Ultrastructural identification of collagen and glycosaminoglycans in notochordal extracellular matrix in vivo and in vitro

Abstract

Notochordal extracellular matrix consists of a continuous basal lamina, amorphous materials and microfibrils embedded in the ground substance of low electron density. Together they comprise the notochord sheath and are of considerable interest because of their suspected role in early embryonic tissue interactions. The notochord is particularly well-suited to morphological investigation of extracellular matrix because it is one of the few embryonic epithelia which produces ultrastructurally recognizable stroma in vitro without the advantage of a collagenous substratum. Furthermore, these matrix components produced in vitro are morphologically identical to those observed in vivo. The present study used ruthenium red staining to demonstrate that notochordal microfibrils exhibit collagen-like cross-banding patterns both in vivo and in vitro. Collagenase and testicular hyaluronidase digestion studies designed to localize collagen and glycosaminoglycans show a reduction of microfibrillar diameters by 30-35%. Furthermore, these enzyme treatments frequently result in enhanced striations of microfibrils. When cis-hydroxyproline (a proline analog) or beta-aminoproprionitrile (BAPN, a lathyrogenic compound) is added to the culture medium, a similar reduction in microfibrillar diameters is seen. Moreover, increased ruthenium red-positive surface coats and large collagen fibrils are frequently present in BAPN-treated cultures, implying a stimulatory metabolic effect. We conclude that most, if not all, notochordal extracellular matrix components are composed of both collagen and glycosaminoglycans and suggest that the entire extracellular matrix should be considered a macromolecular composite which acts in concert to induce or stabilize developmental interactions.