Acid mucopolysaccharide (glycosaminoglycan) has been demostrated at the epithelial-mesenchymal interface of mouse embryo submandibular glands by (a) specific staining for polymeric sulfate with Alcian blue 8 GX at various magnesium concentrations, (b) specific staining for polymeric uronic acid by selective oxidation of these residues to Schiff-reactive compounds, (c) electron microscope localization of ruthenium red staining, (d) radioautographic localization of glucosamine-3H and 35SO4, and (e) by susceptibility of the glucosamine radioactivity at the interface to digestion with protease-free hyaluronidase. Moreover, material labeled with glucosamine-3H and 35SO4 and with chemical characteristics identical with those of acid mucopolysaccharide were isolated from the glands. Acid mucopolysaccharide is distributed over the entire epithelial surface. The amount of acid mucopolysaccharide, as revealed by the staining procedures, is nearly equivalent at all sites. In contrast, the rate of accumulation of glucosamine-labeled mucopolysaccharide is greater at the surface of the distal ends of the growing and branching lobules. This distribution of newly synthesized acid mucopolysaccharide at the sites of incipient cleft formation suggests that surface-associated acid mucopolysaccharide is involved in the morphogenetic process. A mechanism of branching morphogenesis is proposed which accounts for the distribution of collagen fibers and total and newly synthesized acid mucopolysaccharide at the epithelial surface.

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