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Endocrinology Vol. 139, No. 1 340-349
Copyright © 1998 by The Endocrine Society

ARTICLES

Heterogeneity Among Cells That Express Osteoclast-Associated Genes in Developing Bone

Rune Jemtland, Kaechoong Lee and Gino V. Segre

Endocrine Unit, Massachusetts General Hospital, and the Department of Medicine, Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Gino V. Segre, M.D., Endocrine Unit, WEL 501, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail:. segre{at}helix.mgh.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In the present study, we characterized the phenotype of cells in the osteoclast lineage by in situ hybridization, using antisense complementary RNA probes that encode three genes typically expressed by osteoclasts, tartrate-resistant acid phosphatase (TRAP), type IV collagenase (matrix metalloproteinase-9), and c-fms, the receptor for macrophage colony-stimulating factor. By using complementary RNA probes labeled with 35S, digoxygenin, or a combination of the two labeling methods (dual labeling in situ hybridization), we found that each of these genes exhibited a distinct expression pattern during early stages of endochondral bone development [embryonic day 15 (ED15) to ED17] in fetal mouse hind limbs. Type IV collagenase messenger RNA (mRNA) was first expressed in or just outside of the cellular layers that define perichondrium/periosteum, earlier than transcripts for TRAP or c-fms appeared at the same sites (ED15). Although transcripts for TRAP and c-fms colocalized within the skeleton, c-fms was also found in surrounding soft tissue, whereas TRAP mRNA was never detected outside the skeleton (ED16). Type IV collagenase mRNA was uniquely distributed at the chondro-osseous border, being distinct from the distribution of TRAP or c-fms (ED17). At later stages of skeletal development (ED18 to 15-day-old postnatal bone), however, there was more overlap among TRAP, type IV collagenase, and c-fms mRNAs in cells throughout bone, except at the chondro-osseous junction, where type IV collagenase continued to be uniquely localized to some cells at all developmental stages. Whereas the levels of type IV collagenase mRNA expression was most intense at the chondro-osseous margin, the levels of c-fms and TRAP mRNA expression appeared to be more uniform throughout the developing bone. The results indicate that there is considerable heterogeneity among cells expressing osteoclast-associated genes, particularly during early stages of endochondral bone development, but that this difference becomes less pronounced later in the more mature skeleton. Distinct expression patterns of these markers may represent different stages of osteoclastogenesis. Alternatively, type IV collagenase-positive and TRAP/c-fms-positive cells may represent distinct subpopulations of cells of the osteoclast lineage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE OSTEOCLAST is primarily responsible for resorbing calcified tissues and thus plays essential roles in skeletal morphogenesis and bone remodeling (1). Although osteoblasts are derived from a mesenchymal cell lineage, osteoclasts are of hemopoietic origin and are not bone derived (2). However, there is controversy about the specific cell lineage that gives rise to the osteoclast. Several lines of evidence suggest that there is a close relationship between osteoclasts and mononuclear cells of the monocyte/macrophage family (3, 4, 5, 6, 7). For example, in osteopetrotic op/op mice, the development of osteoclasts, macrophages, and monocytes is severely impaired due to the absence of functional macrophage colony-stimulating factor (M-CSF), which is caused by a point mutation in the M-CSF gene (8, 9). c-fms, the receptor for M-CSF, is expressed by both monocytes/macrophages as well as osteoclast progenitors and mature osteoclasts (5). Administration of recombinant human M-CSF to op/op animals largely rescues the osteopetrotic phenotype by inducing osteoclastogenesis and subsequently restoring bone resorption (8, 10). However, as osteoclasts are present in some skeletal sites in untreated op/op mice (11), and the skeletal sclerosis of op/op mice improves spontaneously beginning in the sixth postnatal week (12), the development of some osteoclasts is probably independent of M-CSF. It has been proposed that macrophage/monocyte-derived osteoclasts differentiate from colony-forming units-granulocyte-macrophage through early and late precursor stages (13). Osteoclasts also derive from more differentiated cells of the monocyte-macrophage lineage (3). Thus, the differentiation pathway for the osteoclast appears to have multiple branches.

