Real time large scale in vivo observations by light-sheet microscopy reveal intrinsic synchrony, plasticity and growth cone dynamics of midline crossing axons at the ventral floor plate of the zebrafish spinal cord

Axonal growth and guidance at the ventral floor plate is here followed in vivo in real time at high resolution by light-sheet microscopy along several hundred micrometers of the zebrafish spinal cord. The recordings show the strikingly stereotyped spatio-temporal control that governs midline crossing. Commissural axons are observed crossing the ventral floor plate midline perpendicularly at about 20 microns/h, in a manner dependent on the Robo3 receptor and with a growth rate minimum around the midline, confirming previous observations. At guidance points, commissural axons are seen to decrease their growth rate and growth cones increase in size. Commissural filopodia appear to interact with the nascent neural network, and thereby trigger immediate plastic and reversible sinusoidal-shaped bending movements of neighboring commissural shafts. Ipsilateral axons extend concurrently, but straight and without bends, at three to six times higher growth rates than commissurals, indicating they project their path on a substrate-bound surface rather than relying on diffusible guidance cues. Growing axons appeared to be under stretch, an observation that is of relevance for tension-based models of cortical morphogenesis. The in vivo observations provide for a discussion of the current distinction between substrate-bound and diffusible guidance cues. The study applies the transparent zebrafish model that provides an experimental model system to explore further the cellular, molecular and physical mechanisms involved during axonal growth, guidance and midline crossing through a combination of in vivo and in vitro approaches.