Cultured Schwann cells assemble normal-appearing basal lamina only when they ensheathe axons

Abstract

Previous work demonstrated that Schwann cells (SCs) must interact with nerve cells (NCs) in order to generate their basal lamina (BL) in culture ( M. B. Bunge, A. K. Williams, and P. M. Wood, 1982, Dev. Biol. 92, 449–460 ). The present study was undertaken to determine if this interaction requires proximity of NCs to SCs. Coverslips carrying isolated SCs were placed into culture dishes containing normally contacting SCs+NCs, NCs alone, or SCs alone and were maintained in these dishes for 3–4 weeks in medium known to foster the differentiation of axon-related SCs (BL formation, myelination). The SCs on the coverslip were not allowed to contact the cells in the culture dish. In other experiments, SCs isolated on coverslips were simply cultured in medium conditioned by contacting SCs+NCs, NCs alone, or SCs alone. The accumulation of BL components was monitored by light microscopic immunocytochemistry and the assembly of BL structure was assessed by electron microscopy. When SCs were cocultured with but not contacted by neurons, immunostaining for BL constituents revealed a patchy deposition of material in sharp contrast to the linear deposition observed on axon-related SCs. Electron microscopy of these isolated SCs revealed short segments of BL, strands or clumps of BL-like material extending away from the cell surface, and accumulation of this material between cells. A greater number of isolated SCs were immunostained when grown with contacting SCs+NCs than with NCs or SCs. The conditioned medium experiments yielded similar results; only patchy BL was observed and more immunostaining was detected on isolated SCs when the medium had been conditioned by contacting SCs+NCs than by NCs alone or SCs alone. Immunostaining was less overall in the conditioned medium experiments than in the cell coculture work. In addition, standard SC+NC cultures grown in differentiation-supporting medium were studied by electron microscopy. SCs that were not contacted by axons but were positioned between fascicles of normally contacting SCs+NCs were identified under phase microscopy and then examined for the presence of BL. These SCs exhibited only occasional segments of BL or detached BL-like material. Lastly, within differentiated fascicles, nonensheathing SCs were compared with neighboring myelinating SCs that were in substantial contact with axons. BL-deficient nonensheathing SCs were found directly adjacent to axons and BL-coated myelinating SCs. These studies demonstrate that SCs and NCs need not be in contact for SCs to deposit short segments of BL and clumps of BL-like material but that uncontacted SCs cannot assemble normal-appearing (continuous and consistently adherent) BL. Neither released diffusible neuronal factors nor products released from adjacent BL-assembling SCs are sufficient to engender full SC BL assembly. In the culture systems used here, we observed that only when SCs clearly ensheathe and myelinate axons, and thus are in substantial contact with neurons, is full assembly of normal appearing SC BL achieved. We therefore suggest that substantial contact between axon and SC is required for full BL assembly.