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...

During development of the nervous system, growing axons rely on guidance molecules to direct axon pathfinding. A well-characterized family of guidance molecules are the membrane-associated ephrins, which together with their cognate Eph receptors, direct axon navigation in a contact-mediated fashion. In C. elegans, the ephrin-Eph signaling system is conserved and is best characterized for their roles in neuroblast migration during early embryogenesis. This study demonstrates a role for the C. elegans ephrin EFN-4 in axon guidance. We provide both genetic and biochemical evidence that is consistent with the C. elegans divergent L1 cell adhesion molecule LAD-2 acting as a non-canonical ephrin receptor to EFN-4 to promote axon guidance. We also show that EFN-4 probably functions as a diffusible factor because EFN-4 engineered to be soluble can promote LAD-2-mediated axon guidance. This study thus reveals a potential additional mechanism for ephrins in regulating axon guidance and expands the repertoire of receptors by which ephrins can signal.

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 biosynthesis genes and HSPG core proteins, which function cell autonomously in the AIY neurons. This is the first report of an epidermal ephrin providing a developmental cue to the nervous system. This phenotype is strongly suppressed by mutations in HSPG biosynthetic enzymes and in vitro binding studies have confirmed that KAL-1 can bind the HSPGs sdn-1/syndecan and gpn-1/glypican (Bulow et al. 2002, Hudson et al. 2006 Tornberg et al. 2012). HSPGs are required for many aspects of nervous system development in both vertebrates and invertebrates, including cell migration, axon guidance and synaptogenesis (Rhiner et al. 2005;Van Vactor et al. 2006;Kinnunen 2014). Considering the importance of both HSPGs and the Eph/ephrin signaling during nervous system development, surprisingly little research has been dedicated to possible interactions between these pathways (Irie et al. 2008;Holen et al. 2011). In this study, we focus on the interplay between ephrins and HSPGs in the development of C. elegans AIY interneurons.We show that the ephrin EFN-4 is required non-cell autonomously to promote AIY primary neurite outgrowth, functioning in parallel with SDN-1/syndecan in this process. We also show that in a C. elegans model of X-linked KS, EFN-4 plays a role in promoting AIY ectopic neurite branching, where it again functions non-cell autonomously. Finally, we show that SDN-1/syndecan and GPN-1/glypican have cell autonomous yet mutually antagonistic roles in ectopic 6 neurite formation. This is the first report of an ephrin acting non-cell autonomously from the epidermis to regulate neurite outgrowth and branching. 7 MATERIALS AND METHODS Strains and maintenanceC. elegans strains were grown on nematode growth medium plates (NGM Lite) at 20°C according to Brenner (1974). All analyses were conducted at 20°C unless otherwise noted. The following mutations were used in the course of this embryonic morphogenesis, axon outgrowth and synapse formation Ackley et al. 2005). We found that ptp-3(mu256) mutants also exhibits defects in both primary neurite outgrowth and kal-1(gf) ectopic branching. We did not attempt further analysis of efn-4 and ptp-3 genetic interactions due to the strong synthetic-lethal phenotype observed betwe...