CLEC-38, A Transmembrane Protein with C-Type Lectin-Like Domains, Negatively Regulates UNC-40-Mediated Axon Outgrowth and Promotes Presynaptic Development in Caenorhabditis elegans

Identification and characterization of CLEC-38

A mutation, ur280, was isolated from a genetic screen for mutations that could suppress dorsal guidance defects caused by the unc-6(rh46) mutation. The unc-6(rh46) mutation is a partial loss-of-function allele (Hedgecock et al., 1990; Wadsworth et al., 1996) and mutations were isolated that could improve the ability of DA and DB motor axons to reach the dorsal cord in the unc-6(rh46) background. The ur280 mutation was mapped to a region of chromosome V (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Using cosmids containing sequence corresponding to this region and DNA-mediated transformation rescue, we found that the ur280 locus is within cosmid T25E12 in the gene clec-38. The clec-38 gene is predicted to encode a protein of 382 aa comprising a short cytoplasmic tail with a putative SH2 binding site, a type-II transmembrane domain and two extracellular CTLDs (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). DNA sequence analysis of ur280 identifies a TTT to GTT change, which causes valine 269 within the second CTLD to be substituted for phenylalanine. We also analyzed tm2035, which was isolated in a PCR-based screen for deletions by the National BioResource Project. This clec-38 mutation can also suppress the dorsal guidance defects of unc-6(rh46) (data not shown). The tm2035 mutation is a deletion removing part of the clec-38 promoter and the first five exons, which correspond to the first 222 aa of CLEC-38 (supplemental Fig. 1, available at www.jneurosci.org as supplemental material). Animals deficient for clec-38 by RNAi show a mild dumpy phenotype similar to that caused by the clec-38(ur280) and clec-38(tm2035) mutations. Furthermore, the ur280 phenotypes were not enhanced when the ur280 allele was placed over the deficiency yDf4, which removes the clec-38 gene (data not shown). Together, these observations indicate that clec-38(ur280) is a strong loss-of-function mutation.

To determine cells that express the clec-38 gene, we designed a promoter fusion construct using 2 kb upstream of clec-38 sequence fused to a green fluorescent protein coding sequence (see Materials and Methods). The transgenic animals carrying this construct show GFP fluorescence in the nervous system (Fig. 1). Fluorescence could be detected in early embryos and persists through adulthood. The expression was detected in head neurons of threefold stage embryos (Fig. 1A). Strong expression was also observed in intestine. In the adults, the fluorescence is detected in the ventral cord motor neurons, PLM touch neurons, head neurons, and the intestine (Fig. 1B,C). Attempts to generate strains expressing a GFP-tagged CLEC-38 translational fusion have been unsuccessful.

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Figure 1.

Expression of clec-38. Pattern of clec-38 promoter expression was obtained from an extrachromosomal array containing 2 kb upstream sequence of clec-38 fused with GFP. Expression is observed in a subset of neurons and in the intestine. Fluorescence could be detected in early embryos and persists through adulthood. A, Threefold stage embryo showing clec-38 expression in head neurons (arrowheads) and intestine (arrow). B, Adult animal showing fluorescence in PLM neuron (arrowhead). C, Expression is detected in ventral cord motor neurons (arrowheads) and the intestine (arrow) in an adult. Anterior is to the left in B and C. Scale bars, 20 μm.

CLEC-38 regulates dorsal axon guidance mediated by UNC-40

The clec-38(ur280) allele was isolated as a suppressor of DA and DB motor axon defects caused by the unc-6(rh46) allele. We examined whether the dorsal migration of DA and DB motor neuron axons in different unc-6 mutants are improved by the clec-38(ur280) mutation (Fig. 2). We found that dorsal guidance is improved in unc-6(rh46) and unc-6(e78) mutants, but not in unc-6(ev400) mutants (Fig. 2F). The unc-6(rh46) and unc-6(e78) are reduction-of-function missense mutations that are predicted to encode altered forms of UNC-6, whereas unc-6(ev400) causes an early stop mutation and is considered a null allele (Hedgecock et al., 1990; Wadsworth et al., 1996). These results suggest UNC-6 is required for the improved guidance in the loss of clec-38 function mutant.

