Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Michael Granato

Abstract

Vertebrate peripheral nerves can regenerate, enabling severed axons to reconnect with their original synaptic targets. The interactions between injured nerves with cells in their environment, as well as the functional significance of these interactions, have not been determined in vivo and in real time. Here we provide the first minute-by-minute account of cellular interactions between laser transected motor nerves, macrophages, and Schwann cells in live intact zebrafish using transgenic lines that label each cell type in vivo. We find that axon fragmentation triggers macrophage invasion into the nerve to engulf axonal debris, and that delaying nerve fragmentation in a Wlds model does not alter macrophage recruitment but induces a previously unknown `nerve scanning' behavior, suggesting that macrophage recruitment and subsequent nerve invasion are controlled by separate mechanisms, both independent of Schwann cells. A major challenge for regenerating peripheral axons is to identify their original trajectory; Schwann cells are known to provide regenerating axons with factors that stimulate outgrowth, and an adhesive substrate that axons preferentially extend along during regeneration, yet their role in guiding regenerating axons onto their original trajectory is less clear. We show in mutants lacking Schwann cells that axonal growth cones sprout and extend at rates comparable to wild type, but fail to identify their original path, and instead extend along aberrant trajectories. To determine whether Schwann cells function primarily as an adhesive substrate we tested whether a Schwann cell-less axonal scaffold is sufficient to direct axonal growth. These substrates failed to compensate for the absence of Schwann cells, providing evidence that Schwann cells direct regenerating axons towards their original trajectory. To identify signals that guide regenerating motor axons in vivo, we examined mutants lacking the guidance receptor DCC. We find that in DCC mutants a significant fraction of regenerating axons extend along aberrant trajectories. Collectively, this work details the dynamic activities of macrophages and Schwann cells during axon degeneration and early regeneration, while axons are regrowing and selecting their trajectory, and we propose that Schwann cells and DCC mediated guidance are critical early during regeneration, enabling growth cones to navigate towards their original axonal trajectories.

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