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LIMB SYNOVIAL JOINTS ARE PRODUCED BY A DISTINCT POPULATION OF PROGENITOR CELLS *Koyama, E; *Shibukawa, Y; **Rountree, R B; ***Kingsley, D; *Enomoto-Iwamoto, M; *Iwamoto, M; +*Pacifici, M +Thomas Jefferson University, Philadelphia, PA [email protected] INTRODUCTION Synovial joints and articular cartilage are critically important for skeletal function and quality of life, and much is known about their structure and properties. Surprisingly, quite little is known about how synovial joints actually form in the developing embryo, including how joints are patterned spatio-temporally, what the nature of joint progenitor cells is, and how the cells carry out their multiple joint-forming tasks (12). This type of information would have fundamental value and could also lead to conception of novel cell-based therapies to restore function in affected joints, In the embryonic limb, skeletogenesis initiates with formation of uninterrupted mesenchymal pre-chondrogenic condensations, including the Y-shaped condensation spanning the stylopod and zeugopod regions and the digital rays in autopod region. The first overt sign of joint development is the appearance of a layer of closely-packed mesenchymal cells at each prospective joint site (often referred to as the interzone). Given their spatio-temporal appearance and distribution, the cells are thought to be important for joint formation, but it remains unclear whether they serve a mere boundary function, represent progenitor cells for joint tissues or underlying long bone shaft, or may have other functions. To tackle these fundamental issues, we made use of a genetic cell-fate approach in vivo and cell behavior studies in vitro. Our data show for the first time that the cells serve as progenitors of joint tissues, including articular cartilage. METHODS Gdf-5-Cre transgenic mice generated as described (3) were mated with ROSA R26R Cre-inducible LacZ reporter mice. Genotyping was carried out with tail clip DNA, and β-galactosidase staining was by standard protocols. In situ hybridization was carried out on paraffin sections using 35S-labeled riboprobes. Immunohistochemistry was carried out on paraffin sections that were de-masked by brief treatment with 0.02% pepsin, using a tenascin-C antiserum (provided by Dr. T. Yoshida). Interzonal cells were dissected out by microsurgery following microinjection of vital fluorescent dye DiI serving as marker of the interzonal site. Cells were dissociated and grown in multiwell in serumcontaining medium. Gene expression was assessed by standard RT-PCR. RESULTS To establish joint progenitor cell fate maps, we used ROSA R26R mice in which the reporter β-galactosidase gene is silent, but becomes irreversibly expressed after Cre-recombinase action. ROSA mice were mated with transgenic mice expressing Cre under control of regulatory sequences from the Gdf-5 gene that is specifically expressed in interzonal cells at onset of joint formation. ROSA/Gdf-5-Cre mouse progenies were examined at fetal and postnatal stages to track behavior and developmental fate of β-galactosidase-expressing cells. Whole mount staining showed that reporter activity was restricted to developing metacarpal-phalangeal (m-ph), inter-phalangeal (ph), wrist (w), elbow (e) and shoulder (s) joints at each stage examined (E13.5 through P28), while intervening long bone shafts were devoid of reporter-expressing cells (Fig. 1A). Identical patterns were observed in hindlimb joints, including hip. Histochemical analyses clearly showed that the positive cells constituted articular cartilage, were part of adjacent developing joint structures such as synovial lining (sl) and capsule tissue (ct), but were excluded from underlying shaft and growth plates. To determine the molecular signature of joint forming cells over developmental time, sections were processed for in situ hybridization. At E15.5, the reporter-positive cells expressed Wnt-14, Gdf-5, and transcription factor ERG. By E17.5 and P0, expression of these early markers decreased, while the cells began to express products typical of functional articular chondrocytes, including lubricin and tenascin-C. Wnt-14, Gdf-5, ERG and other genes are widely thought to be important for joint formation, but their specific action on interzonal cells has never been tested directly. Thus, we established a novel method of microsurgical isolation and culturing of interzonal cells and focused on Wnt-14, recently suggested to be pivotal for joint formation (4). The freshly-isolated cells expressed Gli3, CD44 and collagen IIA in addition to Wnt-14 and Gdf-5, as to be expected. Within a few days in culture, the cells rapidly differentiated into chondrocytes. When the cells were made to over-express Wnt-14 retrovirally, they failed to differentiate, remained mesenchymal in appearance, expressed higher levels of interzonal cell markers, and exhibited increased cell adhesion and migration. DISCUSSION It has long been known that articular cartilage is structurally and functionally different from growth plate cartilage. Our data now reveal that these functional differences involve distinct developmental origins. Articular chondrocytes and other joint cells derive from Gdf-5expressing interzonal cells and their descendants, whereas shaft and growth plate cartilages derive for Gdf-5-negative cells likely representing the bulk of the original mesenchymal condensations. The data establish a new paradigm for limb skeletogenesis. Our data demonstrate also that interzonal cells rapidly differentiate into chondrocytes in vitro. This potential appears to be tamed and kept under negative control by Wnt-14. Such negative influence could be instrumental in establishing the initial mesenchymal character of the interzone. Given Wnt-14’s ability to stimulate cell adhesion and migration as well, the factor could also promote the compact nature of the interzone and recruitment/migration of cells into it. What remains unclear is how interzonal cells eventually escape Wnt-14 influences, undergo chondrogenesis and give rise to articular chondrocytes. These and related key issues are under investigation. REFERENCES 1. Archer CW, Dowthwaite GP and Francis-West P. 2003. Development of synovial joints. Birth Defects Res, Pt. C. 69:144-155 2. Pacifici M, Koyama E and Iwamoto M. 2005. Mechanisms of synovial joint and articular cartilage formation: recent advances, but many lingering mysteries. Birth Defects Res, Pt. C. 75:237-248. 3. Rountree RB, Schoor M, Chen M, Marks ME, Mishina Y and Kingsley DM. 2004. BMP receptor signaling is required for postnatal maintenance of articular cartilage. PLoS Biology. 2:1815-1827. 4. Hartmann C and Tabin CJ. 2001. Wnt-14 plays a pivotal role in inducing synovial joint formation in the developing appendicular skeleton. Cell. 104:341-351. AFFILIATED INSTITUTIONS ** Amgen Washington, Seattle, WA; ***Stanford University, Stanford, CA 53rd Annual Meeting of the Orthopaedic Research Society Paper No: 0041