Differential Expression of Genes Associated with Collagen Fibril Growth in the Chicken Tendon: Identification of Structural and Regulatory Genes by Subtractive Hybridization

Collagen fibril growth is a very rapid and abrupt process, resulting in a 4- to 5-fold increase in fibril length between 16 and 18 days of chicken metatarsal tendon development. This fibril growth is due to a postdepositional fusion/association of preformed intermediates, termed fibril segments. We propose that the regulated assembly of collagen fibrils from the segment intermediates is mediated by interactions of structural macromolecules. The cells could modulate this process by responding to cytokines and altering cell–matrix signaling, transcription, and translation. To identify the genes involved in this process a subtractive hybridization procedure was utilized. Genes of cell proliferation were excluded as major contributors to differential gene expression in avian tendon on days 14 and 19 of development after analysis of BrdUr incorporation. The BrdUr incorporation studies revealed little, if any, tendon fibroblast proliferation at both stages. This suggested that observed alterations in gene expression would be related to the pre- and postfibril growth phases in developing tendons. A total of 80 unique up- and down-regulated cDNA fragments were isolated and 26 of these were identified. There was an up-regulation of structural proteins (for example, collagen types I, VI, and XI and fibromodulin), a number of regulatory proteins (including TGF-β2 and IGF-1), as well as other enzymes/proteins. Northern analysis confirmed the up-regulation of mRNAs for all the structural proteins. The observed 20-fold increase of mRNA for the isolated clone corresponding to the 3′ UTR of α1(VI) collagen makes it a possible marker for the postfibril growth stage of collagen fibrillogenesis. The large number of isolated genes differentially expressed during the rapid phase of fibril growth reveals a fine and possibly tissue-specific control of fibrillogenesis.