Figure 4

Down-regulation of MDGA2 causes pathfinding errors of commissural axons. (A) Schematic diagram of the time course of commissural axon pathfinding. Note that midline crossing occurs between stages 22 and 24, and rostral axon turning is initiated around stage 24. (B) Schematic drawing of a stage 26 open-book preparation. The red circle represents the DiI injection site labelling cell bodies of dorsolateral commissural neurons and their axons. (C,D) Confocal images of DiI-injected open-book preparations of an untreated control embryo (C) and an embryo injected and electroporated with a YFP control plasmid (D). Note that in both cases commissural axons make a sharp turn upon leaving the midline area and grow rostrally along the longitudinal axis of the spinal cord. The area between the dashed lines indicates the location of the floor-plate. (E) Electroporation efficiency with YFP. An embryo electroporated with a YFP-plasmid was imaged with epifluorescence. (F,G) RNAi knockdown experiments in which embryos were co-injected with long dsRNA and a YFP expression plasmid. Two independent, non-overlapping fragments were used to produce long dsRNA. Numbers in parentheses indicate the cDNA sequences used to produce dsRNA. Ipsilateral electroporation resulted in a slightly reduced number of axons reaching the contralateral side and a lack of growth of commissural axons along the longitudinal axis. Identical defects were seen with both dsRNA fragments. (H,I) A similar phenotype was observed when MDGA2 peptide antibodies (Ab) were injected into the central canal of stage 20 chicken embryos (H), whereas normal axon growth was seen in embryos injected with a control IgG (I). Scale bars: 100 μm.