The Role of GAP-43 Phosphorylation in Axon Behavior in the Developing Zebrafish Visual System

Abstract

Developing neurons extend processes to specific targets and establish connections that are essential for future function of the nervous system. One of these processes, the axon, has a motile tip called a growth cone that rearranges its membrane-associated actin cytoskeleton to turn toward or away from environmental guidance cues. Growth associated protein 43 (GAP-43) is one of the most abundant proteins associated with axonal growth cone membranes and is known to modulate the formation and stability of the actin cytoskeleton during axon guidance. Protein kinase C (PKC)-mediated phosphorylation of GAP-43 on serine 42 regulates its interactions with actin. Phosphorylated GAP-43 stabilizes actin filaments in growth cones that are actively pathfinding. Using the developing zebrafish visual system to model axon growth and guidance, we determined the role of GAP-43 phosphorylation during retinal ganglion cell (RGC) axon emergence and growth by disrupting phosphorylation in vivo. In zebrafish embryos we induced the expression of a mutant version of GAP-43, GAP43s42, that has a serine to alanine substitution mutation at position 42. This dominant negative mutant GAP-43 cannot be phosphorylated by PKC. Overexpression of the GAP43s42a mutant early in visual system development gave rise to embryos in which the optic nerve appeared thinner. Further investigation revealed this phenotype was caused by defects in RGC differentiation and axon guidance errors within the retina. Overexpression of GAP43s42a later in development, when RGC axons are actively growing in the optic chiasm, resulted in a loss in the close association of axons in the optic nerve leading to defasciculation and aberrant axon trajectories. We conclude from these results that phosphorylation of GAP-43 plays an important role in regulating RGC differentiation, axon guidance, and axon fasciculation.