Immunocytochemical localisation of tenascin during the development of scleral papillae and scleral ossicles in the embryonic chick.

We have used a polyclonal antibody against tenascin (a 240 kDa extracellular matrix glycoprotein) and indirect immunofluorescence to investigate the distribution of tenascin in cryostat sections of the chick sclera during the six stages of scleral papilla development (Murray, 1943) and formation of membrane bone anlagen (mesenchymal condensations between 6-12 days of incubation - HH Stage 29-37). At Stages 1 and 2, when the papilla was a slight thickening in the conjunctival epithelium and mesenchymal condensation formation was initiated, tenascin was sparse in the sclera. At Stage 3, when the papilla had an epithelial mass intruding into the mesenchyme, there was an accumulation of tenascin fibrils along the subsurface of the papilla with fibrils extending from this region into the mesenchymal condensation. Interestingly, tenascin was sparse between papillae and between mesenchymal condensations. The Stage 4 papilla had a similar localisation of tenascin fibrils and in addition fine arborizing fibrils of tenascin were observed within the basal epithelia of the papilla adjacent to the epithelial-mesenchymal interface (which suggested that the fibrils infiltrated through the basement membrane region). The Stage 5 and 6 papillae had a column of vertical tenascin fibrils extending from the subsurface of the papilla to the interior mesenchyme corresponding exactly to the location of the mesenchymal condensation which was then forming the anlage of the ossicle in a flat bed about 100 microns from the conjunctival surface. The column of tenascin disappeared as the osteoid appeared in the ossicular bed on the 12th day of incubation but a dense accumulation of tenascin remained along the subsurface of the papilla. With the exception of tenascin fluorescence in the basal region of the Stage 4 papilla, tenascin fibrils were not observed in the other stages of papilla development in the epithelium covering areas between the mesenchymal condensation. This restricted distribution of tenascin may be important in the morphogenesis of scleral papillae and scleral ossicles.