Midline crossing is not required for subsequent pathfinding decisions in commissural neurons

Abstract

BackgroundGrowth cone navigation across the vertebrate midline is critical in the establishment of nervous system connectivity. While midline crossing is achieved through coordinated signaling of attractive and repulsive cues, this has never been demonstrated at the single cell level. Further, though growth cone responsiveness to guidance cues changes after crossing the midline, it is unclear whether midline crossing itself is required for subsequent guidance decisions in vivo. In the zebrafish, spinal commissures are initially formed by a pioneer neuron called CoPA (Commissural Primary Ascending). Unlike in other vertebrate models, CoPA navigates the midline alone, allowing for single-cell analysis of axon guidance mechanisms.ResultsWe provide evidence that CoPA expresses the known axon guidance receptors dcc, robo3 and robo2. Using loss of function mutants and gene knockdown, we show that the functions of these genes are evolutionarily conserved in teleosts and that they are used consecutively by CoPA neurons. We also reveal novel roles for robo2 and robo3 in maintaining commissure structure. When midline crossing is prevented in robo3 mutants and dcc gene knockdown, ipsilaterally projecting neurons respond to postcrossing guidance cues. Furthermore, DCC inhibits Robo2 function before midline crossing to allow a midline approach and crossing.ConclusionsOur results demonstrate that midline crossing is not required for subsequent guidance decisions by pioneer axons and that this is due, in part, to DCC inhibition of Robo2 function prior to midline crossing.

Robo2–Slit and Dcc–Netrin1 Coordinate Neuron Axonal Pathfinding within the Embryonic Axon Tracts

“…It also highlights the requirement of Netrin1 function in the presence of the Robo2-mediated repulsion. Finally, it is interesting to note that a previous study showed that Dcc promotes ventral growth of zebrafish spinal cord commissural primary ascending neuron axons through inhibition of Robo2 (Bonner et al, 2012). Therefore, the interaction between the Robo2-Slit and the Dcc-Netrin1 signaling pathways may play roles in a variety of guidance choice mechanisms in the CNS.…”

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

“…mRNA probes for islet, gata, and vsx families label specific post-mitotic neurons of consistent position and number within each spinal cord hemisegment 6 . In addition, axon guidance receptors such as those in the robo family are expressed in restricted populations of postmitotic neurons ( Figure 2C) 2,7,8 . Lateral mounting of embryos allows accurate counts of post-mitotic neurons.…”

“…They branch, cross the midline, and exit both the dorsal and ventral spinal cord. When coupled with confocal laser scanning microscopy, these varied cellular behaviors are evident in laterally mounted embryos, using immunocytochemistry and genetically encoded fluorescent proteins such as GFP 2 . In Figure 2D, znp-1 immunocytochemistry was used to label developing motoneurons.…”

“…Neuronal subtype differentiation can be analyzed through expression of islet, vsx, engrailed, and gata genes 1,[4][5][6] . And finally, analysis of axon guidance is possible through antibodies such as anti-acetylated tubulin, 3A10, and znp-1 2,11,12 . Though other vertebrate model systems (mouse and chick) are used to study spinal cord development, only the zebrafish permits simultaneous viewing of all developmental axes in the intact embryo.…”

“…In particular, we have determined that robo2, robo3, and dcc are required for Commissural Primary Ascending (CoPA) axon pathfinding. Using the mounting procedure described here, we were able to examine ventral growth, midline crossing, commissure width, dorsal and anterior growth 2,3 . This mounting procedure may also be applied to DV or AP patterning of the spinal cord.…”