In this work we have set out to better clarify the epigenetic differences between connective tissues using a genomic expression analysis of ACL, PCL, MCL, AT, PT, and synovium, with our underlying hypothesis being that significant differences would exist specifically between tendon and ligament. Our analysis uncovered several genes that are of potential interest in understanding the development and physiology of connective tissues. In particular, we have identified a unique transcript (fibin2
), that exhibits very strong and restricted expression in developing and mature tendons and ligaments. Faithful molecular markers of tendons and ligaments have only recently been identified starting with Scleraxis and Tendin/Tenomodulin in 2001,18,29,30
followed by Mohawk in 2006.26
The identification of molecular markers of tendons and ligaments, particularly of Scleraxis, which is the most faithful to date, has spurred rapid progress and interest in the molecular development of these tissues. Molecular visualization has allowed researchers to track the commitment and fate of tendon progenitors,17,19
describe the effect of systemic mutations on tendon development, and to track the ability of stem cells to develop into tendon precursors in vitro (Pearse et al. unpublished data). The identification of fibin2
provides both an independent marker gene for analysis of tendon and ligament precursors and, as a putative secreted signal, is a candidate gene for cooperative specification and maintenance of tendon and ligament tissues.
Our analysis also highlights several known genes that have not previously been linked to tendon and ligament function. Specifically DKK3, a family member of a group of Wnt inhibitory proteins, Nephroblastoma Overexpressed Gene (NOE), Ccdc3, and Keratocan, were highly expressed in connective tissues relative to control tissues. These particular genes also demonstrated spatially restricted expression in the developing chick implicating a role for them during embryogenesis (). It is, in fact, interesting that most of the genes within this cluster are expressed in patterns that are individually unique from each other but that are all spatially related to aspects of the development of connective tissues, bones and muscles. These expression patterns are compelling because tendons and ligaments will ultimately bind together the complexity of the musculoskeletal system in the adult.
We also saw expression level differences between intraarticular and extraarticular connective tissues in 24 different genes. In each of these cases the observed difference was a matter of level of expression rather than of absolute expression. Our studies, in fact, were unable to identify a single gene demonstrating absolute “on or off” expression differences between subtypes of connective tissues regardless of the genome-wide coverage of the expression array used in our analysis. There could be multiple reasons for this. It is possible that there simply are no factors that are absolutely restricted to a specific subtype of connective tissue and that the differential properties that they exhibit are due to the more subtle alterations we see in this analysis. There could still be embryonic factors that are sufficient to specify subtypes of connective tissues but from our analysis of the adult tissue, the resulting differences largely specify an adjustment up or down in levels of specific cohorts of connective tissue factors.
The analysis of these expression patterns indicates, from a broader perspective, that there is a closer molecular relationship between extraarticular ligaments and tendons than there is between intraarticular ligaments and extraarticular ligaments. That is, from a molecular standpoint, the major subdivision of dense regular connective tissues is not tendon versus ligament, it is intra- versus extraarticular. This conclusion is based on the observation that, among the five different connective tissues, the genome-wide expression profiles of the ACL and PCL tissues were linked to each other (but not to the MCL), by two clusters, containing 24 transcripts, whereas there are only five genes (discussed above), whose expression pattern distinguishes tendons and ligaments. These differentially expressed genes, which are all listed in , are candidates for effectors of the differential biology of intra- and extraarticular connective tissues. Our observations are consistent with recent work demonstrating that intra- and extraarticular connective tissues undergo very different developmental pathways. Specifically, all intraarticular tissues, including ligaments, cartilage, and synovial lining, are descended from the same GDF5 expressing progenitor cells while extraarticular connective tissues are not.31
Additionally, analysis of mice containing null mutations in both the Sox5 and Sox6 genes demonstrated a lack of intraarticular ligaments but normal development of extraarticular tendons and ligaments (V. Lefebvre personal communication), underscoring the unique developmental requirements of intra- and extraarticular connective tissues. It is worth reiterating, however, that all of the gene cohorts we have analyzed (while tracking together) show a spectrum of expression levels across the tissues analyzed. Thus, there is a gradient of expression levels of these genes from intraarticular ligaments, to MCL, to extraarticular tendons.
This study also found that the gene for collagen Type II alpha I (COL2A1) was expressed by tendon and ligament cells. COL2A1 expression is typically associated with chondrocyte function, but the finding here in fibroblasts is consistent with prior work showing type II collagen production by rat renal fibroblasts,32
as well as chick embryo fibroblasts33
Similarly, the matrix metalloproteinase I (MMP-1) expression noted in the synovium is consistent with prior studies of rat synovial fibroblasts where MMP-1 is noted particularly in the lining layer of the synovium,35
and has been shown to be regulated by IL-1b36,37
One of the weaknesses in this study was the small number of tissues examined. This makes it difficult to determine whether the appropriate distinction is between intra- and extraarticular tissues or intra- and extrasynovial tissues. Future studies including analyses of additional tissues (e.g., the extraarticular flexor tendons and additional extraarticular ligaments) are planned to follow up and expand on the initial findings reported here.
In summary, we have identified the molecular signatures of a set of diverse connective tissues. These data have helped to identify at least one novel transcript, Fbn2, that is expressed very specifically in developing and mature tendons and ligaments. They also demonstrate a stronger molecular connection between tendons and extraarticular ligaments than between intra and extraarticular connective tissues.