Immunoelectron Microscopic Localization of Neural Cell Adhesion Molecules (L1, N-CAM, and MAG) and Their Shared Carbohydrate Epitope and Myelin Basic Protein in Developing Sciatic Nerve

The cellular and subcellular localization of the neural cell adhesion molecules L1, N-CAM, and myelin-associated glycoprotein (MAG), their shared carbohydrate epitope L2/HNK-1, and the myelin basic protein (MBP) were studied by preand postembedding immunoelectron microscopic labeling procedures in developing mouse sciatic nerve. L1 and N-CAM showed a similar staining pattern. Both were localized on small, non-myelinated, fasciculating axons and axons ensheathed by non-myelinating Schwann cells. Schwann cells were also positive for L1 and N-CAM in their non-myelinating state and at the onset of myelination, when the Schwann cell processes had turned ~1.5 loops. Thereafter, neither axon nor Schwann cell could be detected to express the L1 antigen, whereas N-CAM was found in the periaxonal area and, more weakly, in compact myelin of myelinated fibers. Compact myelin, Schmidt-Lanterman incisures, paranodal loops, and finger-like processes of Schwann cells at nodes of Ranvier were Ll-negative. At the nodes of Ranvier, the axolemma was also always L1and N-CAM-negative. The L2/HNK-1 carbohydrate epitope coincided in its cellular and subcellular localization most closely to that observed for L1. MAG appeared on Schwann cells at the time L1 expression ceased. MAG was then expressed at sites of axon-myelinating Schwann cell apposition and noncompacted loops of developing myelin. When compaction of myelin occurred, MAG remained present only at the axon-Schwann cell interface; Schmidt-Lanterman incisures, inner and outer mesaxons, and paranodal loops, but not at finger-like processes of Schwann cells at nodes of Ranvier or compacted myelin. All three adhesion molecules and the L2/HNK-1 epitope could be detected in a nonuniform staining pattern in basement membrane of Schwann cells and collagen fibrils of the endoneurium. MBP was detectable in compacted myelin, but not in Schmidt-Lanterman incisures, inner and outer mesaxon, paranodal loops, and finger-like processes at nodes of Ranvier, nor in the periaxonal regions of myelinated fibers, thus showing a complementary distribution to MAG. These studies show that axonSchwann cell interactions are characterized by the sequential appearance of cell adhesion molecules and MBP apparently coordinated in time and space. From this sequence it may be deduced that L1 and N-CAM are involved in fasciculation, initial axon-Schwann cell interaction, and onset of myelination, with MAG to follow and MBP to appear only in compacted myelin. In contrast to L1, N-CAM may be further involved in the maintenance of compact myelin and axon-myelin apposition of larger diameter axons. URING development of peripheral nerves, cell-to-cell interactions follow a sequence of events that involve surface contacts of axons with the mesenchymal environment and peripheral glia, the Schwann cells, which are derived from the neural crest. Proliferating Schwann cells have been observed to migrate along outgrowing axons of peripheral nerves and withdraw from the mitotic cycle to form the myelin sheath or assume their roles as non-myelin-forming Schwann cells (Peters et al., 1976). Formation of myelin occurs around axons of larger diameter, whereas small diameter axons remain unmyelinated either as fascicules or ensheathed singly by Schwann cell processes. Myelin formation involves the ensheathment of the axon by loops of a Schwann cell process whose cytoplasmic faces of membranes are initially separated by Schwann cell cytoplasm but, upon extrusion of the cytoplasm and reduction of extracellular space to 2-2.5 nm between surface membranes, form the tightly apposed membranes of the adult compact myelin (Peters et al., 1976). The formation of compact myelin is the basis for rapid propagation of action potentials in the axon. Propagation of action potentials is saltatory at the nodes of Ranvier, where the internodal myelin is interrupted leaving the axolemma covered loosely by the finger-like processes of adjacent Schwann cells. The cellular and molecular signals © The Rockefeller University Press, 0021-9525/86/12/2439/10 $1.00 The Journal of Cell Biology, Volume 103 (No. 6, Pt. 1), Dec. 1986 2439-2448 2439 on A uust 8, 2017 jcb.rress. org D ow nladed fom Table L List of Primary Antibodies Concentration or dilution used for Antibody* against Reference for production and specificity Pre-embedding Post-embedding Mouse L1 Rathjen and Schachner, 1984; Faissner et al., 1984 0.3 mg IgG/ml 0.6 mg IgG/ml Mouse L1, immunoafiinity purified See Materials and Methods 0 . 2-0.6 mg IgG/ml Mouse N-CAM Goridis et al., 1983 1:50, 1:100, 1:25, 1:50* Mouse MAG Poltorak M. , G. Keilhauer, A. Meyer, C. Landa, 1:50, 1:25, 1:50, and M. Schachner, manuscript submitted for