L1CAM/Neuroglian controls the axon–axon interactions establishing layered and lobular mushroom body architecture

The establishment of neuronal circuits depends on the guidance of axons both along and in between axonal populations of different identity; however, the molecular principles controlling axon–axon interactions in vivo remain largely elusive. We demonstrate that the Drosophila melanogaster L1CAM homologue Neuroglian mediates adhesion between functionally distinct mushroom body axon populations to enforce and control appropriate projections into distinct axonal layers and lobes essential for olfactory learning and memory. We addressed the regulatory mechanisms controlling homophilic Neuroglian-mediated cell adhesion by analyzing targeted mutations of extra- and intracellular Neuroglian domains in combination with cell type–specific rescue assays in vivo. We demonstrate independent and cooperative domain requirements: intercalating growth depends on homophilic adhesion mediated by extracellular Ig domains. For functional cluster formation, intracellular Ankyrin2 association is sufficient on one side of the trans-axonal complex whereas Moesin association is likely required simultaneously in both interacting axonal populations. Together, our results provide novel mechanistic insights into cell adhesion molecule–mediated axon–axon interactions that enable precise assembly of complex neuronal circuits.