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RPM-1 and DLK-1 Regulate Pioneer Axon Outgrowth by Controlling Wnt Signaling Eun Chan Park and Christopher Rongo*

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RPM-1 and DLK-1 Regulate Pioneer Axon Outgrowth by Controlling Wnt Signaling Eun Chan Park and Christopher Rongo* © 2018. Published by The Company of Biologists Ltd | Development (2018) 145, dev164897. doi:10.1242/dev.164897 RESEARCH ARTICLE RPM-1 and DLK-1 regulate pioneer axon outgrowth by controlling Wnt signaling Eun Chan Park and Christopher Rongo* ABSTRACT White et al., 1986). Conserved extracellular guidance cues, including β Axons must correctly reach their targets for proper nervous system Netrin/UNC-6, Slit/SLT-1 and TGF /UNC-129, guide the polarized function, although we do not fully understand the underlying migration of axons (Colavita et al., 1998; Garriga et al., 1993; Hao mechanism, particularly for the first ‘pioneer’ axons. In C. elegans, et al., 2001; Hedgecock et al., 1990; Ishii et al., 1992; Wadsworth, AVG is the first neuron to extend an axon along the ventral midline, 2002). However, few such cues have been identified for pioneers and this pioneer axon facilitates the proper extension and guidance of like AVG (Hutter, 2003). follower axons that comprise the ventral nerve cord. Here, we show Whereas AVG axon guidance does not depend on established that the ubiquitin ligase RPM-1 prevents the overgrowth of the AVG cues, the polarity of the AVG soma and the site of axon sprouting axon by repressing the activity of the DLK-1/p38 MAPK pathway. is regulated by the ubiquitin ligase PLR-1, which inhibits the Unlike in damaged neurons, where this pathway activates CEBP-1, surface expression of the Wnt receptors Ror/CAM-1 and Ryk/ we find that RPM-1 and the DLK-1 pathway instead regulate LIN-18 (Bhat et al., 2015; Moffat et al., 2014). Wnts are secreted the response to extracellular Wnt cues in developing AVG axons. glycoproteins that act as long-range signals in development The Wnt LIN-44 promotes the posterior growth of the AVG axon. In the (Gammons and Bienz, 2017; Loh et al., 2016). In the absence of absence of RPM-1 activity, AVG becomes responsive to a different PLR-1, the AVG neuron inappropriately responds to the Wnts Wnt, EGL-20, through a mechanism that appears to be independent CWN-1 and CWN-2, reorienting its polarity towards these anteriorly of canonical Fz-type receptors. Our results suggest that RPM-1 and expressed cues during embryogenesis (Harterink et al., 2011; the DLK-1 pathway regulate axon guidance and growth by preventing Kennerdell et al., 2009; Song et al., 2010). Unlike for AVG cell Wnt signaling crosstalk. polarity, the factors that regulate AVG anterior-posterior axon guidance remain unknown. KEY WORDS: Axon guidance, Wnt, p38 MAPK, C. elegans, RPM-1 One known regulator of anterior-posterior axon growth is RPM-1, a founding member of the Pam/Highwire/RPM-1 protein family (Grill et al., 2016; Schaefer et al., 2000). Loss of RPM-1 INTRODUCTION function results in abnormal neuronal morphology, axon overgrowth, Proper nervous system development requires that neurons extend impaired presynaptic differentiation, and internalization of postsynaptic axons to precise targets. Groups of neurons exhibit similar patterns of AMPA receptors (Opperman and Grill, 2014; Park et al., 2009; axon outgrowth depending on their spatial position and sequential Schaefer et al., 2000; Zhen et al., 2000). RPM-1 is a signaling hub timing during development. Growing ‘pioneer’ axons often blaze with multiple protein-protein interaction domains. RPM-1 binds the pathways through the nervous system, with ‘follower’ axons extending F-box protein FSN-1, and together they act as a ubiquitin ligase to along these pathways using the pioneers as a guide (Chitnis and target the DLK-1 MAPKKK for degradation (Liao et al., 2004; Kuwada, 1991; Hidalgo and Brand, 1997; Hutter, 2003; Klose and Nakata et al., 2005). In the absence of RPM-1, stabilized DLK-1 Bentley, 1989; McConnell et al., 1989). Extracellular signals act as acts through the MAPKK MKK-4 and the p38 MAPK PMK-3 to guidance cues for growing axons, yet how the nervous system activate the translation of the transcription factor CEBP-1 (Yan employs a small number of guidance cues over a vast array of neuron et al., 2009). This MAP kinase pathway, which is activated in types and developmental time points remains poorly understood response to damage, promotes axon regeneration; however, its role (Kaplan et al., 2014; Morales and Kania, 2017; Petrovic and in normal axon outgrowth is unclear. RPM-1 has a role in normal Schmucker, 2015). Modulation of the signal transduction pathways axon growth, as rpm-1 mutants have PLM mechanosensory neurons downstream of these guidance cues might provide one explanation for with overgrown axons and DA/DB motor neurons with dorsal- how the same cues are used in multiple contexts. ventral axon guidance