Figure 1

Commissural axons fail to reach the contralateral floor-plate border in the absence of RabGDI. Dorsolateral commissural neurons (dI1; shown in red) extend their axons ventromedially toward the floor plate, where they cross the midline. After reaching the contralateral floor-plate border axons turn rostrally into the longitudinal axis, in close contact with the floor-plate border (A). Commissural axon pathfinding was visualized in ‘open-book’ preparations by injecting the lipophilic dye DiI into the area of their cell bodies. At HH25, commissural axons in control-injected (B) and in non-injected embryos (not shown) had crossed the floor plate and extended a considerable distance along the longitudinal axis of the spinal cord. In the absence of RabGDI, commissural axons entered the floor plate (indicated by the dashed lines) but most of them failed to reach the contralateral floor-plate border (arrowheads in C) in age-matched embryos. The same phenotypes were obtained by injection of dsRNA derived from independent, non-overlapping cDNA fragments from the coding region (C) and the 3’ UTR (D). The downregulation of RabGDI was quantified in HEK293 cells using Western blots. RNAi reduced RabGDI protein levels by 77 ± 12%, compared to controls (E). Efficient downregulation of RabGDI mRNA by in ovo RNAi on the electroporated (right side) compared to the control (left) side of the spinal cord could also be observed by in situ hybridization at HH23/24 (F). A vector encoding YFP was co-injected to verify efficient transfection (G). Bar 50 μm. Rostral is to the top in B - D.