ARTICLE |
Correspondence to: Gary P. Dowthwaite, Dept. of Veterinary Basic Sciences, The Royal Veterinary College, University of London, London NW1 0TU, UK.
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Summary |
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We studied the expression of hyaluronan binding proteins (HABPs) during the development of embryonic chick joints, using immunocytochemistry and biotinylated HA. The expression of actin capping proteins and of actin itself was also studied because the cytoskeleton is important in controlling HAHABP interactions. Three cell surface HABPs were localized in the epiphyseal cartilage, articular fibrocartilage, and interzone that comprise the developing joint. Of these three HABPs, CD44 was associated with the articular fibrocartilages and interzone, whereas RHAMM and the IVd4 epitope were associated with all three tissues. Biotinylated HA was localized to interzone and articular fibrocartilages before cavity formation and within epiphyseal chondrocytes post cavitation. Actin filament bundles were observed at the developing joint line, as was the expression of the actin capping protein moesin. Manipulation of joint cavity development, using oligosaccharides of HA, disrupted joint formation and was associated with decreases in CD44 and actin filament expression as well as decreased hyaluronan synthetic capability. These results suggest that HA is actively bound by CD44 at the developing joint line and that HAHABP interactions play a major role in the initial separation events occurring during joint formation. (J Histochem Cytochem 46:641651, 1998)
Key Words: hyaluronan, CD44, joint development, actin capping proteins, chick, immunocytochemistry
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Introduction |
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The development of diarthrodial joints has been divided into two basic phases, the initial formation of the cartilaginous anlagen and the subsequent formation of the joint space and its associated synovial lining between these anlagen (for review see
In adult joints the cavity is filled with viscous synovial fluid, which contains a high concentration of hyaluronan (HA) (
Subsequently, we found that during embryonic joint development, cells at the developing articular surfaces of both human and chick joints resemble those found in adult synovium. They contain elevated UDPGD activity per cell, as well as elevated immunocytochemical labeling for molecules associated with HA synthesis, compared with neighboring epiphyseal chondrocytes (
Previous studies have shown that HA in conjunction with cell surface HA binding proteins (HABPs) is capable of facilitating cell separation in vitro (
The aim of this study was to establish the temporospatial expression of several cell surface HABPs during the process of joint cavitation in developing chick joints, and to determine the potential ligand binding status of these HABPs using biotinylated HA (
Finally, because it is known that small HA chains containing less than six disaccharide repeats are capable of displacing the HA-containing pericellular coat of cultured chondrocytes (
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Materials and Methods |
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Tissue Preparation
Hind limbs (n = 6 from each stage) were removed from white leghorn chick embryos between Days 6 and 18 (stages 3044) (
Immunocytochemical Staining for HABPs and ERM Family Members
Primary Antibodies.
A panel of monoclonal and polyclonal antibodies to various HABPs and ERM family members was used in this study (Table 1). Antibody 30189 (a kind gift from Dr. S. Tsukita, Kyoto University, Japan) was raised to CD44 isolated from baby hamster kidney cells (Tsukita et al. 1994). The monoclonal antibody IVd4 (a kind gift from Prof. Bryan Toole, Tufts University, Boston, MA) was raised against an HABP isolated from embryonic chick brain (
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Immunocytochemistry. Immediately before immunolabeling, cryostat sections were dried under a fan for 30 min at room temperature (RT) and rinsed briefly with Tris-buffered saline (TBS, pH 7.2; Sigma, St Louis, MO) for 5 min. Antibodies were then applied after the optimal antibody dilution and incubation times had been determined (see Table 1). Sections were then washed in TBS (three times for 5 min) and appropriate fluoroscein isothiocyanate (FITC)-conjugated secondary antibodies (Sigma) diluted 20% in heat-inactivated chick serum (Gibco; Paisley, UK) in TBS were applied for 1 hr at RT. Subsequently, sections were washed with TBS (three times for 5 min) and mounted in 0.05 M Tris buffer (pH 8.5), containing 33% glycerol, 15% polyvinyl alcohol (Sigma), and 2.5% DABCO (1,4,diazobicyclo[2.2.2.] octane; Sigma) to reduce bleaching of fluorescence. Controls consisted of sections treated with TBS or appropriate nonimmune immunoglobulins instead of primary antibody.
