Domain-specific mutations in unc-6/Netrin differentially affect dorsal-ventral axon pathfinding in Caenorhabditis elegans

ABSTRACT UNC-6/Netrin is a conserved regulator of dorsal-ventral axon and cell migrations. Here, we identified missense mutations in distinct UNC-6 domains and assessed their roles in dorsal VD/DD motor axon guidance and ventral anterior ventral microtubule (AVM) axon guidance. A missense mutation in a conserved residue of the laminin N-terminal (LN) domain (G289D) resulted in dorsal and ventral axon guidance defects similar to the unc-6 null. A distinct missense mutation in the LN domain (S120F) strongly perturbed ventral AVM axon guidance with minimal effects on dorsal VD/DD axon guidance. Mutations altering cysteine residues involved in disulfide bonding in the epidermal growth factor (EGF) domains were analyzed. EGF1(C321G) and EGF2(C347Y) caused both ventral and dorsal axon guidance defects, whereas EGF3(C410Y) specifically disrupted dorsal axon guidance. The crystal structure of UNC-6 shows conserved N-linked glycosylation at N114 and N128. These sites were not solely required for axon guidance, but mutations interacted genetically with unc-40 and unc-5 mutations, indicating that these residues have a role in UNC-6 signaling. Our results reveal the effects of UNC-6 domains on dorsal-ventral axon guidance and will inform studies on how these distinct UNC-6 domains interact with guidance receptors (e.g. UNC-40/DCC and UNC-5) and other extracellular molecules to mediate dorsal-ventral axon guidance.