The Slit-binding Ig1 domain is required for multiple axon guidance activities of Drosophila Robo2

Drosophila Robo2 is a member of the evolutionarily conserved Roundabout (Robo) family of axon guidance receptors. The canonical role of Robo receptors is to signal midline repulsion in response to their cognate Slit ligands, which bind to the N-terminal Ig1 domain in most Robo family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 cooperate to signal Slit-dependent midline repulsion, while Robo2 also regulates the medial-lateral position of longitudinal axon pathways and acts non-autonomously to promote midline crossing of commissural axons. Although it is clear that Robo2 signals midline repulsion in response to Slit, it is less clear whether Robo2's other activities are also Slit-dependent. To determine which of Robo2's axon guidance roles depend on its Slit-binding Ig1 domain, we have used a CRISPR/Cas9-based strategy replace the endogenous robo2 gene with a robo2 variant from which the Ig1 domain has been deleted (robo2ΔIg1). We compare the expression and localization of Robo2ΔIg1 protein with that of full-length Robo2 in embryonic neurons in vivo, and examine its ability to substitute for Robo2 to mediate midline repulsion and lateral axon pathway formation. We find that removal of the Ig1 domain from Robo2ΔIg1 disrupts both of these axon guidance activities. In addition, we find that the Ig1 domain of Robo2 is required for its proper subcellular localization in embryonic neurons, a role that is not shared by the Ig1 domain of Robo1. Finally, we report that although FasII-positive lateral axons are misguided in embryos expressing Robo2ΔIg1, the axons that normally express Robo2 are correctly guided to the lateral zone, suggesting that Robo2 may guide lateral longitudinal axons through a cell non-autonomous mechanism.

Proteolytic cleavage of Slit by the Tolkin protease converts an axon repulsion cue to an axon growth cue in vivo

ABSTRACTSlit 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.

Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.