ABSTRACT
Slit is a secreted protein that has a canonical function of repelling growing axons from the CNS midline. The full-length Slit (Slit-FL) is cleaved into Slit-N and Slit-C fragments, which have potentially distinct functions via different receptors. Here, we report that the BMP-1/Tolloid family metalloprotease Tolkin (Tok) is responsible for Slit proteolysis in vivo and in vitro. In Drosophilatok mutants lacking Slit cleavage, midline repulsion of axons occurs normally, confirming that Slit-FL is sufficient to repel axons. However, longitudinal axon guidance is highly disrupted in tok mutants and can be rescued by midline expression of Slit-N, suggesting that Slit is the primary substrate for Tok in the embryonic CNS. Transgenic restoration of Slit-N or Slit-C does not repel axons in Slit-null flies. Slit-FL and Slit-N are both biologically active cues with distinct axon guidance functions in vivo Slit signaling is used in diverse biological processes; therefore, differentiating between Slit-FL and Slit fragments will be essential for evaluating Slit function in broader contexts.
Subject(s)
Axons/metabolism , Bone Morphogenetic Protein 1/metabolism , Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , Nerve Tissue Proteins/metabolism , Proteolysis , Amino Acid Sequence , Animals , Axon Guidance , Cell Membrane/metabolism , Drosophila Proteins/chemistry , Epistasis, Genetic , Extracellular Space/metabolism , Models, Biological , Mutation/genetics , Nerve Tissue Proteins/chemistry , Phenotype , Protein BindingABSTRACT
The Slit protein is a major midline repellent for central nervous system (CNS) axons. In vivo, Slit is proteolytically cleaved into N- and C-terminal fragments, but the biological significance of this is unknown. Analysis in the Drosophila ventral nerve cord of a slit allele (slit-UC) that cannot be cleaved revealed that midline repulsion is still present but longitudinal axon guidance is disrupted, particularly across segment boundaries. Double mutants for the Slit receptors Dscam1 and robo1 strongly resemble the slit-UC phenotype, suggesting they cooperate in longitudinal axon guidance, and through biochemical approaches, we found that Dscam1 and Robo1 form a complex dependent on Slit-N. In contrast, Robo1 binding alone shows a preference for full-length Slit, whereas Dscam1 only binds Slit-N. Using a variety of transgenes, we demonstrated that Dscam1 appears to modify the output of Robo/Slit complexes so that signaling is no longer repulsive. Our data suggest that the complex is promoting longitudinal axon growth across the segment boundary. The ability of Dscam1 to modify the output of other receptors in a ligand-dependent fashion may be a general principle for Dscam proteins.