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Figure 2.

clec-38 affects unc-6-dependent dorsal guidance. A, Schematic diagram of the migration of DA and DB motor axons. These axons migrate away from ventral UNC-6 sources. B–E, DA and DB axon guidance in wild-type and mutant L4 larva. DA and DB axons in L4 stage animals were visualized with evIs82a [unc-129::gfp] in wild-type (C) and unc-6(rh46) (E) animals. Axons between the pharynx and vulva were observed, and dorsal guidance was scored as defective if all the axons failed to reach the dorsal cord. In this image, axons have only extended as far as the dorsal sublateral nerve. F, clec-38(ur280) suppresses the dorsal guidance defects caused by the unc-6(rh46) and unc-6(e78) partial loss-of-function alleles but not the null allele, unc-6(ev400). The suppression of dorsal guidance defects in clec-38(ur280);unc-6(rh46) is inhibited by the loss of unc-40 function, as observed in the triple mutant, clec-38(ur280);unc-6(rh46);unc-40(e1430). This suggests that the suppressor activity of clec-38(ur280) is dependent on unc-40 function. Error bars represent SEs of proportions, and asterisks indicate statistically significant difference (**p < 0.005; ***p < 0.0005). Anterior is to the left, and dorsal is up. vc, Ventral cord; dc, dorsal cord; dsl, dorsal-sublateral cord. Scale bars, 20 μm.

The dorsal guidance of DA and DB motor neurons in response to UNC-6 is mediated by UNC-5 and UNC-40 receptors (Leung-Hagesteijn et al., 1992; Chan et al., 1996). To determine whether the improved dorsal guidance observed in the clec-38(ur280) mutants are affected by loss of the receptors, we examined animals with unc-5 and unc-40 loss-of-function mutations (Fig. 2F). Loss of unc-5, but not unc-40, function causes dorsal axon guidance defective by the criteria of our assay, which scored dorsal migration as defective only if none of the axons from the neuron cell bodies situated along the ventral nerve cord in the region between the pharynx and vulva failed to reach the dorsal cord. Results from clec-38; unc-40; unc-6(rh46) and clec-38; unc-5; unc-6(rh46) triple mutants show that the clec-38 mutation cannot suppress the unc-6(rh46) dorsal guidance phenotype in the absence of UNC-40, indicating that the suppression by loss of clec-38 function requires UNC-40 signaling. The dorsal guidance defects are the same in clec-38; unc-5(e53); unc-6(rh46) and unc-5(e53) single mutants; however, this result is not too informative because dorsal guidance is severely impaired by the loss of unc-5 function alone. Together, the results suggest that the loss of clec-38 function affects UNC-40 activity in the DA and DB motor neurons and that this change improves the ability of the axons to respond to the dorsal guidance information provided by UNC-6.

CLEC-38 regulates ventral guidance mediated by UNC-40

The ventral migration of the AVM axon is guided by the UNC-6 and SLT-1 guidance cues through signals mediated by the UNC-40 and SAX-3 receptors (Hedgecock et al., 1990; Wadsworth et al., 1996; Hao et al., 2001; Yu et al., 2002; Gitai et al., 2003). The AVM axon travels away from dorsal sources of SLT-1 and toward ventral sources of UNC-6 (Fig. 3). Loss of both cues results in almost complete failure of AVM axon ventral guidance, whereas the loss of either slt-1 or unc-6 function results in ∼40% failure (Fig. 3D–F). There is no significant difference in the penetrance of the defect between unc-6(ev400) and clec-38;unc-6(ev400) double mutants or between unc-6(rh46) and clec-38;unc-6(rh46) double mutants (Fig. 3F). However, the ventral guidance defect observed in slt-1 mutants is suppressed in clec-38; slt-1 double mutants. These results suggest that loss of clec-38 function can improve the ability of UNC-6 to provide ventral guidance information. Consistent with the finding that UNC-40 is required for improving the dorsal guidance of the DA and DB motor neuron axons, the loss of clec-38 function could similarly improve the AVM response to UNC-6 by enhancing UNC-40 activity. The ability of clec-38(ur280) to improve the ventral AVM axon guidance in the slt-1 loss-of-function background further indicates that the effect of the clec-38 mutation is not dependent on altered forms of UNC-6 but can also improve guidance by the wild type UNC-6 protein.

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Figure 3.

