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.
Highlights
Growth cone navigation across the vertebrate midline is critical in the establishment of nervous system connectivity
Our study directly addresses whether pioneer neurons are responsive to guidance cues in the absence of midline crossing, and carefully characterizes the pathfinding of pioneer neurons in various Robo/Deleted in colorectal cancer (DCC) mutant conditions
Expression of Robo and DCC guidance receptors during zebrafish primary spinal neurogenesis To determine if pioneer commissural neurons utilize guidance systems sequentially and cell autonomously, we characterized expression of known guidance receptors in the zebrafish spinal cord
Summary
Growth cone navigation across the vertebrate midline is critical in the establishment of nervous system connectivity. As Slit signals are present on both sides of the ventral spinal cord, [6,7,8,9] responsiveness to Slitmediated repulsion in the midline is tightly regulated to Reduction of Robo function can result in appropriate targeting in the absence of crossing and intact function in both mice and humans. This is evident in precerebellar neurons [18,19,20]. As a pioneer that is temporally separated from its followers, CoPA is an excellent single-cell model of commissural pathfinding
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