A non-signaling role of Robo2 in tendons is essential for Slit processing and muscle patterning

Coordinated locomotion of an organism relies on the development of proper musculoskeletal connections. In Drosophila, the Slit-Robo signaling pathway guides muscles to tendons. Here, we show that the Slit receptor Roundabout 2 (Robo2) plays a non-cell-autonomous role in directing muscles to their corresponding tendons. Robo2 is expressed by tendons, and its non-signaling activity in these cells promotes Slit cleavage, producing a cleaved Slit N-terminal guidance signal that provides short-range signaling into muscles. Consistently, robo2 mutant embryos exhibited a muscle phenotype similar to that of slit, which could not be rescued by muscle-specific Robo2 expression but rather by ectodermally derived Robo2. Alternatively, this muscle phenotype could be induced by tendon-specific robo2 RNAi. We further show that membrane immobilization of Slit or its N-terminal cleaved form (Slit-N) on tendons bypasses the functional requirement for Robo2 in tendons, verifying that the major role of Robo2 is to promote the association of Slit with the tendon cell membrane. Slit-N tends to oligomerize whereas full-length uncleavable Slit does not. It is therefore proposed that Slit-N oligomers, produced at the tendon membrane by Robo2, signal to the approaching muscle by combined Robo1 and Robo3 activity. These findings establish a Robo2-mediated mechanism, independent of signaling, that is essential to limiting Slit distribution and which might be relevant to the regulation of Slit-mediated short-range signaling in additional systems.