CLEC-38 and ventral guidance mediated by UNC-40. A, Schematic diagram of the AVM axon. The AVM axon is repelled from dorsal SLT-1 sources and is attracted toward ventral UNC-6 sources. B–E, AVM axon guidance in wild-type and mutant larva. The AVM axons in L4 stage animals were visualized with zdIs5 [mec-4::gfp] in wild-type (C) and mutant (E) animals. F, The AVM guidance defects caused by slt-1 and sax-3 loss-of-function mutations are suppressed by the loss of clec-38 function, suggesting enhancement of UNC-6 signaling is likely through improved UNC-40 activity. This effect is reversed when clec-38 is expressed in AVM, suggesting that clec-38 acts cell autonomously. Error bars represent SEs of proportions, and asterisks indicate statistically significant difference (**p < 0.005; ***p < 0.0005). Anterior is to the left, and dorsal is up. Scale bars, 20 μm.

We wanted to determine whether clec-38 expression by AVM is important for the phenotype. When the clec-38 promoter was used to express GFP, we did not specifically detect expression in AVM (Fig. 1); however, mosaic or low-level expression by the transgene could cause AVM expression to be missed. To test whether clec-38 acts cell autonomously in AVM, we expressed a clec-38 transgene under the control of the mec-4 promoter to drive expression in the touch neurons, which includes AVM. This expression was able to reverse the suppression of the AVM guidance defects caused by the clec-38 mutation in clec-38; slt-1double mutants (Fig. 3F). This indicates that AVM expression of clec-38 can account for the axon guidance phenotypes.

CLEC-38 is required for presynaptic terminal organization

From the same genetic screen used to isolate the clec-38(ur280) mutation, we isolated an allele of rpm-1, which encodes a protein that regulates presynaptic terminal (Schaefer et al., 2000; Zhen et al., 2000). To determine whether mutations in clec-38 also disrupt presynaptic terminals, we analyzed the presynaptic terminals of the GABAergic DD and VD motor neurons using a transgene expressing synaptobrevin fused to GFP (SNB-1::GFP) and driven by the unc-25 promoter (Hallam and Jin, 1998; Nonet, 1999).

In wild-type animals, SNB-1::GFP puncta are uniform in shape and are evenly distributed along the processes (Fig. 4A,B). In 86% of clec-38 mutants, the distribution of SNB-1::GFP puncta is irregular, leaving clear areas in the dorsal and ventral cord (Fig. 4C,D). The number of dorsal and ventral cord puncta is reduced in clec-38 mutants (Fig. 4E). In addition, the remaining puncta are of irregular shape and size (Fig. 4C,D). To determine whether these synaptic defects could be a result of gross axon guidance defects, we checked the morphology of DD and VD axons in clec-38 mutants. We did not observe mispositioned axons (data not shown), suggesting that axon guidance defects are not the cause of the irregular SNB-1::GFP pattern in clec-38(ur280) mutants.

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Figure 4.

CLEC-38 affects motor synapses. Expression of synaptic vesicle marker SNB-1::GFP driven by unc-25 promoter in DD (A, C) and VD (B, D) motor neurons in wild-type and clec-38 mutant animals, respectively. The SNB-1::GFP puncta are uniformly distributed in wild-type animals (A, B, arrowheads). In clec-38 mutants, these puncta are reduced leaving gaps in DC and VC (C, D, arrows), and the remaining puncta show diffused pattern (C, D, arrowheads). Anterior is to the left. Scale bars, 10 μm. E, Quantization of SNB-1::GFP puncta in dorsal and ventral cord in wild-type and clec-38 mutant animals.

clec-38 genetically interacts with rpm-1, which encodes a presynaptic terminal protein

RPM-1 is a large protein localized to the presynaptic periactive zone in the mature nervous system. Loss of rpm-1 function causes a range of defects affecting neuronal morphology and synaptic organization (Schaefer et al., 2000; Zhen et al., 2000; Nakata et al., 2005). In both clec-38 and rpm-1 mutants similar GABAergic motor neuron synapse defects are observed. In addition, loss of rpm-1 function similarly affects axon outgrowth mediated by guidance receptors, although RPM-1 negatively regulates the UNC-5 and SAX-3 receptors rather than UNC-40 (Li et al., 2008). Furthermore, RPM-1 appears to regulate the UNC-5 and SAX-3 receptors through a pathway involving the Rab GEF GLO-4; however, the guidance phenotypes of glo-4 and clec-38 mutations are different, suggesting that clec-38 do not act through this pathway.

