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Genetic analysis of a novel family of repulsive guidance cues acting through Unc5 receptors

Final Report Summary - FLRT IN NEUROBIOLOGY (Genetic analysis of a novel family of repulsive guidance cues acting through Unc5 receptors)

The elaboration of precise connectivity patterns during the establishment of neuronal connections is a complex process which involves interaction between guidance receptors and extracellular signals. These signals, called guidance cues, are normally presented in an overlapping fashion and often trigger opposed effects such as repulsion and adhesion. A major puzzle in development is how a small number of guidance cues direct the vast repertoire of cellular behavior and how simultaneous inputs of several cues are interpret in order to guide axons to the appropriate target.

We focus on FLRT proteins, an emerging protein family implicated in neuronal development, and addressed this challenge together with Guillermina López-Bendito (Instituto de Neurociencias de Alicante, CSIC & Universidad Miguel Hernández, Spain) and Joaquim Egea (Institut de Recerca Biomedica, University of Lleida, Spain). We have found that FLRT3 is a novel co-receptor for Robo1 that is essential to regulate the cooperation between Netrin and Slit, acting as a modulator of Netrin-1/DCC attraction through Robo1. When rostral thalamic (rTh) axons are stimulated with Slit1 and Netrin-1, convergent activation of Robo1 and FLRT3 induces Netrin-1 attraction by upregulation of the netrin receptor DCC. We found that increased surface expression of DCC depends on the activation of PKA which is downstream Robo1 and FLRT3. Intermediate thalamic (iTh) axons, which express Robo1 but not FLRT3, do not show PKA activation and DCC upregulation when stimulated with Slit1 and Netrin-1. Given that Robo1 is expressed in both rTh and iTh, we asked whether ectopic expression of FLRT3 in non-responsive iTh may be sufficient to trigger DCC upregulation and therefore Netrin-1 attraction. Indeed, overexpression of FLRT3 in iTh neurons is sufficient to modulate the response to Netrin-1 and to drive their axons to a more rostral positions. Altogether, these results indicate that FLRT3 is sufficient to transform the behavior of iTh to that of rTh axons. These findings implicate, for the first time, FLRT3 as an important modulator in axon guidance.

FLRTs have the unique property of acting as homophilic cell adhesion molecules and as repulsive ligands of Unc5 receptors. How these functions direct cell behavior and the molecular mechanisms involved remain largely unclear. We examined these questions in detail in a second study together with Yvonne Jones (Division of Structural Biology, University of Oxford, UK) and Amparo Acker-Palmer (Neural & Vascular Guidance Group, Goethe University Frankfurt, Germany). Based on crystal structures for FLRTs and Unc5s, we designed mutants that specifically disrupt adhesive or repulsive functions of FLRT. These mutants allow us to dissect specifically the functions of FLRTs in both neuronal and cardiac development.
In stripe assays we found that FLRT3 acts as repulsive guidance cue on thalamocortical axons (TCAs) originating from the intermediate thalamus where Unc5B, but not FLRT3 is expressed. TCAs from the rostral thalamus co-express Unc5B and FLRT3. Here, we found that FLRT3 can signal through both Unc5B and FLRT3 in a parallel fashion, and that the adhesive FLRT interaction is responsible for attenuating the Unc5B-dependent repulsive signal.
We also found that FLRTs control distinct processes during cortical migration by repulsive and adhesive signals. FLRT2 expressed in the cortical plate delays the radial migration of neurons by binding Unc5D. In contrast, FLRT3 is expressed by migrating pyramidal neurons and modulates their tangential distribution, a phenomenon that is poorly understood. Interestingly, in the cortex FLRT3 acts through a distinct mechanism using adhesive molecular surfaces rather than interactions with Unc5 receptors. We further found that the fundamental adhesive/repulsive functions of FLRTs are conserved in cultures of vascular endothelial cells expressing FLRT3 and Unc5B. FLRT3 repels Unc5B-expressing endothelial tip cells and controls vascular branching in the developing mouse eye, which is an established system for vascular development.
Altogether, these results will be of direct value for researchers in the neurobiology and vascular biology fields. The conserved functional mechanisms we uncover also directly impact other fields concerned with FLRT-expressing tissues (heart, kidney, muscle, pancreas, lung etc.). Beyond mammalian biology, we showcase the importance of collaboration to effectively address the most challenging scientific questions