Removal of Heparan Sulfate Chains Halted Epithelial Branching Morphogenesis of the Developing Mouse Submandibular Gland in vitro

Abstract

The physiological function of heparan sulfate chains in the mouse embryonic submandibular gland was studied by the use of heparitinases purified from Flavobacteriu heparinum. Heparitinase I, which catalyzes the cleavage of specific glycosaminidic linkages adjacent to non‐or monosulfated disaccharides of heparan sulfate chains, in the culture medium of the mid and late 12‐day gland inhibited the branch‐initiation and changed their round epithelial shape to elongated one, together with a concommitant reduction in lobular growth. [³H]Thymidine incorporation experiments indicated that heparitinase I treatment blocked 24% of the DNA synthesis compared with controls. Analysis of ³⁵S‐inorganic sulfate labeled glycosaminoglycans extracted from cultured rudiments revealed that the glands with heparitinase I contained no heparan sulfate, while in the glands without the enzyme more than 20% of total glycosaminoglycans was heparan sulfate. The heparitinase effect on morphogenesis was mimicked by the addition of heparan sulfate (1 mg ml⁻¹) or heparin (75 μg ml⁻¹), but not by chondroitin sulfate (1 mg ml⁻¹) in the culture medium. Transmission electron microscopic study indicated that at the epithelial‐mesenchymal interface close contacts between the fibroblast and epithelial cells were much fewer in heparitinase‐treated glands than in controls. Immunohistochemical analysis demonstrated that the core protein of basement membrane heparan sulfate proteoglycan and type IV collagen accumulated abnormally inside the epithelial lobules of glands cultured with heparitinase I. These results strongly suggested that glycosaminoglycan chains of heparan sulfate or heparin is involved in the epithelial morphogenesis of the mouse embryonic submandibular gland.