To further explore a relationship between RPM-1 and CLEC-38 functions, we determined whether there are genetic interactions between clec-38 and rpm-1. For the DA and DB motor neurons, the axon guidance phenotype caused by unc-6(rh46) is suppressed by both clec-38 and rpm-1 mutations (Fig. 5A) (Li et al., 2008). In clec-38; rpm-1; unc-6(rh46) triple mutants, the penetrance is similar to mutants with unc-6(rh46) alone (Fig. 5A). Thus, the loss of clec-38 function reverses the effects of the loss of rpm-1. For the ventral migration of the AVM axon, we find a 90% penetrant defect in the rpm-1;slt-1double mutants, but only a 21% penetrant defect in rpm-1;slt-1;clec-38 mutants (Fig. 5B). Again, the loss of clec-38 function reverses the effects of the loss of rpm-1 function.

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Figure 5.

CLEC-38 regulates UNC-40 and RPM-1-dependent axon outgrowth. A, The DA and DB motor neuron dorsal guidance defects caused by the unc-6(rh46) mutation are partially suppressed by either the clec38(ur280) or the rpm-1(ur299) mutation. However, this effect is reversed in the triple mutant, clec-38(ur280);rpm-1(ur299);unc-6(rh46), suggesting that the suppressor activity of both mutations is dependent on each other. B, The rpm-1;slt-1 double mutants are as severe as slt-1;unc-6 mutants, suggesting that loss of rpm-1 function prevents UNC-6 signaling, likely by inhibiting UNC-40 signaling. This silencing effect is dependent on the SAX-3 receptor (Li et al., 2008). The silencing effect observed in rpm-1;slt-1 mutants is reversed in the triple clec-38;rpm-1;slt-1 mutants, suggesting that the increased UNC-40 activity by loss of clec-38 function reverses the inhibition caused by the rpm-1 mutation. Error bars indicate SE of proportions, and asterisks indicate statistically significant difference (**p < 0.005; ***p < 0.0005).

We also observe that double mutations have an effect on the control of axon extension. In rpm-1 mutants, the PLM anterior process axon often extends beyond the normal termination point (Fig. 6A–E) (Schaefer et al., 2000; Li et al., 2008). We find that the overextension caused by the loss of rpm-1 function is affected by the loss of clec-38 function. Interestingly, the penetrance of the overextension phenotype in clec-38; rpm-1 mutants is not greater than that of rpm-1 mutants; rather, the length of the extension increases (Fig. 6G,H). Whereas in rpm-1(ur299) mutants only 15% of the PLM axons extend beyond the position of the AVM cell body, 83% of the axons in clec-38; rpm-1 mutants extend beyond the AVM cell body (Fig. 6H). This effect is suppressed by the loss of UNC-40 in unc-40; clec-38; rpm-1 triple mutants, suggesting that the additional extension is likely attributable to the effect on UNC-40 by loss of clec-38 function. The effect is also suppressed by expression of the clec-38 transgene under the control of the mec-4 promoter, suggesting that clec-38 acts cell autonomously in the PLM touch neuron (data not shown).

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Figure 6.

CLEC-38 and RPM-1-dependent PLM axon outgrowth. Loss of clec-38 function can affect PLM axon outgrowth. A, Schematic diagram of the PLM longitudinal axon. B, C, In wild-type animals, the PLM extend to a position near the vulva. D, E, In rpm-1 mutants, the PLM axon overextends, passing the ALM cell body. F, G, In clec-38;rpm-1 mutants, the PLM axon extends beyond the AVM cell body position. The neurons were visualized with zdIs5 [mec-4::gfp] in C, E, and G. H, Loss of rpm-1 function causes overextension of PLM axon. Loss of both clec-38 and rpm-1 function cause longer extension of PLM, which crosses the AVM. This phenotype is dependent on unc-40 because the triple mutant, clec-38;rpm-1;unc-40, phenocopies the rpm-1 single mutant. Error bars indicate SE of proportions, and asterisks indicate statistically significant difference (***p < 0.0005). Anterior is to the left, and dorsal is up. Scale bars, 20 μm.

