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Functional studies of grasshopper semaphorin-2A Boileau, Ève

Abstract

One family of neuronal guidance cues used by growth cones to pathfind correctly is the semaphorin family. Although most members function as inhibitory guidance cues, the grasshopper semaphorin-1a is attractive. Previous experiments in Drosophila and grasshopper have suggested that semaphorin-2a is a neuronal repulsive guidance cue, however, in both cases the results have been equivocal. In this thesis, I directly test the function of grasshopper semaphorin-2a in vivo, while presenting ectopic sources of semaphorin-2a during the neuroembryonic development of grasshoppers. In addition, as a first approximation to determine protein regions used for function, I analysed whether a chimeric semaphorin that contains the transmembrane and the cytoplasmic domains of semaphorin-1a and the semaphorin domain of semaphorin-2a functions like the wild type semaphorin-2a. I found that the addition of cells expressing both the chimera and the wild type Sema-2a significantly increases the proportion of misguided growth cones in the developing peripheral nervous system. Grasshoppers cultured with semaphorin-2a expressing cells showed a significant increase in errors with misguidance apparent in 79% of the pathway analyzed. Similarly, chimera expressing cells induced misguidance in 84% of the cases. Misguidance-errors were categorised into four groups: dorsal projections, axon defasciculation, growth impairment and emergence of multiple axon sprouts directed away from expressing cells. The proportion of the phenotypes-induced are hot significantly different whether full length of semaphorin-2a or chimera expressing cells were placed in the limb and all phenotypes suggest that semaphorin-2a and the chimera protein both function as repulsive guidance cues in the developing peripheral nervous system. This data shows that the semaphorin domain of Sema-2a is sufficient for its repulsive function and that the generation of a transmembrane version of this protein does not perturb this function.

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