). These genetic results argue against a common effect of CLR-1 on the netrin ligand. By implication, they suggest that, in AVM ventral guidance, CLR-1 normally limits the activity of the UNC-40 receptor.

Fig. 2.

CLR-1 potentiates UNC-5–dependent repulsion from UNC-6/netrin. (A) Schematic diagram of wild-type (wt) and mutant DA/DB motor axon guidance. (B) clr-1 potentiates DA/DB dorsal guidance. unc-5 data are from (34). Asterisks, data significantly different from clr-1(+) control (P < 0.001). DA and DB neurons in L4 stage animals visualized with evIs82[unc-129::gfp] in (C) unc-40 and (D) clr-1; unc-40. DA/DB axons in the dorsal nerve cord (red arrowheads) are misrouted to dorsolateral positions (white arrowheads). White arrows indicate lateral seam cells. Open arrowheads, ventral DA/DB cell bodies. Dorsal is up and anterior at left. Scale bar, 20 μm.

CLR-1 could inhibit UNC-40 by acting either nonautonomously, for example as a transmembrane ligand for UNC-40, or autonomously in the netrin-responsive cell. A clr-1::gfp fusion gene is expressed in many cells whose normal guidance requires unc-40, including AVM, HSN, DD, VD, and mesodermal cells, suggesting an autonomous role (fig. S2). To ask where CLR-1 acts to regulate AVM attraction to netrin, we expressed a clr-1 cDNA under the mec-7 promoter, which is expressed only in AVM, ALM, PVM, and PLM neurons. mec-7::clr-1 rescued the effect of the clr-1 mutation in a clr-1; slt-1 double mutant, indicating that CLR-1 acts cell-autonomously in AVM to inhibit UNC-40 activity (Fig. 3A). mec-7::clr-1 did not cause AVM defects by itself (Fig. 3A) or in a slt-1(eh15)/+ background, indicating that overexpression of clr-1 did not disregulate its activity.

Fig. 3.

CLR-1 acts through UNC-34 in AVM. (A) clr-1 acts cell-autonomously in AVM. Animals expressing cyEx[mec-7::clr-1, odr-1::dsred]; zdIs5[mec-4::gfp] were significantly different from sibling animals that spontaneously lost the mec-7::clr-1 transgene (asterisk, P < 0.001). (B) CLR-1 requires UNC-34 to inhibit UNC-40 signaling. AVM was visualized with zdIs5[mec-4::gfp]. Asterisks indicate data significantly different from clr-1(+) controls (P < 0.001).

DCC signaling in AVM is mediated by two downstream signaling pathways, one involving UNC-34, an enabled (Ena) homolog, and the other CED-10, a Rac guanosine triphosphatase, and UNC-115, an actin-binding protein similar to human abLIM/limatin (26, 27). To address whether either of these pathways might mediate the CLR-1 effect, we disrupted each branch in a slt-1 mutant background, where only unc-40 guidance signaling is active in AVM, and asked whether the clr-1 mutation could still modify the AVM phenotype. The clr-1 mutation significantly suppressed the AVM defects of slt-1; ced-10 and slt-1; unc-115 but not slt-1; unc-34 double mutants (Fig. 3B). Thus the slt-1; ced-10 double mutants retain a pathway that is inhibited by CLR-1 and can respond to the mutation, whereas the slt-1; unc-34 double mutants have lost the CLR-1-sensitive signaling pathway. These results therefore suggest that clr-1 exerts its negative effect in netrin attraction through the unc-34–dependent pathway. In the case of DA and DB axon dorsal guidance, the clr-1 mutation enhanced unc-40 mutant phenotypes but not those of unc-34 mutants (Fig. 2B), indicating that clr-1 also exerts its positive effect in netrin repulsion through an unc-34–dependent pathway. In contrast to its role in attractive and repulsive axon guidance, clr-1 did not affect an outgrowth-promoting activity of unc-40 (fig. S3).

Thus, the RPTP CLR-1 functions as a negative regulator of the netrin attractive guidance pathway, acting with or downstream of the netrin receptor UNC-40/DCC in the UNC-34/enabled pathway.

The suppression of ventral guidance defects in slt-1mutants by clr-1 is remarkably complete: Nearly all AVM axons are guided normally in clr-1; slt-1 double mutants. This result indicates that UNC-6/netrin is sufficient for accurate guidance of AVM even without the dorsal repellent SLT-1, provided the CLR-1 negative regulatory influence is removed. Reducing clr-1 activity in a wild-type background did not cause guidance defects, but the null phenotype of clr-1 is lethal, so only nonnull alleles were examined; stronger axon defects might be observed if clr-1 function was eliminated. The rate of axon extension is enhanced in neurons from RPTPσ–/– mice (28) and in Xenopus retinal ganglion cell neurons expressing a dominant negative CRYP (RPTPσ) (29), suggesting a conserved role for RPTPs in inhibiting axon growth.

Our results suggest a model in which a protein tyrosine kinase is a positive regulator of netrin-mediated attraction and CLR-1 is a negative regulator. Such antagonistic effects might reflect opposite effects on the tyrosine phosphorylation state of UNC-40 or of an UNC-40 effector, perhaps UNC-34/enabled (Fig. 4) (30). The enhancement of UNC-40–mediated guidance by removal of CLR-1 identifies a mechanism for negatively regulating netrin attraction and suggests that effects of RPTPs in axon guidance might result from regulation of key axon guidance receptors.

Fig. 4.

Model for CLR-1 regulation of netrin attraction. Arrows indicate a positive effect and bars a negative effect. UNC-40, UNC-34, or an unknown effector is phosphorylated by an unknown tyrosine kinase during netrin signaling and dephosphorylated by CLR-1 to inhibit signaling. The cytoplasmic domain of CLR-1 produced in vitro can associate with GST:UNC-40 cytoplasmic domain, consistent with a direct interaction of the proteins (33). EGF, epidermal growth factor; PTK, protein tyrosine kinase; PXXP, proline motif. Question marks indicate undefined signaling components.

Supporting Online Material

Materials and Methods

Figs. S1 to S4


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