Effects of enzyme isolation on the basal lamina of developing avian limb bud epithelia.

The developing avian limb bud is a classic example of an epithelial-mesenchymal interaction. Numerous attempts at maintenance of the epithelia in culture have been predominantly unsuccessful. The fate of the isolated epithelial sheet of the limb bud [including the apical ectodermal ridge (AER)] in culture may depend at least in part on the integrity of its basal lamina following isolation. In this study the distal epithelium of the stage 23 limb bud was isolated utilizing trypsin and Dispase II in a variety of procedures. The integrity of the basal lamina of limb epithelium immediately upon isolation and after 2 h in culture was determined by immunofluorescent staining for laminin, and electron microscopy. In epithelial sheets isolated with Dispase II a direct relationship was observed between maintenance of the extracellular matrix at isolation and the preservation of the tissue structure and cytoarchitecture following 2 h in culture. In contrast, there was an accelerated deterioration during incubation of the tissue isolated with trypsin, independent of isolation conditions and integrity of basal lamina after isolation. Short-term maintenance of limb bud epithelial structure and cytoarchitecture after enzymatic isolation seems correlative to the maintenance of extracellular matrix at the epithelial basal surface.