Nogo‐B is the major form of Nogo at the floor plate and likely mediates crossing of commissural axons in the mouse spinal cord

Using Nogo antibodies with defined binding specificity, Nogo‐B, but not Nogo‐A, was localized on radial glia in the floor plate of mouse embryos. The presence of Nogo‐B was confirmed in Nogo‐A knockout mice. In explant cultures of embryonic day (E) 11 and E12 spinal cord, blocking of NgR function with antagonist peptide NEP1‐40 reduced the crossing of newly arrived commissural axons, resulting in an accumulation of growth cones in the floor plate. Analysis of growth cone morphology demonstrated an increase in size of growth cones in the floor plate after peptide treatment, which was not detected in axons growing toward the midline. In knockout embryos, midline crossing was not affected by absence of Nogo‐A. In co‐culture experiments using collagen gel, floor plate showed a strong inhibitory effect on the extension of post‐commissural neurites from the spinal cord. This effect was abolished by NEP1‐40, and was observed neither in pre‐commissural neurites, nor in post‐commissural neurites grown with floor plate derived from Nogo‐A knockout embryo. Furthermore, western blot analysis of conditioned medium from floor plates showed a truncated form of Nogo with molecular weight of 37 kDa, which could mediate the diffusible effect to axon growth. We conclude that Nogo‐B is expressed in the floor plate of mouse embryo, which probably mediates axon crossing in the spinal cord by repelling axons out of the midline when they start upregulate NgR. Nogo acts on axon growth not only through a contact‐mediated mechanism, but also through a diffusible mechanism. The Nogo‐A specific antibody 11C7 only showed a slight staining at the floor plate (A), while the Bianca antibody that recognizes both Nogo‐A and ‐B strongly labeled the floor plate (B), indicating that Nogo‐B is the isoform specifically expressed in this midline structure. The outgrowth of post‐commissural neurites was inhibited significantly when cocultured with floor plate (C). This inhibition was abolished by NEP1‐40, a Nogo receptor antagonist, but not by floor plate isolated from Nogo‐A knockout (KO) embryos (C). These findings indicate that Nogo‐B mediates suppressive effect of floor plate on post‐commissural axon growth.