Abstract
During neural circuit formation, axons navigate several choice points to reach their final target. At each one of these intermediate targets, growth cones need to switch responsiveness from attraction to repulsion in order to move on. Molecular mechanisms that allow for the precise timing of surface expression of a new set of receptors that support the switch in responsiveness are difficult to study in vivo. Mostly, mechanisms are inferred from the observation of snapshots of many different growth cones analyzed in different preparations of tissue harvested at distinct time points. However, to really understand the behavior of growth cones at choice points, a single growth cone should be followed arriving at and leaving the intermediate target. Existing ex vivo preparations, like cultures of an “open‐book” preparation of the spinal cord have been successfully used to study floor plate entry and exit, but artifacts prevent the analysis of growth cone behavior at the floor plate exit site. Here, we describe a novel spinal cord preparation that allows for live imaging of individual axons during navigation in their intact environment. When comparing growth cone behavior in our ex vivo system with snapshots from in vivo navigation, we do not see any differences. The possibility to observe the dynamics of single growth cones navigating their intermediate target allows for measuring growth speed, changes in morphology, or aberrant behavior, like stalling and wrong turning. Moreover, observation of the intermediate target—the floor plate—revealed its active participation and interaction with commissural axons during midline crossing.
Highlights
Commissural axons in the developing spinal cord have been used for over two decades to learn fundamental molecular mechanisms of axon guidance (Stoeckli, 2018)
Mechanisms are inferred from the observation of snapshots of many different growth cones analyzed in different preparations of tissue harvested at distinct time points
The comparison between axons in open-book preparations of control and experimentally manipulated spinal cords, dissected at specific time points, offered a solid understanding of molecules involved in axonal midline crossing and subsequent turning in higher vertebrates
Summary
Commissural axons in the developing spinal cord have been used for over two decades to learn fundamental molecular mechanisms of axon guidance (Stoeckli, 2018). The comparison between axons in open-book preparations of control and experimentally manipulated spinal cords, dissected at specific time points, offered a solid understanding of molecules involved in axonal midline crossing and subsequent turning in higher vertebrates. The information about mechanisms that can be extracted from such experiments is limited, as it is deduced from snapshots of axons taken from different animals sacrificed at a specific time point. For this reason, we have established a live-imaging approach that allows for visualization of dI1 axonal behavior at the FP, while axons are in the process of crossing the midline and turning rostrally. Our results on the dynamic behavior of growth cones at the spinal cord midline allowed us to compare axonal behavior at different choice points, such as the optic chiasm, reported previously (Godement et al, 1994; Sretavan & Reichardt, 1993)
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