Evolution of NELL binding by dual-ligand-responsive axon guidance receptor Robo

Robo receptors are conserved across bilaterians and best known for their ability to mediate axonal repulsion in response to Slit family ligands. In mammals, this applies to Robo1 and Robo2, but mammalian Robo3 binds NELL proteins instead of Slits. The evolutionary origin of NELL-Robo interactions and the possible existence of dual-ligand responsiveness across species remain unknown. Here, we systematically analyzed Robo and NELL homologs across bilaterians and found that NELL-Robo binding is conserved among chordate Robos, but not in protostomes. We show that cephalochordate Robo and NELL can mediate axon repulsion in vitro, suggesting conserved functionality. We observed that conformational masking of the NELL-binding site is prevalent among chordate Robos, modulating NELL-Robo affinity. We also demonstrate that NELL-Robo complexes undergo liquid-liquid phase separation in vitro, a property preserved from cephalochordates to mammals. Our findings support a model in which an ancestral chordate Robo receptor was dual-responsive to Slit and NELL, still the case for some extant Robos, and vertebrate paralogs subfunctionalized, with full ligand specialization emerging in mammals.