Differential expression of type XII collagen in developing chicken metatarsal tendons.

Type XII collagen is a fibril-associated collagen with multiple functional domains. The purpose of this work was to determine its role in regulating tendon matrix assembly. The temporal and spatial expression patterns of both collagen and mRNA were analysed in developing chicken metatarsal tendons using immunofluorescence microscopy, in situ hybridization and real-time quantitative PCR. Temporally, type XII collagen was present during all stages of development (day 14-hatch). However, spatially, type XII collagen expression shifted from the entire tendon at day 14, when the tendon is immature and fascicles are not well developed, to the interfacial matrix (endotendinium) associated with developing fascicles. This shift was obvious beginning at day 17, becoming prominent at day 19. Associated with this shift was a gradual decrease in type XII collagen reactivity in the tendon proper (non-sheath). By hatching, the reactivity was sequestered almost exclusively to the sheaths with some reactivity remaining at the fibroblast-matrix interface within the fascicle. In situ hybridization indicated that fibroblasts in the tendon expressed type XII collagen mRNA homogeneously at day 14. However, by hatching, when the tendon matures, type XII collagen is restricted primarily to the sheath cells. Quantitative PCR analyses, of NC3 splice variants, demonstrated highest expression levels for the short splice variant mRNA at days 14-17, followed by a significant decrease at day 19 with levels remaining constant to adult. Long variant mRNA expression was highest at day 14 then decreased and was constant from day 17 to adult. These changing patterns may be related to the spatial shift in type XII collagen expression to the sheaths. Differential temporal and spatial expression patterns indicate that type XII collagen functions to integrate the developing tendon matrices and fascicles into a functional unit.

The roles of types XII and XIV collagen in fibrillogenesis and matrix assembly in the developing cornea.

Corneal transparency depends on the architecture of the stromal extracellular matrix, including fibril diameter, packing, and lamellar organization. The roles of collagen types XII and XIV in regulation of corneal fibrillogenesis and development were examined. The temporal and spatial expression patterns were analyzed using semi-quantitative RT-PCR, in situ hybridization, Western analysis, and immunohistochemistry. Expression of types XII and XIV collagens in cornea development demonstrated that type XII collagen mRNA levels are constant throughout development (10D-adult) while type XIV mRNA is highest in early embryonic stages (10D-14D), decreasing significantly by hatching. The spatial expression patterns of types XII and XIV collagens demonstrated a homogeneous signal in the stroma for type XIV collagen, while type XII collagen shows segregation to the sub-epithelial and sub-endothelial stroma during embryonic stages. The type XII collagen in the anterior stroma was an epithelial product during development while fibroblasts contributed in the adult. Type XIV collagen expression was highest early in development and was absent by hatching. Both types XII and type XIV collagen have different isoforms generated by alternative splicing that may alter specific interactions important in fibrillogenesis, fibril-fibril interactions, and higher order matrix assembly. Analysis of these splice variants demonstrated that the long XII mRNA levels were constant throughout development, while the short XII NC3 mRNA levels peaked early (12D) followed by a decrease. Both type XIV collagen NC1 splice variants are highest during early stages (12D-14D) decreasing by 17D of development. These data suggest type XII collagen may have a role in development of stromal architecture and maintenance of fibril organization, while type XIV collagen may have a role in regulation of fibrillogenesis.