The resorptive process is believed to involve both recruiting osteoclast precursor cells to the bone surface and regulating the activity of preexisting mature osteoclasts (14). At an early stage of skeletal development, mononucleated osteoclast precursors are carried by the bloodstream and take up residence in the mesenchyme surrounding the limb primordium (15). Subsequently, mononucleated osteoclast progenitor cells proliferate, migrate to bone resorption sites, fuse to become multinucleated, and attach to specific bone surfaces, where they assume their roles in the replacement of cartilage by bone and in the remodeling of bone (2, 16).

The present study was undertaken to characterize cells that express osteoclast-associated genes by in situ hybridization with complementary RNAs (cRNAs) encoding phenotypic markers for cells of the osteoclast and macrophage lineage. Mononucleated (pre)osteoclasts and multinucleated osteoclasts characteristically synthesize lysosomal tartrate-resistant acid phosphatase (TRAP), whereas earlier committed progenitors and most mononuclear phagocytes do not (17). Transcripts encoding a 92-kDa type IV collagenase [gelatinase B or matrix metalloproteinase-9 (MMP-9)] are selectively expressed in cells of the osteoclast lineage, and activation of this gene has been proposed to be a relatively early event during osteoclastogenesis (18, 19). Here, we used molecular phenotyping to characterize osteoclasts, their progenitors, and related cells in the context of morphological changes occurring in the skeleton from early endochondral bone formation through late stages of skeletal development. The appearance of TRAP-, type IV collagenase-, and c-fms messenger RNA (mRNA)-expressing cells along bone surfaces in the developing mouse hindlimbs was investigated at stages when osteoclast precursors migrate across the bone collar [embryonic day 15 (ED15) to ED16], when the primary ossification center is formed (ED16 to ED17), at later embryonic stages (ED18), and in postnatal bone. Moreover, the relationship between cells expressing different osteoclast-associated mRNAs was assessed by dual labeling in situ hybridization, which simultaneously detected transcripts for two mRNAs that were labeled with 35S or digoxygenin (DIG), respectively, on the same section. Our data show that there is considerable heterogeneity among cells expressing osteoclast-associated genes at earlier stages of endochondral bone formation, but that this difference becomes less pronounced at later stages of skeletal development. They also suggest that type IV collagenase may have a unique role in both the invasion of bone by osteoclast/chondroclast precursors and the replacement of cartilage by bone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue preparation
Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals. Pregnant Swiss-Webster mice were killed by CO2, and fetuses from E15–E18 were removed from the uteri and dipped into freshly prepared 4% paraformaldehyde (PFA)-PBS (pH 7.4) for at least 1 h. Tibias from 5- to 15-day-old postnatal pups were decalcified (20% EDTA-10% formalin) for 1 week. In both cases bones were dehydrated and embedded in paraffin wax. Bone sections of 5-µm thickness were prepared by standard histological procedures and mounted on Superfrost/Plus glass slides (Fisher, Pittsburgh, PA).

Preparation of riboprobes
Complementary DNA encoding mouse c-fms (20) was obtained from American Type Culture Collection, mouse 92-kDa type IV collagenase (18) was obtained from Dr. K. Tryggvason, and rat TRAP (21) was obtained from Dr. G. Andersson. Antisense 35S- or DIG-labeled RNAs were synthesized from the linearized complementary DNAs using the Gemini Transcription kit (Promega, Madison, WI) and [35S]UTP (1289 Ci/mmol; New England Nuclear Corp., Boston, MA) or DIG RNA labeling mixture (Boehringer Mannheim, Indianapolis, IN). The specificity of in situ hybridization was demonstrated by the absence of hybridization signal when adjacent tissue sections were subjected to identical conditions with radiolabeled or DIG-labeled sense RNA probes (data not shown). For dual labeling in situ hybridization, a cocktail of 35S- and DIG-labeled cRNA probes was added to the hybridization mixture as described in the figure legends.

