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. Robo receptors signal midline repulsion in response to Slit ligands, which bind to the N‐terminal Ig1 domain in most family members. In the Drosophila embryonic ventral nerve cord, Robo1 and Robo2 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. While 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 used a clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐based strategy to replace the endogenous robo2 gene with a robo2 variant lacking the Ig1 domain (robo2∆Ig1). We compare the expression and localization of Robo2∆Ig1 protein with 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 the removal of the Ig1 domain from Robo2∆Ig1 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 Robo2∆Ig1, 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.