Chick corneal development in vitro: diverse effects of pH on collagen assembly

In vivo, the embryonic chick corneal epithelium lays down a stroma of collagen and proteoglycans whose fibrils are unusual as their diameter distribution peaks sharply about a mean of 20 nm. Such epithelia cultured on Nuclepore filters will also lay down a stroma containing 20 nm diameter fibrils, although there is only limited orthogonal organisation. We report here that collagen fibril morphology is critically dependent on the pH of the medium in which the corneal epithelium is cultured and that normal 20 nm diameter fibrils only assemble in a narrow band around neutral pH (approx. 6.9-7.4). At higher pH (7.6-8.1), fibrils in the distal region of the stroma more closely resemble those seen in non-corneal stroma as their diameters can be up to 200 nm even though fibrils near the basal lamina are only about 10 nm in diameter. At low pH (approx. 6.5), there are again wide fibrils, but with the hieroglyphic cross-sections typical of those seen in heritable disorders of N-terminal procollagen processing. Biochemical analysis by SDS-PAGE and fluorography confirms that N-terminal procollagen processing is deficient at this pH. At very low pH (approx. 5.8-6.2), there is little processing of procollagen and the stroma comprises filamentous material with the occasional banded structures typical of those formed by unprocessed procollagen at high concentration. Gel electrophoresis and peptide mapping showed that the collagens produced by the corneal epithelium of the primary stroma included types I, II and V and that total collagen production, as assessed by incorporation of [3H]proline, increased with pH, although the relative amounts of the different collagens produced remained essentially unchanged. While the biochemical data can account for the altered morphologies in the pH range 5.8 to 7.0, the sensitivity of fibril diameter to small changes around neutral pH remains unexplained, but points to the subtle, charge-based interactions necessary for the formation of 20 nm diameter fibrils in the developing cornea.