The Caenorhabditis elegans Ephrin EFN-4 Functions Non-cell Autonomously with Heparan Sulfate Proteoglycans to Promote Axon Outgrowth and Branching

Abstract

The Eph receptors and their cognate ephrin ligands play key roles in many aspects of nervous system development. These interactions typically occur within an individual tissue type, serving either to guide axons to their terminal targets or to define boundaries between the rhombomeres of the hindbrain. We have identified a novel role for the Caenorhabditis elegans ephrin EFN-4 in promoting primary neurite outgrowth in AIY interneurons and D-class motor neurons. Rescue experiments reveal that EFN-4 functions non-cell autonomously in the epidermis to promote primary neurite outgrowth. We also find that EFN-4 plays a role in promoting ectopic axon branching in a C. elegans model of X-linked Kallmann syndrome. In this context, EFN-4 functions non-cell autonomously in the body-wall muscle and in parallel with HS modification genes and HSPG core proteins. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system. KEYWORDS ephrin; Eph receptor; heparan sulfate proteoglycan; axon outgrowth; axon branching A CCURATE development of the central nervous system requires contributions both from the extracellular environment in the form of guidepost cues and secreted guidance molecules and from contact with adjacent neurons or other tissues in the form of cell-surface receptors that can detect and transduce navigation cues. Guideposts typically take the form of specific tissue types such as the ventral midline of Caenorhabditis elegans or Drosophila, which provides a permissive environment for neurons to migrate along, while secreted guidance molecules provide spatial information and navigation instructions in the form of repulsive or attractive cues (Chilton 2006;Killeen and Sybingco 2008). Many studies have identified how individual neurons and guidance cues act to provide spatial and navigation information, but how does a single cell that possesses multiple axon guidance receptors find its target when exposed to multiple guidance cues? The nervous system of the nematode C. elegans provides a simple and defined model to examine the interplay between multiple neuronal guidance systems. The morphology and connectivity of C. elegans neurons have been established by serial-section electron microscopy, and the C. elegans genome possesses orthologs of most vertebrate axon guidance and guidepost genes (White et al. 1986;Bargmann 1998;Chisholm and Jin 2005;Ackley 2014). One of the most important classes of axon guidance molecules is the Eph receptor tyrosine kinases and their cognate ligands, the ephrins (Flanagan 2006;Lisabeth et al. 2013;Cayuso et al. 2015). Eph receptors and ephrins are required for the accurate connectivity of many parts of the vertebrate brain and also have roles in cell adhesion and embryonic morphogenesis (George et al. 1998;Chin-Sang et al. 1999;Chin-Sang et al. 2002;Klein 2012). We previously showed that the C. elegans ephrin EFN-4 functions in concert with the KAL-1/anosmin-heparan sulfate proteoglycan (HSPG) pathway to regulate neuroblast migration during...

Diverse roles for glycosaminoglycans in neural patterning

“…First, there is the combinatorial use of different HSPG core proteins that mediate a particular developmental process (e.g., (Kinnunen, ; Saied‐Santiago et al, ). Second, the core proteins may function from different tissues (e.g., Díaz‐Balzac et al, ; Schwieterman et al, ). Third, the combinatorial use of defined modifications may results in unique, cell‐specific glycan patterns.…”

“…a) (Kinnunen, ; Saied‐Santiago et al, ). Moreover, an axon branching phenotype in C. elegans that is dependent on the expression of the extracellular cell adhesion protein KAL‐1/Anosmin‐1 (mutant in Kallmann syndrome) (Bülow et al, ) is mediated by specific HSPG core proteins that act in a partially redundant manner to mediate branching (Díaz‐Balzac et al, ; Schwieterman et al, ).…”

“…First, the genetic redundancy observed in double mutants of HSPG core proteins (see above), some of which are known to act in different tissues during neuronal migration and patterning, suggest that HSPGs and their attached HS chains may act from different tissues (Díaz‐Balzac et al, ). Second, rescue experiments of mutations in HSPG core proteins and modifying enzymes with different tissue specific heterologous promoters argue that HS from different tissues is involved in mediating the kal‐1‐ dependent neuronal branching paradigm in C. elegans (Díaz‐Balzac et al, ; Schwieterman et al, ). Lastly, recent findings suggest that two genes involved in GAG biosynthesis, namely the chondroitin synthase ( sqv‐5 ) and an UDP‐sugar transporter ( sqv‐7 ) function cell nonautonomously to pattern the hermaphrodite specific neurons (HSN) (Pedersen et al, ).…”

ngn-1/neurogenin Activates Transcription of Multiple Terminal Selector Transcription Factors in theCaenorhabditis elegansNervous System

“…To further examine the genetic interactions between candidate axon guidance genes and ngn-1 during axon outgrowth, ngn-1; candidate gene double mutants were assayed for axon outgrowth and guidance defects in the AIY interneurons (Wang et al, 2008;Christensen et al, 2011;Hartin et al, 2015;Schwieterman et al, 2016). Each double mutant showed similar average AIY axon lengths when compared to ngn-1(ok2200) alone, suggesting that the ok2200 phenotype masks defects seen in each single mutant (Supplemental Figure 3).…”

Deciphering functional glycosaminoglycan motifs in development

“…In C. elegans , KAL-1-dependent neurite branching relies on HS 6- O- sulfation, HS C5- epimerization (i.e. iduronic acid), HS 3 -O -sulfation and, to a lesser extent, on HS 2- O- sulfation [13••,14,15•,32,33•]. Thus, KAL-1 neuronal function is coordinated by an HS motif comprising iduronic acid and glucosamine with sulfation on the C6 carbon atom (Figure 2).…”