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Mechanisms underlying neuronal growth cone pathfinding Isbister, Carolyn Marie


From the initial stages of axon outgrowth to the formation of a functioning synapse, growth cones of developing neurons integrate and respond to multiple environmental guidance cues. To explore growth cone guidance mechanisms in vivo, we have taken advantage of the ease of access and manipulation of the well-characterized model system, the developing grasshopper embryo. To elucidate the role of substrate adhesivity in growth cone pathfinding, we developed an in vivo assay for measuring filopodial-substrate adhesivity using the Til pioneer neuron pathway of the embryonic grasshopper limb. Using time lapse imaging and a combination of rhodamine-phalloidin injections and Dil labeling, we demonstrate that filopodial retraction rate following treatment with cytochalasin D or elastase reflects the degree of filopodia-substrate adhesivity. There is no difference between retraction rates of filopodia extending towards the correct target and filopodia extending away from the correct target. The homogeneity in filopodial retraction rates, even among turning growth cones where differential adhesivity might be expected to be greatest, strongly indicates that differential adhesion does not govern Til pioneer neuron pathfinding. In addition to contact-mediated mechanisms, secreted molecules, such as semaphorins, can also steer growth cones. We demonstrate that a novel secreted semaphorin in grasshopper, gSema 2a, functions as a chemorepulsive guidance molecule critical for Til pioneer axon fasciculation and for determining both the initial direction and subsequent pathfinding events of the Til axon projection. Simultaneous perturbation experiments indicated that different semaphorin family members can provide functionally distinct guidance information to the same growth cone in vivo. Furthermore, Sema 2a is expressed in two overlapping perpendicular gradients: a steep distal-proximal and a shallow dorsal-ventral gradient. Although the Til growth cone will sample both the steep and shallow gradients, the majority of pathfinding errors occur within the shallow dorsal-ventral gradient. Although the gradient steepness differs, the absolute levels of Sema 2a at the stereotyped growth cone decision points are comparable. Ourtresults indicate that it is the steepness, or shape, of the gradient that encodes the degree of chemorepulsion. Finally, using these gradient data we propose theoretical mechanisms of growth cone gradient detection in vivo.

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