Cell adhesion molecules regulate guidance of dorsal root ganglion axons in the marginal zone and their invasion into the mantle layer of embryonic spinal cord

In order to elucidate the mechanisms regulating the projections of dorsal root ganglion (DRG) axons in the dorsal funiculus and invasion into target regions in the mantle layer (prospective gray matter) of the spinal cord, we examined the interactions between DRG axons and spinal cord. DRG neurons were dissociated from chick embryos and cultured for 1-2 days on cryostat sections of the spinal cord at embryonic day 5 (E5) or at E9. E5 and E9 DRG neurons extended neurites onto both marginal zone (prospective white matter) and mantle layer (prospective gray matter) of the spinal cord, suggesting that both of these regions are permissive for neurite growth. When E5 DRG neurites approached cryosections of E5 spinal cord from outside, most of them ran in the marginal zone without invading the mantle layer. In contrast, about half of E9 DRG neurites entered the mantle layer after crossing the marginal zone of E9 spinal cord. These growth patterns of DRG neurites on spinal marginal zone and mantle layer are similar to the pathway formation of DRG axons at comparable stages in vivo; DRG axons run exclusively in the prospective dorsal funiculus before E6, and enter the mantle layer (prospective dorsal horn) to reach the target regions by E9. Perturbation of functions of Ng-CAM, Nr-CAM, and axonin-1/SC2 by adding the specific antibodies in the culture medium increased the ratio of DRG neurites entering the mantle layer of E5 spinal cord, suggesting that these cell adhesion molecules are involved in keeping DRG neurites in the marginal zone. Taken together with the expression of Ng-CAM, Nr-CAM, and axonin-1/SC2, these CAMs on DRG axons may regulate the guidance of these axons in the marginal zone before E6, and the subsequent decrease in the relative levels of these CAMs might allow DRG axons to invade the target mantle layer.