The long-term objective of this proposal is to understand the intrinsic mechanisms that regulate axon development and neuronal morphology. The proposal focuses on the role of neurofilament expression in the growth and morphology of axons. The experiments in this proposal test the hypothesis that changes in neurofilament subunit composition directly modulate axon growth. All experiments will be carried out in Xenopus laevis embryos or primary cultures derived from embryos. In studies that follow-up observations made in the previous project period, in which an antibody to the neurofilament protein NF-M eliminated neurofilaments from growing axons and stunted their growth, time-lapse video microscopy will be used to determine what aspect of axonal growth is compromised by lack of neurofilaments. Specifically, the rate of elongation and retraction phases of axonal growth and the development of collaterals will be compared between axons that do not contain neurofilaments and those that do (Aim 1).
In Aims 2 and 3, RNAs encoding frog neurofilament subunits will be injected into developing frog embryos to determine the effect of prematurely expressing or overexpressing specific subunits. The consequences of these perturbations on axon growth will be studied in vivo in wholemounts of intact tadpoles, and in single neurons in spinal cord cultures derived from injected animals. These experiments are designed to obtain information about how axons grow and recover from injury.