In situ hybridization
Radiolabeled cRNAs. In situ hybridization using radiolabeled probes was performed as described previously (22, 23, 24). In brief, sections were dewaxed with xylene, dehydrated with increasing concentrations of ethanol, and postfixed with 4% PFA-PBS for 15 min. After washing with PBS, sections were digested with 1 µg/ml proteinase K (37 C; 15 min) in PBS and again treated with 4% PFA-PBS for 10 min. Sections then were sequentially washed with PBS, incubated with 0.2 N HCl (10 min), again washed with PBS, acetylated with 0.25% acetic anhydride in the presence of triethanolamine (0.1 M; 10 min), dehydrated with increasing concentrations of ethanol, and air-dried. Hybridizations with 35S-labeled complementary RNAs (cRNAs; 5 x 107 cpm/ml) were performed in a humidified chamber in a solution containing 50% formamide, 10% dextran sulfate, 1 x Denhardt’s solution (0.02% Ficoll 400, 0.02% polyvinylpyrrolidone, 0.02% BSA), 600 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 50 mM dithiothreitol, 0.25% SDS, and 200 µg/ml transfer RNA (18 h; 55 C).

After hybridization, sections were washed briefly with 5 x SSC (standard saline citrate) at 50 C, with 50% formamide-2 x SSC at 50 C for 30 min, and then with 10 mM Tris-HCl (pH 7.6)-500 mM NaCl-1 mM EDTA (TNE) at 37 C for 30 min. Sections were treated with 10 µg/ml ribonuclease A in TNE (37 C; 30 min). After being washed with TNE, sections were incubated once with 2 x SSC (50 C; 20 min) and twice with 0.2 x SSC (50 C; 20 min). Sections were dehydrated with increasing concentrations of ethanol and air-dried. Slides were then placed on x-ray films (Hyperfilm ß-max, Amersham, Arlington Heights, IL), and film autoradiographs were obtained after overnight exposure. Slides were dipped into NTB-2 (Eastman Kodak, New Haven, CT) and stored at 4 C for the times estimated from the intensity of expression on x-ray film (1–7 days). After development, sections were counterstained with hematoxylin and eosin and mounted.

Digoxygenin-labeled cRNAs. In situ hybridization using riboprobes labeled with digoxygenin was performed as described previously (24). The dehydration step after incubation with 0.2 x SSC was omitted, and the sections were transferred directly into TBS (50 mM Tris, pH 7.2, and 140 mM NaCl) buffer. Then, the hybridized probe was detected with alkaline phosphatase-conjugated anti-DIG antibody (1:500 to 1:1000), and a solution of nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate was used as substrate in the color reaction according to the manufacturer’s instructions (Boehringer Mannheim).

Dual labeling in situ hybridization
Sections, probed with a cocktail of DIG- and 35S-labeled RNA, were first processed for detection of the DIG-labeled probe as described above. After development of the color reaction, the slides were photographed and dehydrated by washing in increasing concentrations of ethanol. The sections were subsequently dipped into a solution of 3% (wt/vol) parlodion (25) in isoamyl acetate and allowed to dry overnight. Finally, the sections were developed for emulsion autoradiography, as described above, and mounted.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of osteoclast-associated mRNAs in developing endochondral bone (ED15)
In developing tibia from ED15 mouse embryos, type IV collagenase mRNA was expressed earlier than either c-fms or TRAP mRNAs (Fig. 1Go, b and c, respectively); its expression was in cells immediately outside or in the cellular layers of the periosteum/perichondrium (Fig. 1Go, d–f). Signals for c-fms mRNA were scattered in cells surrounding the mesenchyme; however, they were not expressed in the periosteum/perichondrium, and few, if any, cells expressed TRAP mRNA. Transcripts for M-CSF (Fig. 1gGo), the ligand for c-fms, were expressed in hypertrophic chondrocytes in the cartilage core.



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  		Figure 1. Expression of osteoclast-associated mRNAs in developing endochondral bone (ED15). Hindlimb serial sections were hybridized with 35S-labeled cRNAs for c-fms (b), TRAP (c), type IV collagenase (d–f), and M-CSF (g). Sections were stained with hematoxylin and eosin (a and e) and darkfield views (b–d, f, and g) are shown. The cartilage core is composed of hypertrophic chondrocytes (hc) flanked by several layers of cells defining the perichondrium/periosteum (p). Note scattered expression of c-fms mRNA in surrounding soft tissue, but not in the perichondrium/periosteum (b), whereas M-CSF transcripts are located in the zone of hypertrophic chondrocytes (g). Type IV collagenase mRNA is highly expressed in discrete cells in or immediately outside the perichondrium/periosteum (d). A higher magnification is shown of the area indicated by the rectangle in d showing type IV collagenase-positive cells (depicted with arrows) in the proximity of the newly formed bone collar (e, hematoxylin/eosin-stained section; f, darkfield view). Bars = 60 µm.

