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Functional studies of grasshopper semaphorin-2A

University of British Columbia

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.

Thesis/Dissertation

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2009-10-19

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http://hdl.handle.net/2429/14068

Neuroscience

University of British Columbia

UBCV

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