Extracellular matrix production by embryonic epithelium cultured on type IV collagen. Deposition of a primary corneal stroma-like structure containing large irregular type I fibrils without type II collagen.

The corneal stroma of the chick embryo is deposited in two steps. The primary stroma is laid down by the corneal epithelium and it contains type I, type II and type IX collagens. Its formation is subsequent to the presumptive epithelial cells' migration onto the lens capsule (which is rich in type IV collagen). The secondary, ultimate stroma is synthesized by fibroblasts which, on day 5 of development, invade the swollen primary stroma. It is composed of a matrix of thin (25 nm), regular fibrils containing type I and type V collagens. We found that a chick corneal epithelium isolated from either a 6-day or a 14-day embryo was able to produce, in vitro, stroma-containing type I collagen fibrils. However, the amount of collagen deposited and its organization were highly dependent on the substratum used. Plastic or purified bovine type I collagen substrata led to the release of very few fibrils. Purified human type IV collagen induced the production of an abundant matrix made of large irregular collagen fibrils. When compared to native corneal stroma, there were two aspects in which this matrix differed: (1) it contained only type I collagen, as shown by indirect immunofluorescence, and (2) there were numerous large, irregular fibrils of about 100 to 130 nm in diameter. In conclusion, it is suggested that purified type IV collagen substitutes, in part, for the basement membrane and allows the production of a corneal stroma-like matrix by an embryonic corneal epithelium in culture. This production is possible even with a 14-day epithelium which, in vivo, is no more involved in the synthesis of the stroma collagens. Moreover, the regulatory effect of type II collagen, previously suggested by in vivo observations, may be confirmed in this in vitro system by the appearance of large fibrils in the newly deposited stroma that are made only by type I collagen.