Study of the spatio-temporal dynamics of guidance receptors during commissural axon navigation in the spinal cord

Abstract : During embryonic development, commissural axons are guided through the midline, crossing from one side of the CNS to the other one at specific time points and positions to project onto contralateral neurons. Several sources of attractive cues regulate their navigation. In addition, repulsive forces act at different steps to keep the axons along their path. In the developing spinal cord, commissural axons cross the midline in a ventral territory, the floor plate (FP). Commissural axons gain sensitivity to repellents present in the FP after their crossing. The setting of these novel properties is necessary for preventing the axons to cross back and also for pushing them towards FP exit. Various ligand/receptor couples have been reported to mediate these repulsive forces. Whether commissural axons gain response to all the repulsive cues at the same time is not known. Whether these repulsive cascades have specific functions is suggested by different outcome of their invalidation in mouse models, but how are set these differences also remains unknown. We hypothesized that the generation of functional specificities could be achieved though specific controls of the spatial and temporal dynamics of guidance receptors at the growth cone surface. During my PhD, I developed a set up for time-lapse imaging of “open book” spinal cords, to monitor the dynamics of guidance receptors in axons experiencing native guidance decisions across the midline. To visualize their cell surface sorting, receptors were fused to the pH-sensitive GFP, pHLuorin, whose fluorescence at neutral pH reports membrane protein pools (Nawabi et al, 2010; Delloye-Bourgeois et al, 2014), and were expressed in spinal commissural neurons through in ovo electroporation. This paradigm revealed striking differences in the temporal dynamics of Nrp2, Robo1, Robo2 and PlexinA1, the receptors known to mediate the responsiveness to the major midline repellents referenced in vertebrates: Slit-Ns, Slit-Cs and Semaphorin3B. Moreover, using super-resolution microscopy, I could evidence that PlexinA1 and Robo1 are sorted in distinct subdomains of commissural growth cones navigating the floor plate. I also introduced the pHLuo-tagged receptors in the mouse embryo. These experiments showed that the temporal sequences established in the chick are conserved in the mouse, and that FP crossing in Robo1/2 mutant embryos was rescued in growth cones that could achieve cell surface sorting of Robo1. Thus, my results show that guidance receptors for midline repellents have highly specific spatial and temporal dynamics. The generation of a temporal sequences of cell surface sorting thus represents a mechanism whereby commissural growth cones discriminate concomitant signals by functionalizing them at different timing of their spinal cord navigation
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Aurora Pignata. Study of the spatio-temporal dynamics of guidance receptors during commissural axon navigation in the spinal cord. Neurobiology. Université de Lyon, 2018. English. ⟨NNT : 2018LYSE1286⟩. ⟨tel-02309060⟩

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