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 cult ured on Nuclepore filters will also lay down a stroma containing 20 nm diameter fibrils, although there is only limited orthogonal organisat ion. We report here that collagen fibril morphology is critically depe ndent on the pH of the medium in which the corneal epithelium is cultu red and that normal 20 nm diameter fibrils only assemble in a narrow b and around neutral pH (approx. 6.9-7.4). At higher pH (7.6-8.1), fibri ls 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 tho ugh 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 hier oglyphic cross-sections typical of those seen in heritable disorders o f N-terminal procollagen processing. Biochemical analysis by SDS-PAGE and fluorography confirms that N-terminal procollagen processing is de ficient at this pH. At very low pH (approx. 5.8-6.2), there is little processing of procollagen and the stroma comprises filamentous materia l with the occasional banded structures typical of those formed by unp rocessed procollagen at high concentration. Gel electrophoresis and pe ptide mapping showed that the collagens produced by the corneal epithe lium of the primary stroma included types I, II and V and that total c ollagen production, as assessed by incorporation of [H-3]proline, incr eased with pH, although the relative amounts of the different collagen s produced remained essentially unchanged. While the biochemical data can account for the altered morphologies in the pH range 5.8 to 7.0, t he sensitivity of fibril diameter to small changes around neutral pH r emains unexplained, but points to the subtle, charge-based interaction s necessary for the formation of 20 nm diameter fibrils in the develop ing cornea.