Download limb synovial joints are produced by a distinct population of

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