Localization of Actin Filaments
Polymerized actin filaments were localized using 1 µg/ml FITC-conjugated phalloidin (in TBS; Sigma) applied to sections for 30 min at RT and washed in TBS (three times for 5 minutes). Thereafter, nuclei were counterstained by incubating sections with propidium iodide (0.5 µg/ml in TBS; Sigma) for 2 min and were subsequently washed in TBS (three times for 5 minutes) and mounted in DABCO. Dual labeled sections were examined using a Molecular Dynamics (Sunnyvale, CA) Sarastro 2000 laser scanning confocal microscope.
Detection of HA Binding Sites Using Biotinylated HA
Biotinylated HA (bHA) was a kind gift from Dr. Jim Melrose (University of Sydney, Australia) (
Manipulation of Joint Cavity Formation Using Oligosaccharides of HA
HA oligosaccharides (HAO) with no more than six disaccharides per chain were prepared as previously described (
White Leghorn chick embryos were windowed at Stage 24, the windows resealed using transparent adhesive tape, and replaced in the incubator at 39C. At Stage 30, windows were reopened and agarose beads, previously soaked for 1 hr in 50 µl of 1 mg/ml HAO and washed in PBS containing 0.1% phenol red for 15 min, were applied to wounds cut in the presumptive knee joints of the right leg after exposure of the limb by careful dissection of the extraembryonic membranes. Control experiments consisted of contralateral limbs, limbs into which a PBS-soaked bead were implanted, sham-operated limbs, and unoperated (i.e., normal) limbs. After application of the bead, the extraembryonic membranes were carefully closed over the embryo and the window resealed with transparent adhesive tape. Embryos were replaced in the incubator at 39C and allowed to develop for a further 3 days (up to Stage 36), after which knee joints were removed, chilled, and sectioned as described above.
Sagittal cryosections were stained with 0.1% toluidine blue in 0.1 M acetate buffer (pH 6.0) and examined by routine microscopy to histologically assess the effect of HAO bead application on joint cavitation compared with controls. Sections were also labeled with antibody (30189) to CD44, and actin filament expression was observed using FITC-conjugated phalloidin as described above.
Assessment of UDPGD Activity in HAO Beaded Limbs and Controls
Limbs were reacted and assesssed microdensitometrically for UDPGD activity as described by
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Results |
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Distribution of Cell Surface HABPs
All joints examined (knee, MTP, and interphalangeal joints) exhibited the same distribution of CD44 (as shown by immunolabeling with antibody 30189) during the process of joint cavitation. Before and during joint cavitation, intense labeling for CD44 was observed on cells of the interzone and developing articular fibrocartilages, with the most intense label associated with the developing articular fibrocartilages, whereas the epiphyseal chondrocytes were weakly labeled (Figure 1A). On completion of the cavitation process, a decreasing gradient of labeling intensity was evident, from the most intensely labeled surface cells of the articular fibrocartilage to less intense labeling in the deeper regions of articular fibrocartilage and finally weak labeling in epiphyseal chondrocytes (Figure 1D).
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Before, during, and after formation of the joint cavity, label for the RHAMM epitope (antibody R7.2) was also intense in the cells of the interzone and developing articular surfaces (Figure 1B and Figure 1E). However, in contrast to the marked differential labeling intensity evident for CD44, labeling for RHAMM in the epiphyseal chondrocytes was only slightly less intense than in the articular fibrocartilage (Figure 1E).
The IVd4 epitope was also detected within the cells of the interzone and developing articular fibrocartilage before, during, and after cavitation (Figure 1C and Figure 1F). As with the RHAMM antibody, labeling in the epihyseal chondrocytes was only slightly less intense than that seen in the articular fibrocartilage (Figure 1F). Therefore, the differential labeling intensity observed between the cells bordering the developing joint cavity and their neighboring epiphyseal chondrocytes was most marked for CD44. Control sections treated with the appropriate nonimmune serum or without primary antibody in the initial incubation and examined using routine microscopy showed no labeling (data not shown).
