The mature, functioning cornea results from the synthesis and assembly of several collagenous matrices, such as Bowman's membrane, the stroma, and Descemet's membrane. Transparency depends on the small uniform diameter and regular spacing of collagen fibrils in the stroma, and is acquired by compacting the stroma and coalescing the collagen fibril bundles. This compaction is accompanied by an increase in the fibril-associated collagen, type XIV. This collagen has recently been shown to facilitate the movement of fibrils in hydrated collagen gels in vitro, and therefore in vivo it may have a similar function in facilitating compaction of the stroma. Our studies suggest that all tissues express type XIV collagen mRNA (e.g., corneal epithelium and stroma), but not all translate the mRNA (e.g., the protein is not expressed by corneal epithelium). To examine how transcription is regulated by the corneal epithelium (a non-expressor of the protein), and stroma (an expressor of protein) we will measure transcriptional activity using gene transfections with CAT constructs containing the type XIV collagen gene promoter. We will also examine how translational regulation is conferred by isolating and analyzing polysomes (intracellular structures containing translatable mRNAs) and monosomes (structures with non-translatable mRNAs) from corneal epithelia and stromas for the presence of type XIV collagen mRNAs. The expression of corneal proteoglycans varies during development. We have found 4 splice variants of type XIV collagen that may bind these proteoglycans with different affinities. We will test this using affinity coelectrophoresis. We will also use quantitative PCR to determine which variants are prevalent at different developmental stages, especially around the time that transparency is acquired. To assess the functional role of type XIV collagen in compaction, early corneas will be infected in ovo with a retroviral construct that synthesizes a truncated type XIV collagen polypeptide. This will create a dominant negative phenotype for the collagen XIV molecule, which should interfere with its normal function. Lastly, we have found that another fibril-associated collagen, type XII, is prevalent in the interfacial matrices of the cornea, where Bowman's membrane and Descemet's membrane adjoin the stroma. Immunohistochemistry and gene structural analysis suggest that in cornea a new isoform of the molecule exists, one which may provide structural stability to these matrices. We will use a PCR based strategy to obtain a cDNA for the corneal type XII isoform, to elucidate its structure.
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