RPM-1, a Caenorhabditis elegans protein that functions in presynaptic differentiation, negatively regulates axon outgrowth by controlling SAX-3/robo and UNC-5/UNC5 activity

Abstract

Changes in axon outgrowth patterns are often associated with synaptogenesis. Members of the conserved Pam/Highwire/RPM-1 protein family have essential functions in presynaptic differentiation. Here, we show that Caenorhabditis elegans RPM-1 negatively regulates axon outgrowth mediated by the guidance receptors SAX-3/robo and UNC-5/UNC5. Loss-of-function rpm-1 mutations cause a failure to terminate axon outgrowth, resulting in an overextension of the longitudinal PLM axon. We observe that PLM overextension in rpm-1 mutants is suppressed by sax-3 and unc-5 loss-of-function mutations. PLM axon overextension is also induced by SAX-3 overexpression, and the length of extension is enhanced by loss of rpm-1 function or suppressed by loss of unc-5 function. We also observe that loss of rpm-1 function in genetic backgrounds sensitized for guidance defects disrupts ventral AVM axon guidance in a SAX-3-dependent manner and enhances dorsal guidance of DA and DB motor axons in an UNC-5-dependent manner. Loss of rpm-1 function alters expression of the green fluorescent protein (GFP)-tagged proteins, SAX-3::GFP and UNC-5::GFP. RPM-1 is known to regulate axon termination through two parallel genetic pathways; one involves the Rab GEF (guanine nucleotide exchange factor) GLO-4, which regulates vesicular trafficking, and another that involves the F-box protein FSN-1, which mediates RPM-1 ubiquitin ligase activity. We show that glo-4 but not fsn-1 mutations affect axon guidance in a manner similar to loss of rpm-1 function. Together, the results suggest that RPM-1 regulates axon outgrowth affecting axon guidance and termination by controlling the trafficking of the UNC-5 and SAX-3 receptors to cell membranes.

Figure 1.

rpm-1(ur299) affects guidance receptor activities for dorsal axon guidance. A, Schematic diagram of the migration of DA/DB motorneurons. These neurons extend axons dorsally away from ventral UNC-6 sources. B–E, The phenotypes of DA/DB axon guidance in wild-type (B, C) and unc-6(rh46) mutant larva (D, E). DA and DB motor axons in L4-stage animals were visualized with evIs82a [unc-129::gfp]. In the image of the unc-6(rh46) mutant larva, some axons have extended and joined the dorsal sublateral nerve. F, Quantification of DA/DB dorsal guidance defect in various mutants. rpm-1(ur299) suppresses the dorsal guidance defects caused by the unc-6(rh46) and unc-6(e78) hypomorphic alleles but not the null allele, unc-6(ev400). Triple-mutant analysis suggests that suppression by rpm-1(ur299) is dependent on unc-5 function. Asterisks indicate statistically significant difference (*p < 0.05). Anterior is left and dorsal is up; vc, ventral cord; dc, dorsal cord; dsl, dorsal–sublateral cord. Error bars represent SEs of proportions. Scale bars, 20 μm.

Figure 2.

rpm-1(ur299) affects guidance receptor activities for ventral axon migration. 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 (C) and mutant larva (E). The AVM axons in L4 stage animals were visualized with zdIs5 [mec-4::gfp]. F, Quantification of the AVM guidance defects in different mutant backgrounds. 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 effect is not observed in rpm-1;sax-3 double mutants indicating that the ability of the rpm-1 mutation to inhibit UNC-6 signaling is dependent on the SAX-3 receptor. This is consistent with biochemical evidence indicating that an interaction between UNC-40/DCC and SAX-3/robo silences the guidance effects of UNC-6/netrin (Stein and Tessier-Lavigne, 2001). The silencing effect occurs in rpm-1;slt-1 mutants, indicating that the SLT-1 ligand is not required. The silencing effect observed in rpm-1;slt-1 mutants is reversed in the triple, rpm-1;slt-1;sax-3 showing that the silencing effect is mediated by SAX-3 receptor. Also, the AVM defects caused by unc-6 or unc-40 mutants are suppressed by loss of rpm-1 function suggesting the upregulation of SAX-3 improves SLT-1 signaling. Asterisks indicate statistically significant difference (*p < 0.05; **p < 0.005). Anterior is left and dorsal is up. Error bars represent SEs of proportions. Scale bars, 20 μm.

