Calsyntenin 1-mediated trafficking of axon guidance receptors regulates the switch in axonal responsiveness at a choice point.

Axon guidance at choice points depends on the precise regulation of guidance receptors on the growth cone surface. Upon arrival at the intermediate target or choice point, a switch from attraction to repulsion is required for the axon to move on. Dorsal commissural (dI1) axons crossing the ventral midline of the spinal cord in the floor plate represent a convenient model for the analysis of the molecular mechanism underlying the switch in axonal behavior. We identified in chick a role for calsyntenin 1 in the regulation of vesicular trafficking of guidance receptors in dI1 axons at choice points. In cooperation with RabGDI, calsyntenin 1 shuttles Rab11-positive vesicles containing Robo1 to the growth cone surface in a precisely regulated manner. By contrast, calsyntenin 1-mediated trafficking of frizzled 3, a guidance receptor in the Wnt pathway, is independent of RabGDI. Thus, tightly regulated insertion of guidance receptors, which is required for midline crossing and the subsequent turn into the longitudinal axis, is achieved by specific trafficking.

Calsyntenins Are Expressed in a Dynamic and Partially Overlapping Manner during Neural Development.

Calsyntenins form a family of linker proteins between distinct populations of vesicles and kinesin motors for axonal transport. They were implicated in synapse formation and synaptic plasticity by findings in worms, mice and humans. These findings were in accordance with the postsynaptic localization of the Calsyntenins in the adult brain. However, they also affect the formation of neural circuits, as loss of Calsyntenin-1 (Clstn1) was shown to interfere with axonal branching and axon guidance. Despite the fact that Calsyntenins were discovered originally in embryonic chicken motoneurons, their distribution in the developing nervous system has not been analyzed in detail so far. Here, we summarize our analysis of the temporal and spatial expression patterns of the cargo-docking proteins Clstn1, Clstn2 and Clstn3 during neural development by comparing the dynamic distribution of their mRNAs by in situ hybridization in the spinal cord, the cerebellum, the retina and the tectum, as well as in the dorsal root ganglia (DRG).

RabGDI controls axonal midline crossing by regulating Robo1 surface expression.

BACKGROUND: Axons navigate to their future synaptic targets with the help of choice points, intermediate targets that express axon guidance cues. Once they reach a choice point, axons need to switch their response from attraction to repulsion in order to move on with the next stage of their journey. The mechanisms underlying the change in axonal responsiveness are poorly understood. Commissural axons become sensitive to the repulsive activity of Slits when they cross the ventral midline of the CNS. Responsiveness to Slits depends on surface expression of Robo receptors. In Drosophila, Commissureless (Comm) plays a crucial regulatory role in midline crossing by keeping Robo levels low on precommissural axons. Interestingly, to date no vertebrate homolog of comm has been identified. Robo3/Rig1 has been shown to control Slit sensitivity before the midline, but without affecting Robo1 surface expression. RESULTS: We had identified RabGDI, a gene linked to human mental retardation and an essential component of the vesicle fusion machinery, in a screen for differentially expressed floor-plate genes. Downregulation of RabGDI by in ovo RNAi caused commissural axons to stall in the floor plate, phenocopying the effect observed after downregulation of Robo1. Conversely, premature expression of RabGDI prevented commissural axons from entering the floor plate. Furthermore, RabGDI triggered Robo1 surface expression in cultured commissural neurons. Taken together, our results identify RabGDI as a component of the switching mechanism that is required for commissural axons to change their response from attraction to repulsion at the intermediate target. CONCLUSION: RabGDI takes over the functional role of fly Comm by regulating the surface expression of Robo1 on commissural axons in vertebrates. This in turn allows commissural axons to switch from attraction to repulsion at the midline of the spinal cord.