Immunocytochemical Labeling for ERM Family Members
Within the developing anlagen, immunocytochemical labeling using a pan-specific antibody (CR22) recognizing all members of the ERM family was concentrated at the presumptive joint line in the interzone and developing fibrocartilage before cavitation, and this differential distribution was maintained in articular fibrocartilage during subsequent stages of joint morphogenesis (data not shown). Using antibodies specific to individual members of the ERM family of proteins, we found that moesin (antibodies M22 and 454) was preferentially expressed by cells of the interzone and articular fibrocartilage before cavitation (Figure 2C) and that expression of moesin was maintained in the articular fibrocartilage after cavitation (Figure 2D). At all stages during cavitation, weak labeling was evident for moesin in neighboring epiphyseal chondrocytes (Figure 2C and Figure 2D). Although failure to detect specific proteins immunocytochemically is not necessarily absolute evidence of their absence, label for the other members of the ERM family, i.e., ezrin and radixin (antibodies 464 and 457, respectively), was not present at the joint line or within epiphyseal chondrocytes (Figure 2A and Figure 2B).
Localization of Hyaluronan Binding Sites
Disclosure of occupied HA binding sites after hyaluronidase pretreatment of sections (total HA binding sites) showed intense cell-associated pericellular labeling within the developing fibrocartilaginous articular surfaces and, to a lesser extent, in the interzone before cavitation (Figure 3A). There was very weak labeling with bHA in the epiphyseal chondrocytes after hyaluronidase pretreatment (Figure 3A). After cavitation, binding of bHA in hyaluronidase-pretreated sections was most prominent in chondrocytes of the epiphysis, which showed progressively weaker labeling intensity in the articular fibrocartilage, although some label was apparent on the surface cells of the fibrocartilage (Figure 3B). In contrast, sections treated with bHA without prior hyaluronidase treatment showed very little if any staining for freely available (unoccupied) HA binding sites within the interzone or the fibrocartilage at any stage examined before, during, or after cavitation. Similarly, under these conditions extremely low levels of bHA binding were observed in the cartilage matrix (data not shown). Control sections treated with TBS instead of bHA showed no staining (data not shown).
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Actin Filament Distribution in Cavitating Joints
Using dual labeled sections and confocal microscopy, intense staining for filamentous actin was observed in the cells of the developing interzone and fibrocartilaginous articular surfaces before, during, and after cavitation (Figure 4A and Figure 4B). Epiphyseal chondrocytes also stained for actin, but the staining intensity in these cells was much weaker than in the cells at the joint line (Figure 4A and Figure 4B). Label within interzone and fibrocartilage cells appeared linear, extending away from the nuclei and following the plane of joint cavitation, whereas the weak labeling in chondrocytes extended away from the nuclei but did not appear to follow the plane of cavitation (Figure 4A and Figure 4B).
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Effect of Oligosaccharides of HA on Joint Cavitation
From a total of 24 embryos implanted with HAO-soaked beads, seven embryos survived to Stage 36/37 (29% survival). This low survival rate is probably due to the late stage at which the manipulations were performed because PBS-beaded and sham-operated embryos both had similarly low survival rates (30 and 31%, respectively). Histological examination of the seven HAO-treated embryos revealed disruption of cavitation in six embryos and no effect in the other one (85% joint disruption). This disruption was noted histologically as a loss of the demarcation between the developing articular fibrocartilage and the interzone and the lack of an obvious cavity between the femur and the intervening menisci (Figure 5A and Figure 5B). Compared with control limbs (PBS-beaded, sham-operated, and contralaterals), the cartilage anlagen of the femur and tibia of manipulated limbs were separated by a loose mass of "mesenchymal cells" with no distinct developing articular fibrocartilage and no development of the joint cavity. There was little evidence of differentiation of the menisci in manipulated limbs.
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With the anti-CD44 antibody 30189, there was less intense immunolabeling for CD44 in manipulated limbs compared with control limbs (Figure 5C and Figure 5F), and there was decreased labeling of actin filaments with FITCphalloidin in manipulated limbs (Figure 5D and Figure 5G).
