TheC. elegansephrin 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 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...