Within the corneal stroma, the striated collagen fibrils have small uniform diameters and regular interfibrillar spacings. Both characteristics are necessary for transparency. Our observations show that some corneal collagens become co-assembled into """"""""heterotypic"""""""" striated fibrils, whereas others may confer matrix stability. Our double-label immunoelectron microscopic studies employing monoclonal antibodies will be continued to determine precisely the arrangement of collagen types within heterotypic fibrils. Our studies suggest that type V collagen is involved in determining fibril diameter. Molecular biology will be employed to investigate the developmental regulation of the alpha 1(V) chain of this collagen. The appearance of mRNA for this chain during corneal development will be examined. The sites within the 1(V) chain gene occupied by DNA- binding proteins (putative transcription factors) will be determined with an in situ approach which utilizes the sensitivity of the polymerase chain reaction. The DNA-binding proteins themselves, some which may be corneal- specific, will be isolated, and the involvement of transcriptional regulators will be examined in corneal-scleral cell heterokaryons. A matrix-stabilizing role for type IX collagen is suggested by our observation that the early corneal stroma is dense and compact when type IX collagen is present, but undergoes rapid swelling when the molecule becomes undetectable. The relationship of collagen type IX to other matrix components will be examined by immunoelectron microscopy. Two different, alternately-spliced forms of type IX collagen mRNA exist within the developing cornea. The developmental changes in these mRNAs and their protein products will be examined, as will the tissue interactions involved in these changes. The involvement of matrix metalloproteases (MMPs) in the cleavage of type of IX collagen will be investigated using substrate gels, and by addition of recombinant TIMP (a naturally-occurring inhibitor of MMPs) to organ cultures of corneas. Lastly, we have isolated cDNAs which encode two different forms of a new type of collagenous molecule. One form has a corneal-specific domain. The cDNAs show a unique structural arrangement in which numerous short collagenous regions are interrupted by non-collagenous ones. Possibly the molecules interact with adjacent fibrils, serving as an interfibrillar spacer. The complete primary structure of each form will be obtained by cDNA sequencing, and immunoelectron microscopy will be performed employing antibodies specific for each form.
