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.