The Role of Macropinocytosis in Sonic Hedgehog-Induced Axon Growth and Guidance: A Dissertation

Abstract

Axon pathfinding is an important process required for the establishment of proper neuronal connections during development. An increasing number of secreted and membrane-anchored molecules have been identified as axon guidance cues, which can act as positive or negative factors to increase or decrease the growth of axons and influence the direction of axonal growth. These axon guidance factors present in the extracellular environment interact with receptors present on the growth cone, a structure located at the tip of the axon which functions as the motor unit for the axon. Upon binding to their receptors on the growth cone, the guidance factors then elicit an intracellular signaling cascade within the axon that ultimately influences the direction of axon growth, often through a direct, non-transcriptional mechanism. In this dissertation, we show that Sonic hedgehog (Shh) acts as an axon guidance factor for chick retinal ganglion cell (RGC) axons in a concentration-dependent manner. At a low concentration, Shh functions as a positive factor that induces axon growth and attractive turning while, at a high concentration, Shh functions as a negative factor that induces axon retraction and repulsive axon turning. We further characterized the effects of Shh on macropinocytosis, a fluid-phase type of endocytosis, in the axons. A high concentration of Shh significantly increased macropinocytosis in the axons. Macropinocytosis resulted in the generation of large, dextran-positive, clathrinindependent vesicles in the axonal growth cones, prior to growth cone collapse, axon retraction and repulsive axon turning. These vesicles were found to require dynamic F-actin, nonmuscle myosin II and dynamin for their formation but were formed independently of PI3 kinase signaling. Interestingly, a low concentration of Shh had an opposite effect on macropinocytosis. A low concentration of Shh and soluble laminin decreased macropinocytosis and additionally increased the turnover of these vesicles within the axons, suggesting positive axon guidance factors can additionally regulate downstream processing or maturation of these vesicles. The effect of Shh on regulating the motility of macropinosomes within the axons was investigated. A low concentration of Shh appeared to increase the motility of these vesicles along axonal microtubules in a cAMPdependent manner. However, a high concentration of Shh did not appear to affect the motility of the macropinosomes, suggesting that it likely plays a more predominant role in the formation of these vesicles within the growth cone. When we began this work, a large body of research existed describing the effects of guidance factors on regulating the cytoskeleton during axon motility. However, the role of membrane trafficking events during axon growth and guidance were very poorly characterized. Since we began this project, an increasing number of reports have shown that endo- and exocytosis are important for axon growth and, here, we show that macropinocytosis induced by negative axon guidance factors plays a critical role in growth cone collapse, axon retraction and repulsive axon turning. Positive axon guidance factors also affect macropinocytosis within the axons and additionally regulate their maturation, suggesting that membrane trafficking events mediated by axon guidance factors are important for regulating axon growth and pathfinding.