clec-38 regulates the UNC-40::GFP expression pattern

To further explore the relationship between UNC-40 and CLEC-38, we used transgenic strains expressing functional UNC-40::GFP. In the first case, we used a transgene that contains the unc-40 promoter sequence to drive UNC-40::GFP expression (Chan et al., 1996). Although the construct used in making this transgenes may have an in-frame deletion of 40 aa in the third Ig domain of the protein (Joe Culotti, personal communication), it is able to rescue unc-40 null mutant phenotypes (Chan et al., 1996). In this strain, the GFP signal is detectable in all cells during early gastrulation and then gradually decreases, with later expression observed in neurons and motile cells (Chan et al., 1996). We observe that 20% (n = 50) of the clec-38(+) animals with UNC-40::GFP expression have neurons with abnormal morphologies, including multiple processes (Fig. 7). Excess axon outgrowth and the abnormal neuronal morphology is likely caused by higher levels of UNC-40 because similar phenotypes are associated with the expression of constitutively active forms of UNC-40 in neurons (Gitai et al., 2003). Compared with the clec-38(+) animals, 80% (n = 50) of mutants heterozygous for the clec-38 mutation have severe morphological defects (Fig. 7) and 100% (n = 50) of mutants homozygous for the clec-38 mutation die as embryos. In some individual neurons, differences in the intensity of the GFP signal were noticeable. For example, in ventral cord motor neurons, only weak expression was observed in 5% (n = 50) of the clec-38(+) animals, whereas 45% (n = 50) of clec-38 heterozygous mutants showed strong UNC-40::GFP expression (Fig. 7A,B). From these observations, we infer that high levels of UNC-40::GFP cause morphological changes and that loss of clec-38 activity influences the levels or cellular distribution of UNC-40. Analyses of UNC-5::GFP and SAX-3::GFP expression patterns in clec-38 mutants did not reveal major differences from the patterns seen in the wild-type background (data not shown).

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Figure 7.

CLEC-38 affects UNC-40 expression. A, B, Ventral cord (vc) motor neurons of evEx66[unc-40::GFP] and clec-38(+/−);evEx66[unc-40::GFP] animals, respectively. Arrowheads indicate the neuronal cell bodies. UNC-40::GFP signal is weak in the wild-type neurons and is strong in clec-38(+/−) background. Anterior is to the left, and dorsal is up. C, D, Ventral cord (vc) and tail neurons (arrows) of evEx66[unc-40::GFP] and clec-38(+/−);evEx66[unc40::GFP] animals, respectively. Note a higher fluorescence signal is observed in clec-38(+/−) animals compared with wild-type animals. Also, in clec-38(+/−) animals, there are extra branches from ventral cord (arrowheads). Ventrolateral view with anterior to the top. n = 50 for each group. Scale bars, 20 μm. E, F, HSN neuron of wild-type and clec-38 mutant animals expressing a transgene, kyEx1212[punc-86::UNC-40::GFP]. In wild-type HSN, UNC-40::GFP is localized to membranes at low level, whereas in clec-38 mutants, cell morphology is changed (arrow) with strong GFP signal and multipolar outgrowths (arrowheads). Shown is a lateral view with anterior on left. Scale bars, 10 μm.

We observe that overexpression of the above unc-40::gfp transgene causes severe morphological changes throughout the animal. However, because of the possible effects of the deletion within the unc-40 sequence and because the identification of individual neurons is difficult in these strains (Fig. 7C,D), we also examined the effects of a different unc-40 transgene that is expressed in fewer cells. We used a transgene that expresses UNC-40:GFP under the control of the unc-86 promoter, which drives expression in a subset of cells, which include the HSN neuron (Adler et al., 2006) (Fig. 7E). We observe that expression of this transgene in clec-38 mutants results in strong UNC-40::GFP expression and HSN morphological changes, including multiple axon outgrowths (Fig. 7F). In 78% of clec-38(ur280) mutants (n = 115), there is strong GFP signal in HSN compared with 25% (n = 105) in clec-38(+) animals. Furthermore, in 95% of the mutants, the HSN cell morphology is abnormal and has excessive axon outgrowth. These morphological defects are observed in all larval stages (data not shown). Together, these results suggest that loss of clec-38 function enhances UNC-40::GFP expression. This is consistent with the genetic results that indicate clec-38 negatively regulates UNC-40 activity.