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 structures consisting of at least two different fibrillar collagen types (I and II) and probably a fibril-associated collagen (type IX). The epithelial derivation and vectorial secretion of the components of this matrix provide an advantageous system to study the steps in the assembly of this developmentally regulated matrix, as well as in the assembly and maturation of heterotypic collagen fibrils in general. To ascertain when and where two of the collagens of the primary stroma (collagen types II and IX) are assembled into fibrils, monoclonal antibodies were used that bind to the newly deposited, "immature" (non-cross-linked) forms of these molecules, but not to ones that have been assembled into fibrils and have become cross-linked. METHODS: The patterns of immunoreactivity for newly deposited versus total collagens were compared 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. RESULTS: In early corneas, immunoreactivity for the newly deposited forms of both collagen types II and IX was confined to a narrow subepithelial zone, their epitopes being masked in the deeper layers of the stroma. The masked immunoreactivity could be exposed in these layers by inhibiting cross-link formation with beta-aminopropionitrile. At later developmental 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 period, 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 "unmasked" in deeper stromal layers by brief digestions with bacterial collagenase as well as by inhibition of cross-link formation. The extent of such unmasking of type II, however, is more limited in older corneas, suggesting that some of the putatively masked epitopes at these stages may in fact be proteolytically degraded. CONCLUSIONS: These results conform with previous studies suggesting that the type II and type IX collagens of the primary stroma are derived from the epithelium. They also suggest that (1) the assembly and maturation of the heterotypic fibrils, including the addition of the fibril-associated collagen type IX and covalent cross-link formation, occur shortly after synthesis and secretion of the molecules; (2) most, if not all, of the corneal type IX collagen becomes fibril-associated; and (3) during much of corneal development the N-telopeptide epitope in type II collagen is largely retained in a sterically masked form, but in later stages, during remodeling it may be removed by proteolytic degradation.
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 structures consisting of at least two different fibrillar collagen types (I and II) and probably a fibril-associated collagen (type IX). The epithelial derivation and vectorial secretion of the components of this matrix provide an advantageous system to study the steps in the assembly of this developmentally regulated matrix, as well as in the assembly and maturation of heterotypic collagen fibrils in general. To ascertain when and where two of the collagens of the primary stroma (collagen types II and IX) are assembled into fibrils, monoclonal antibodies were used that bind to the newly deposited, "immature" (non-cross-linked) forms of these molecules, but not to ones that have been assembled into fibrils and have become cross-linked. METHODS: The patterns of immunoreactivity for newly deposited versus total collagens were compared 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. RESULTS: In early corneas, immunoreactivity for the newly deposited forms of both collagen types II and IX was confined to a narrow subepithelial zone, their epitopes being masked in the deeper layers of the stroma. The masked immunoreactivity could be exposed in these layers by inhibiting cross-link formation with beta-aminopropionitrile. At later developmental 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 period, 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 "unmasked" in deeper stromal layers by brief digestions with bacterial collagenase as well as by inhibition of cross-link formation. The extent of such unmasking of type II, however, is more limited in older corneas, suggesting that some of the putatively masked epitopes at these stages may in fact be proteolytically degraded. CONCLUSIONS: These results conform with previous studies suggesting that the type II and type IX collagens of the primary stroma are derived from the epithelium. They also suggest that (1) the assembly and maturation of the heterotypic fibrils, including the addition of the fibril-associated collagen type IX and covalent cross-link formation, occur shortly after synthesis and secretion of the molecules; (2) most, if not all, of the corneal type IX collagen becomes fibril-associated; and (3) during much of corneal development the N-telopeptide epitope in type II collagen is largely retained in a sterically masked form, but in later stages, during remodeling it may be removed by proteolytic degradation.