Figure 3.

rpm-1(ur299) affects guidance receptor activities for longitudinal axon migration. A, Schematic diagram of the PLM longitudinal axon. B, C, In wild-type animals, PLM axons extend to a region near the vulva (arrowhead). D–G, In rpm-1(ur299) (D, E) and rpm-1(ur299);gmIs28 (F, G) animals, the PLM axon overextends, passing the ALM cell body or AVM cell body (arrowhead). H–J, Quantification of the PLM overextension in various mutant backgrounds. The PLM axons were visualized with zdIs5[mec-4::gfp]. Loss of rpm-1 function causes overextension of PLM axon. H, The penetrance of the overextension phenotype of rpm-1 is reduced by loss of unc-5, slt-1, or sax-3 function suggesting the same pathways that control directed dorsal and ventral axon outgrowth also control the longitudinal PLM axon extension. I, J, Overexpression of SAX-3 in mechanosensory neurons, which includes PLM, causes the PLM axon overextension phenotype. SAX-3 overexpression was obtained using a transgenic line, which carried the integrated Pmec7::SAX-3::GFP transgene (gmIs28) (Watari-Goshima et al., 2007). The overextension phenotype caused by overexpression of SAX-3 is suppressed by loss of unc-5 function, but not by the loss of unc-40 function. Overexpression of SAX-3 in rpm-1(ur299) mutant causes even longer extensions than the rpm-1 mutation alone, suggesting rpm-1 influences the SAX-3 function that regulates PLM axon extension. Asterisks indicate statistically significant difference (*p < 0.05; **p < 0.005; ***p < 0.0005). Anterior is left and dorsal is up. Error bars represent SEs of proportions. Scale bars, 20 μm.

Figure 4.

Overexpression of RPM-1 causes short PLM axon extension. The PLM axons were visualized with zdIs5 [mec-4::gfp]. RPM-1 overexpression was obtained using a transgenic line, which carried the integrated rpm-1::GFP transgene (juIs58). A, In wild-type animals, PLM axons stop their anterior extension near the vulva (arrow). B, In juIs58 worms, PLM displays short extension (arrowhead). C, Quantification of the PLM short extension in different mutant backgrounds. The PLM is also shorter in sax-3 and unc-5 mutants, suggesting that RPM-1 might negatively regulate SAX-3 and UNC-5 activity to affect PLM extension. Asterisks indicate statistically significant difference (*p < 0.05; **p < 0.005). Anterior is left and dorsal is up; stars indicate the position of vulva. Error bars represent SEs of proportions. Scale bars, 20 μm.

Figure 5.

RPM-1 affects SAX-3 and UNC-5 expression. SAX-3 and UNC-5 overexpression were obtained using transgenic animals kyEx253 [sax-3::GFP] (Zallen et al., 1998) and evIs98 [unc-5::GFP] (Killeen et al., 2002), respectively. A, Example of the posterior lateral region in kyEx253 [sax-3::GFP] larvae. No expression is detected in PLM neurons. B, Compared with A, in kyEx253 [sax-3::GFP] larvae with the rpm-1(ur299) mutation a strong GFP signal is observed in PLM neurons. Multiple processes are often present (arrowheads). C, Compared with A, in kyEx253 [sax-3::GFP] larvae with the rpm-1(ur299) mutation a strong GFP signal is observed in ventral cord motor neurons and their circumferential axons (arrowheads). D, Example of the lateral midbody region in evIs98 [unc-5::GFP] animals. E, Compared with D, in evIs98 [unc-5::GFP] larvae with the rpm-1(ur299) mutation the GFP signal is detected in PLM (arrowhead). Axons often show multiple short branches; shown are abnormal branches from the dorsal nerve cord (arrows). Anterior is left; dorsal is up; dc, dorsal cord; vc, ventral cord; vmn, ventral motor neurons. Stars indicate the position of vulva. Scale bars, 20 μm.

Figure 6.

RPM-1 affects the localization of SAX-3::GFP in ALM and PLM neurons. SAX-3 overexpression was observed in transgenic animals carrying an integrated Pmec7::SAX-3::GFP transgene (gmIs28) (Watari-Goshima et al., 2007). A, C, In wild-type animals, SAX-3::GFP is localized in the AVM and PLM cell bodies and axons in a punctate pattern. Cell bodies are magnified in the left image in each panel. B, D, In rpm-1 mutants, the number of SAX-3::GFP puncta are reduced and they appear more localized to the cell surface rather than to the cytoplasm. Anterior is left and dorsal is up. Arrows indicate the SAX-3::GFP puncta. Scale bars, 20 μm.

Figure 7.

A model for RPM-1 regulation of guidance receptor functions. A, We hypothesize that RPM-1 negatively regulates SAX-3 and UNC-5 activity at complexes on the cell surface membrane that promote outgrowth. Lack of RPM-1 or GLO-4 activity increases receptor axon outgrowth-promoting activity. B, RPM-1 is proposed to bind GLO-4 and positively regulate a Rab GEF GLO-4 pathway to promote vesicular trafficking for synaptogenesis (Grill et al., 2007). This RPM-1 activity may be activated by signals that promote synaptogenesis. The subsequent regulation of vesicular trafficking for presynaptic development might inhibit the trafficking of SAX-3 and UNC-5 receptors to the locations where complexes could form to promote axon outgrowth.