Minimal structural elements required for midline repulsive signaling and regulation ofDrosophilaRobo1

The Roundabout (Robo) family of axon guidance receptors has a conserved ectodomain arrangement of five immunoglobulin-like (Ig) domains plus three fibronectin type III (Fn) repeats. Based on the strong evolutionary conservation of this domain structure among Robo receptors, as well asin vitrostructural and domain-domain interaction studies of Robo family members, this ectodomain arrangement is predicted to be important for Robo receptor signaling in response to Slit ligands. Here, we define the minimal ectodomain structure required for Slit binding and midline repulsive signalingin vivobyDrosophilaRobo1. We find that the majority of the Robo1 ectodomain is dispensable for both Slit binding and repulsive signaling. We show that a significant level of midline repulsive signaling activity is retained when all Robo1 ectodomain elements apart from Ig1 are deleted, and that the combination of Ig1 plus one additional ectodomain element (Ig2, Ig5, or Fn3) is sufficient to restore midline repulsion to wild type levels. Further, we find that deleting four out of five Robo1 Ig domains (Delta Ig2-5) does not affect negative regulation of Robo1 by Commissureless (Comm) or Robo2, while variants lacking all three fibronectin repeats (Delta Fn1-3 and Delta Ig2-Fn3) are insensitive to regulation by both Comm and Robo2, signifying a novel regulatory role for Robo1's Fn repeats. Our results provide anin vivoperspective on the importance of the conserved 5+3 ectodomain structure of Robo receptors, and suggest that specific biochemical properties and/or ectodomain structural conformations observedin vitrofor domains other than Ig1 may have limited significance forin vivosignaling in the context of midline repulsion.