COLLAGEN FIBRIL ASSEMBLY IN THE DEVELOPING AVIAN PRIMARY CORNEAL STROMA

Purpose. The primary stroma of the developing avian cornea is a highly organized extracellular matrix composed largely of striated collagen fibrils synthesized by the epithelium. These fibrils are heterotypic s tructures consisting of at least two different fibrillar collagen type s (I and II) and probably a fibril-associated collagen (type IX). The epithelial derivation and vectorial secretion of the components of thi s matrix provide an advantageous system to study the steps in the asse mbly of this developmentally regulated matrix, as well as in the assem bly and maturation of heterotypic collagen fibrils in general. To asce rtain when and where two of the collagens of the primary stroma (colla gen types II and IX) are assembled into fibrils, monoclonal antibodies were used that bind to the newly deposited, ''immature'' (non-cross-l inked) forms of these molecules, but not to ones that have been assemb led into fibrils and have become cross-linked. Methods. The patterns o f immunoreactivity for newly deposited versus total collagens were com pared by immunofluorescence histochemistry on sections of corneas from developing chicken embryos. Pretreatments that affect collagen cross- linking and enzymatic digestions with collagenase also were used. Resu lts. In early corneas, immunoreactivity for the newly deposited forms of both collagen types II and IX was confined to a narrow subepithelia l zone, their epitopes being masked in the deeper layers of the stroma . The masked immunoreactivity could be exposed in these layers by inhi biting cross-link formation with beta-aminopropionitrile. At later dev elopmental stages, after the stroma has swollen and become invaded by mesenchymal cells, type IX collagen is no longer detectable either as an ''immature'' or as a fibril-associated form. During most of this pe riod, the distribution of ''immature'' type II collagen is noticeably more restricted to the subepithelial zone than is total type II. Much of the undetectable immunoreactivity for collagen type II could be ''u nmasked'' in deeper stromal layers by brief digestions with bacterial collagenase as well as by inhibition of cross-link formation. The exte nt of such unmasking of type II, however, is more limited in older cor neas, suggesting that some of the putatively masked epitopes at these stages may in fact be proteolytically degraded. Conclusions. These res ults conform with previous studies suggesting that the type II and typ e IX collagens of the primary stroma are derived from the epithelium. They also suggest that (1) the assembly and maturation of the heteroty pic fibrils, including the addition of the fibril-associated collagen type IX and covalent cross-link formation, occur shortly after synthes is and secretion of the molecules; (2) most, if not ail, of the cornea l type IX collagen becomes fibril-associated; and (3) during much of c orneal development the N-telopeptide epitope in type II collagen is la rgely retained in a sterically masked form, but in later stages, durin g remodeling it may be removed by proteolytic degradation.