defects (Li et al., 2008; Schaefer et al., 2000). In the nematode Caenorhabditis elegans, one well-studied RPM-1 regulates these axons by reducing the surface levels of pioneer neuron is AVG. The AVG soma is in the retrovesicular Robo/SAX-3 and UNC-5 guidance receptors (Li et al., 2008). This ganglion, is the first neuron to extend an axon along the ventral function is not mediated through FSN-1 or the DLK-1 MAP kinase midline, and is required for the guidance of the follower axons cascade; rather, RPM-1 separately binds to the Rab GEF GLO-4, that comprise the ventral nerve cord (Durbin, 1987; Hutter, 2003; which activates the Rab GLO-1 to regulate axon guidance and outgrowth (Grill et al., 2007; Li et al., 2008). Here, we reveal a new function for RPM-1: guidance and growth The Waksman Institute, Department of Genetics, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA. of the AVG pioneer axon through regulation of Wnt signaling crosstalk. Normally, AVG makes specific turns in the posterior and *Author for correspondence ([email protected]) arrests growth at the lumbar ganglion. In the absence of RPM-1, E.C.P., 0000-0002-6147-4088; C.R., 0000-0002-1361-5288 AVG overgrows past the lumbar ganglion and into the tip of the tail. AVG overgrowth in rpm-1 mutants is not mediated through GLO-4, Received 22 February 2018; Accepted 27 July 2018 but through the overactivation of the DLK-1/p38 MAPK pathway. DEVELOPMENT 1 RESEARCH ARTICLE Development (2018) 145, dev164897. doi:10.1242/dev.164897 Surprisingly, the downstream target of this pathway, CEBP-1, is not required. Instead, the Wnt LIN-44 and its Fz-type receptor LIN-17 promote posterior growth of the AVG axon, and in loss-of-function rpm-1 mutants, AVG responds to an additional Wnt ligand, EGL- 20, resulting in overgrowth. Our results indicate that RPM-1 inhibits AVG axon growth by modulating Wnt/Fz signaling, controlling the timing and specificity of ligand/receptor sensitivity. RESULTS RPM-1 and DLK-1 signaling regulates AVG axon outgrowth To analyze AVG axon growth, we examined kyIs51[Podr-2b::GFP] and otIs182[Pinx-18::GFP] transgenic animals, both of which express GFP in AVG and a handful of other neurons (Chou et al., 2001; Sarin et al., 2009). In wild type, AVG extends its axon during embryogenesis (Durbin, 1987). By the L1 larval stage, its axon has extended beyond the end of the intestine and preanal ganglion, exited the ventral midline, and migrated dorsally towards the dorsorectal ganglion (Fig. 1A). When AVG reaches the DVA soma in the dorsorectal ganglion, it then turns and migrates posteriorly to the end of the lumbar ganglion before stopping (Fig. 1B-G). The cues for this guidance are unknown. We previously identified alleles of rpm-1 in a screen for mutants with defective GLR-1 synaptic localization (Park et al., 2009). GLR-1 is expressed in AVG, and we noticed that rpm-1 mutants expressing GLR-1::GFP had an elongated axon that extended to the posterior tip of the tail (Fig. 1H,I). We classified animals based on whether AVG terminated growth at the preanal ganglion or dorsal rectal ganglion (indicative of undergrowth), the lumbar ganglion (the normal termination point in wild type), or the most posterior tip of the tail (indicative of overgrowth) (Fig. 1J). All rpm-1 mutants have AVG axons that overgrow to the tail tip. Expression of a wild-type rpm-1 cDNA from the glr-1 promoter, which is expressed in AVG and a handful of other interneurons (Hart et al., 1995; Maricq et al., 1995), restored normal AVG migration in these mutants (Fig. 1J). To characterize AVG axon guidance more thoroughly, we introduced kyIs51 into rpm-1(ok364) deletion (molecular null) mutants (Chou et al., 2001). We used DAPI staining to visualize neuronal and hypodermal nuclei in the tail, using conditions that Fig. 1. RPM-1 prevents AVG axon posterior overgrowth. (A) Diagram of the C. elegans posterior, including the position of neuron cell bodies (circles) for preserved GFP fluorescence. Unlike in wild type, the AVG axon in the preanal ganglion (dark blue), dorsorectal ganglion (light blue) and lumbar rpm-1(ok364) null mutants extends posteriorly to the tail tip ganglion (gray). The secretion of Wnt/LIN-44 from the tail hypodermis is (Fig. 2A-C), similar to what we observed in rpm-1(od14) mutants indicated by red shading. The green line indicates the wild-type AVG after the expressing GLR-1::GFP. Interestingly, we often observed the AVG completion of growth and guidance during embryogenesis. (B-G) L4-stage axon making a U-turn and migrating anteriorly back along the same nematodes expressing GFP in the AVG axon (arrow) (B,E) and DsRed2 in the ventral midline. Taken together, these results indicate that RPM-1 dorsorectal ganglion (C,F) from the indicated transgenes. Images in B-D and E-G are from slightly different focal planes to allow the separate visualization of acts cell autonomously to stop AVG axon migration.
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