In Vivo Functional Analysis of Drosophila Robo1 Fibronectin Type-III Repeats

The repellant ligand Slit and its Roundabout (Robo) family receptors regulate midline crossing of axons during development of the embryonic central nervous system (CNS). Slit proteins are produced at the midline and signal through Robo receptors to repel axons from the midline. Disruption of Slit-Robo signaling causes ectopic midline-crossing phenotypes in the CNS of a broad range of animals, including insects and vertebrates. While previous studies have investigated the roles of Drosophila melanogaster Robo1's five Immunoglobulin-like (Ig) domains, little is known about the importance of the three evolutionarily conserved Fibronectin (Fn) type-III repeats. We have individually deleted each of Drosophila Robo1's three Fn repeats, and then tested these Robo1 variants in vitro to determine their ability to bind Slit in cultured Drosophila cells and in vivo to investigate the requirement for each domain in regulating Robo1's embryonic expression pattern, axonal localization, midline repulsive function, and sensitivity to Commissureless (Comm) downregulation. We demonstrate that the Fn repeats are not required for Robo1 to bind Slit or for proper expression of Robo1 in Drosophila embryonic neurons. When expressed in a robo1 mutant background, these variants are able to restore midline repulsion to an extent equivalent to full-length Robo1. We identify a novel requirement for Fn3 in the exclusion of Robo1 from commissures and downregulation of Robo1 by Comm. Our results indicate that each of the Drosophila Robo1 Fn repeats are individually dispensable for the protein's role in midline repulsion, despite the evolutionarily conserved "5 + 3" protein structure.