Search

The anterior avian cornea possesses several distinct cellular and extracellular regions including the epithelial basal lamina, Bowman's layer and the interfacial matrix that separates Bowman's layer from the stroma. These unique regions differ biochemically, physically and morphologically but all contain type XII collagen. Previously, the collagen fibrils of several of these interfacial regions were shown to be stable to thermal and enzymatic denaturation. We reasoned that type XII collagen, a fibril-associated collagen, would be a good candidate to confer such stabilizing properties. The studies described herein were performed to localize type XII collagen and to assess its role in the interfacial matrices (IM). Using antibodies that react with both the short and long type XII collagen isoforms and that react specifically with the long isoform, we demonstrate that it is the short isoform that is present in Bowman's layer and the associated interfacial matrix lying between Bowman's and the stroma proper. In situ hybridization analyses demonstrate that both the epithelial and endothelial cells synthesize type XII collagen. In vitro cell culture analyses, however, demonstrate that in addition to epithelial cell synthesis, the stromal fibroblasts are capable of synthesizing type XII collagen as well. Immunofluorescence analyses performed at elevated temperature demonstrate that type XII collagen is thermally stable in Bowman's layer, but not in the anterior interfacial matrix or Descemet's layer. In addition, we observed that the distribution of type XII collagen during the development of the anterior extracellular matrices correlates precisely with an elevated density of keratocytes populating the interfacial matrix just deep to Bowman's layer. We show that this cellular density is developmentally regulated and does not arise from a localized increase in cell proliferation. These data demonstrate that Bowman's layer and the anterior interfacial matrix have unique biochemical and morphologic properties. Type XII collagen is thermally stable in Bowman's layer and, as a surface component of type I collagen fibrils, may contribute to the stability of the fibrils in this region. Neither type XII nor type I collagen is stable in the adjacent interfacial matrix, suggesting that differences in the type I-XII collagen fibril organization may exist between Bowman's layer and IM.

Corneal development involves the synthesis and assembly of a number of specialized extracellular matrices. These matrices have distinctive properties derived from a unique assembly of collagens, proteoglycans and glycoproteins. The synthesis of each of these requires a number of enzymes. By probing a corneal cDNA library for genes that appeared to be up-regulated in cornea we have isolated a cDNA that represents an mRNA encoding the enzyme beta-1,4-galactosyltransferase. In cornea, a major function for this enzyme is likely to be in the synthesis of the keratan sulfate proteoglycan, lumican. Employing quantitative reverse transcript-polymerase chain reaction, we have observed that the steady-state level of mRNA for the molecule is elevated during certain stages of corneal development. It is also elevated in corneal fibroblasts in culture that have a greatly decreased synthesis of the mature lumican molecule. These data are consistent with, and complement, studies by others that show a corresponding regulation of the lumican core protein during development and in corneal fibroblast cultures.