Basal lamina persistence during epithelial-mesenchymal interactions in murine tooth development in vitro.

Numerous investigations have demonstrated the necessity of mesenchymal instruction for epithelial differentiation during epidermal organogenesis. In the specific case of tooth formation, cap-stage tooth organ mesenchyme instructs epithelial differentiation into ameloblasts with production of enamel extracellular matrix. The "instructive event" is presumed to be direct cell contact. Mesenchyme-mediated cell contact with adjacent epithelia is assumed to "instruct" epithelial differentiation into ameloblasts. If this were true, basal lamina removal and mesenchyme cell contact with epithelia would be prerequisites for epithelial cytodifferentiation and morphogenesis in the developing tooth system. To test this hypothesis, we designed experiments to evaluate basal lamina stability during epithelial differentiation into ameloblasts. Our studies utilized cap-stage murine molar tooth organs, a serumless and chemically defined medium (PYMS), metabolic isotopic labeling of basal lamina constituents, biochemical methods to analyze macromolecular stability throughout 10 days of organ culture in vitro, and immunological methods to localize the distribution of laminin and fibronectin. Our results indicate that (3H)glucosamine is incorporated into basement membranes present in Theiler stage 25 mandibular mouse molar tooth organ. At this stage, the isotope was incorporated into high molecular weight macromolecules. Specific enzyme methods coupled with electrophoresis and fluorography demonstrated that (3H)glucosamine was incorporated into proteoglycans containing chondroitin sulfates, dermatan sulfate, and hyaluronate. After 10 days in vitro the radiolabeled material remained localized in these same molecules, indicating stability of these constituents within basement membranes. Ultrastructural observations indicated that the basal lamina was not removed during ameloblast differentiation in vitro using PYMS medium. Laminin and fibronectin were localized in the basement membranes during cap stages and did not disappear during subsequent morphogenesis and differentiation. Mesenchymal cells appear to mediate epithelial differentiation in vitro using PYMS medium without a removal of the basal lamina.