Assessment of UDPGD Activity in Manipulated and Control Limbs
The UDPGD activity/cell at various sites in experimental and control limbs is shown in Figure 6. Control limbs showed significantly (p=0.01) higher UDPGD activity in articular fibrocartilage cells than in the femoral or tibial chondrocytes (Figure 5E and Figure 5H), whereas UDPGD activity in these control epiphyseal chondrocytes was not significantly different from that in epiphyseal chondrocytes of HAO-treated limbs (p>0.05). However, although UDPGD activity in sites of joint fusion in HAO-treated limbs was significantly higher than the UDPGD activity in the epiphyseal chondrocytes of the same limbs (p=0.001), it was also significantly decreased compared with UDPGD activity found in control articular fibrocartilage cells (p=0.01).
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Discussion |
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Our findings indicate that HABPs play a central role during synovial joint formation and that their interaction with HA may exert a primary influence coordinating tissue separation at these sites. We have demonstrated (a) that three cell surface HABPs, i.e., CD44, RHAMM, and the IVd4 epitope, are present at sites of joint formation in embryonic chick limbs, (b) that CD44 is co-expressed with moesin and linear bundles of filamentous actin in a joint line-selective distribution that is closely associated with cavitation, (c) that occupied HA binding sites are present at these locations during but not after cavitation, and finally (d) that application of HAOs close to sites of presump-tive cavitation disrupts joint cavity formation and is associated with local decreases in UDPGD activity/cell and with the expression of CD44 and filamentous actin.
It has been shown that low HA concentrations may promote adhesive interactions between adjacent HABP-expressing cells. Conversely, increased HA concentrations may facilitate cellcell separation by a process involving saturation of their cell surface-associated HABPs (
Of these HABPs, only CD44 contains an aggrecan-like link module, HA binding domain (
CD44 can exist in one of three distinct HA binding states: inactive, activatable, and constitutively active (
The "active" HA binding role for CD44 at sites of initial tissue separation is also strengthened by our findings regarding the distribution of the ERM protein family members and filamentous actin. We have shown that interzone cells label strongly with a pan-specific antibody to these actin capping ERMs, and that one ERM family member in particular, i.e., moesin, exhibits a marked differential expression at presumptive and forming joint lines. Moreover, our confocal microscopic analyses using FITC-conjugated phalloidin also revealed a specific alignment of polymerized filamentous actin parallel to developing joint surfaces, and indicate that the intensity of staining as well as the degree of specific alignment is most obvious in cells directly bordering sites of presumptive and recently formed cavities. In contrast, the neighboring epiphyseal chondrocytes show reduced levels of staining that lacks such regular or specific alignment.
Many studies suggest that interactions between actin cytoskeletal elements and cell surface HABPs are a prerequisite for effective ligand binding (
Using chondrocytes, fibrosarcoma and endothelial cells (
Our results indicate that HAO application also results in structural loss of coherence in developing fibrocartilaginous articular surfaces. However, because these cells have neither HA binding capacity (even after hyaluronidase digestion) nor an association with an ECM containing high HA levels, it is apparent that HAOs produce loss of fibrocartilage structural integrity by a mechanism distinct from that apparent at the site of cavity formation. Interestingly, HAO beading failed to disrupt the integrity of epiphyseal cartilage, suggesting that HAOs either failed to reach the epiphyseal region or that HAHABP interactions in these epiphyses are less sensitive to HAO-mediated disruption. Indeed, the size of the HAOs used in this study (less than 12 disaccharides) is insufficient to disrupt HAaggrecan interactions (
In conclusion, on the basis of a close temporospatial association between increased local HA synthesis (
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Acknowledgments |
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We wish to thank Prof Bryan Toole, Prof Eva Turley, Dr Sochiro Tsukita, and Dr Frank Solomon for kindly supplying primary antibodies, Dr Jim Melrose for supplying biotinylated hyaluronan, and Dr Mike Bayliss for the supply of HAOs. GPD was supported by the Arthritis and Rheumatism Council of Great Britain. For the use of the confocal microscopy facilities at the CTBL, University of Wales, Cardiff, we wish to thank Dr Jim Ralphs.
Received for publication June 11, 1997; accepted December 10, 1997.
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