title: Association of type XII collagen with regions of increased stability and keratocyte density in the cornea.
abstract: 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.
authors.affiliation: Department of Anatomy and Cellular Biology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA. jeffrey.marchant@tufts.edu
authors.forename: Jeffrey K
authors.initials: JK
authors.lastname: Marchant
authors.forename: Guiyun
authors.initials: G
authors.lastname: Zhang
authors.forename: David E
authors.initials: DE
authors.lastname: Birk
mesh_terms: D000818:Animals; D001717:Birds / Q000196:embryology / Q000378:metabolism*; D018929:Cell Culture Techniques; D024082:Collagen Type XII / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D003315:Cornea / Q000196:embryology / Q000378:metabolism* / Q000648:ultrastructure; D003319:Corneal Stroma / Q000378:metabolism; D004728:Endothelium, Corneal / Q000378:metabolism; D019573:Epithelium, Corneal / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism / Q000648:ultrastructure; D015870:Gene Expression; D006358:Hot Temperature; D017403:In Situ Hybridization; D011489:Protein Denaturation; D012333:RNA, Messenger / Q000235:genetics
chemical_list: D024082:Collagen Type XII; D012333:RNA, Messenger