Neurofilament regulation of sensory neuron axon caliber - PROJECT SUMMARY The cross-sectional diameter, or caliber, of axons and dendrites varies dramatically (>100 fold). Caliber influences the electric properties of neurites, their capacity for intracellular trafficking, and their vulnerability to damage. Many branching neurite arbors have tapering calibers: The primary branch is the thickest and each higher branch order is successively thinner. Although intense efforts have focused on studying mechanisms of neurite outgrowth, branching, guidance, and synapse formation, much less attention has focused on the regulation of neurite caliber. One reason for the relative lack of attention to caliber is that it is difficult to measure with light microscopy. Relatedly, although much effort has focused on understanding how microtubules and actin filaments contribute to neuronal structure, the third major cytoskeletal components of neurons, neurofilaments (NFs), have been relatively neglected. NF abundance correlates with axon caliber, and NF deficiency reduces average axon caliber, but it is not known if NFs contribute to neurite tapering. The goals of this project are to establish a zebrafish model for studying axon caliber regulation in vivo, create tools to study NF distribution and function in zebrafish, and use those methods and tools to determine if NFs contribute to axon tapering. These studies use a new method to label single zebrafish sensory neurons, called Rohon-Beard (RB) neurons, in live larvae and measure the caliber of their peripheral axons using super- resolution microscopy. Preliminary experiments confirm that axon caliber tapers in RB peripheral axon arbors. Unexpectedly, tapering at the first branch point is usually asymmetric: the primary branch gives rise to one thick and one thin secondary branch. Aim 1 of this proposal uses live imaging to determine how microtubule dynamics, intracellular trafficking, and axon structure are affected by caliber asymmetry. Aim 2 uses a novel strategy is to make fluorescent reporters for endogenous NF proteins and use them to document the distribution of NFs in peripheral arbors of RB neurons. Aim 3 characterizes the effect of NF deficiency on the tapering morphology of RB neurons. Collectively, these studies will establish zebrafish as a powerful new model for studying axon caliber regulation and NF function, and test the hypothesis that NF distribution regulates neurite tapering.
