SummaryFLRTs are broadly expressed proteins with the unique property of acting as homophilic cell adhesion molecules and as heterophilic repulsive ligands of Unc5/Netrin receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. Here we use X-ray crystallography to reveal the distinct structural bases for FLRT-mediated cell adhesion and repulsion in neurons. We apply this knowledge to elucidate FLRT functions during cortical development. We show that FLRTs regulate both the radial migration of pyramidal neurons, as well as their tangential spread. Mechanistically, radial migration is controlled by repulsive FLRT2-Unc5D interactions, while spatial organization in the tangential axis involves adhesive FLRT-FLRT interactions. Further, we show that the fundamental mechanisms of FLRT adhesion and repulsion are conserved between neurons and vascular endothelial cells. Our results reveal FLRTs as powerful guidance factors with structurally encoded repulsive and adhesive surfaces.

Netrin-1 induces repulsive axon guidance by binding to the mammalian Unc5 receptor family (Unc5A-Unc5D). Mouse genetic analysis of selected members of the Unc5 family, however, revealed essential functions independent of Netrin-1, suggesting the presence of other ligands. Unc5B was recently shown to bind fibronectin and leucine-rich transmembrane protein-3 (FLRT3), although the relevance of this interaction for nervous system development remained unclear. Here, we show that the related Unc5D receptor binds specifically to another FLRT protein, FLRT2. During development, FLRT2/3 ectodomains (ECDs) are shed from neurons and act as repulsive guidance molecules for axons and somata of Unc5-positive neurons. In the developing mammalian neocortex, Unc5D is expressed by neurons in the subventricular zone (SVZ), which display delayed migration to the FLRT2-expressing cortical plate (CP). Deletion of either FLRT2 or Unc5D causes a subset of SVZ-derived neurons to prematurely migrate towards the CP, whereas overexpression of Unc5D has opposite effects. Hence, the shed FLRT2 and FLRT3 ECDs represent a novel family of chemorepellents for Unc5-positive neurons and FLRT2/ Unc5D signalling modulates cortical neuron migration.

Highlights d Crystal structures reveal binding site for Latrophilin on the Teneurin YD shell d A ternary Latrophilin-Teneurin-FLRT complex forms in vitro and in vivo d Latrophilin controls cortical migration by binding to Teneurins and FLRTs d Latrophilin elicits repulsion of cortical cell bodies/small neurites but not axons