Robo2 shapes Slit-dependent muscle repulsion by altering the association of Slit to tendon cell surface. Elly Ordan¹, Marko Brankatschk², Frank Schnorrer³, Talila Volk¹. 1) molecular genetics, Weizmann Institute of Science, Rehovot, Israel; 2) Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany; 3) Muscle Dynamics,Max-Planck-Institute for Biochemistry, Munich,Germany.

   Every segment of the Drosophila embryo contains a highly reproducible pattern of muscles, which is the result of muscle specification, fusion, migration, and attachment. The information regarding the control of muscle migration and targeting to tendon cells is limited. The Robo-Slit pathway has emerged in a number of studies concerning muscle pattern formation. This study focuses on Robo2-mediated Slit signaling in muscle migration. We found that Robo2, although expressed by the Slit-secreting tendon cells and not by muscles, was essential for proper migration of a subset of muscles. Robo2 and Slit genetically interact in the context of muscle migration, and overexpression of Robo2 by the ectoderm induced Slit cleavage. Moreover, consistent with a functional significance of Slit cleavage we found that uncleavable Slit, inserted by homologous recombination into the slit locus did not rescue the effects of Slit loss of function on muscle migration. However, membrane-bound, uncleavable Slit did rescue slit phenotype, suggesting that Slit cleavage is essential for its association with the tendon cell membrane. Consistent with this idea, membrane-bound uncleavable Slit is primarily detected on tendon cells membrane and can partially rescue the muscle phenotype of robo2. Live imaging has shown that the muscles migrate in close proximity to Slit- and Robo2-expressing cells but avoid entering their expression domain. Based on these findings, we propose a model whereby tendon-cell-expressed Slit is secreted, trapped by Robo2 and undergoes cleavage, which is necessary for Slit tethering to the membrane. The membrane-tethered Slit is then presented to the migrating muscle and serves as a short-range repellent of the migrating muscle.