Building neuronal circuits: spatio-temporal dynamics and compartmentalization of axon guidance receptors – YADDLE

This proposal aims at exploring key molecular mechanisms controlling the wiring of neuronal circuits. A first critical step of this developmental program is axon navigation, during which all populations of projection neurons must extend their axons along very specific pathways to connect their proper target territory and start building their synapses. Altering the topography of axon navigation has dramatic consequences, leading to abnormal patterns of neuronal circuits. Functional consequences are obvious in strong cases where circuits are simply not formed or strongly impaired, but increasing contexts are reported, which link more subtle but defective axon navigation to neurological diseases, such autism spectrum disorders. Therefore, understanding the rules of axon navigation is a major goal and an absolute prerequisite for future therapeutic strategies of neuronal circuit repair.
Two decades of extensive work established that axon trajectories are controlled by environmental cues. Hence, focal sources and gradients of diffusible and membrane-attached cues having repulsive and attractive properties act in synergy to shape stereotypic axonal pathways. Nevertheless, generating the requested pathway diversity is an incredibly complex task, given that billions of neurons must be connected. How is this diversity encoded remains one of the most fascinating questions in neurodevelopment. Studies that revealed the remarkable potential of axon terminals, the growth cones, to vary their sensitivity to the guidance cues built the current view that diversity might arise from specific spatial and temporal control of guidance receptors and downstream signaling machinery. Despite many advances, information is still sparse on the dynamics of guidance receptors in the growth cones, their cell surface sorting, their spatial distribution and their rearrangement during guidance decisions.
This project brings together to highly complementary partners: V. Castellani (Partner 1, Lyon), a neurobiologist with years of experience in the mechanisms of axon guidance; and O. Thoumine (Partner 2, Bordeaux), a biophysicist with strong expertise in single molecule-based super-resolution microscopy applied to growth cones and synaptic receptors. V. Castellani has developed a unique experimental tissue culture set-up in chick embryo for live imaging recapitulating the physiological path-finding of a population of axons, and generated molecular tools to explore at subcellular scales the dynamics of guidance receptors in axons facing key guidance decisions for their navigation. O. Thoumine brings newly developed monomeric ligands conjugated to photostable organic dyes to study the dynamics and nanoscale distribution of those guidance receptors using super-resolution microscopy. The specific biological model is the navigation of commissural axons across the midline separating the two halves sides of the central nervous system, which is one of the most recognized contexts for exploring the modulations of axon responses to guidance cues. Commissural axons are initially attracted towards the midline. After the crossing, they gain responsiveness to local repellents which they did not perceive before. This switch prevents the axons from crossing back and expels them away. Slit proteins, processed into C-ter and N-ter fragments, and the Semaphorin3B (Sema3B) are the major spinal cord midline repellents. The current view supports that at least three repulsive signaling are set after midline crossing: Slit-N acting via Robo receptors, Slit-C acting via PlexinA1 receptor, and Sema3B acting via Neuropilin2/PlexinA1 receptor complex.
Using cutting-edge technologies, the consortium will explore the temporal changes of guidance receptor dynamics, the insertion of guidance receptors at the growth cone surface and their spatial compartmentalization in axon subdomains, as well as the mechanisms controlling these spatial and temporal sequences.