 
Expression of osteoclast-associated mRNAs in developing endochondral bone (ED16)
By ED16, whereas transcripts for c-fms (Fig. 2Go, a–d) and TRAP (Fig. 2Go, e–h) were highly expressed at similar locations inside the bone collar, the former also continued to be expressed in the surrounding soft tissue. TRAP mRNA expression, however, was restricted to cells in the skeleton. Type IV collagenase mRNA was detected in cells along both inner and outer layers of the bone collar; positive cells at the latter site appeared smaller and/or were labeled less intensely than cells at the former site (Fig. 2Go, i–l). M-CSF mRNA expression was most intense in the area where the future marrow cavity was to be formed and extended into the region of hypertrophic chondrocytes (Fig. 2Go, m and n).



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  		Figure 2. Expression of osteoclast-associated mRNAs in developing endochondral bone (ED16). Hindlimb serial sections were hybridized with 35S-labeled probes for c-fms (a–d), TRAP (e–h), type IV collagenase (i–l), and M-CSF (m and n). Both hematoxylin and eosin staining (a, c, e, g, i, k, and m) and darkfield views (b, d, f, h, j, l, and n) are shown. Higher magnifications of the area indicated by the rectangle in b showing c-fms-positive cells (c and d), the rectangle in f showing TRAP-positive cells (g and h), and the rectangle in j showing type IV collagenase-positive cells (k and l) are shown. Transcripts for both c-fms (a–d) and TRAP (e–h) are expressed in cells (arrows) along the inner layers of the bone collar (arrowheads). Note c-fms expression in soft tissue also. Type IV collagenase mRNA expression is most extensive and includes the bone collar and flanking perichondrium/periosteum (i–l). M-CSF mRNA expression is widely distributed inside the skeleton and includes the region where the future marrow cavity will soon form and the lower part of the hypertrophic chondrocyte (hc) zone. Bars = 100 µm.

 
Expression of osteoclast-associated mRNAs in developing endochondral bone (ED17)
By ED17, the primary ossification center was formed in the tibia, giving rise to areas consisting of epiphyseal cartilage at the ends of the developing bone that were separated by a central marrow cavity. c-fms and TRAP mRNAs had similar distribution patterns at scattered locations in the marrow cavity (Fig. 3Go, b and c, respectively). Type IV collagenase had a distinct distribution in cells at the margin between growth plate chondrocytes and bone and was expressed in some cells in the marrow cavity (Fig. 3dGo). M-CSF mRNA was predominantly expressed by cells located in the marrow cavity and in the proximal half of the hypertrophic zone (Fig. 3Go, e and f).



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  		Figure 3. Expression of osteoclast-associated mRNAs in developing endochondral bone (ED17). c-fms (b) and TRAP (c) mRNAs continue to have a similar distribution throughout the bone. Type IV collagenase mRNA (d), however, has a distinct distribution, being highest at the chondro-osseous junction (arrows) between growth plate hypertrophic chondrocytes (hc) and bone (bm). M-CSF mRNA expression is seen in cells in the bone marrow and along bone surfaces, partly overlapping the zone of hypertrophic chondrocytes (e and f). Representative sections stained with hematoxylin and eosin to show histology (a and e) and darkfield views (b–d and f) are shown. Bar = 100 µm.

 
Relationship among cells expressing various osteoclast-associated mRNAs in fetal and postnatal bone using dual labeling in situ cRNA hybridization
Using serial sections and single radiolabeled cRNA probes, however, did not allow us to definitively identify whether the same cell coexpressed these osteoclast-associated genes. To assess the relationship among cells expressing c-fms, TRAP, and type IV collagenase mRNAs at these stages, we, therefore, used dual labeling in situ cRNA hybridization (Fig. 4Go). First, a cocktail of DIG-labeled TRAP- and 35S-labeled c-fms cRNA probes was hybridized to the same section, and the transcripts were detected sequentially (Fig. 4Go, a–c). Resolution with DIG-labeled probes was much better than that with radiolabeled probes, allowing multinucleated and mononucleated cells to be more readily distinguished. Thus, transcripts for TRAP were seen in numerous large multinucleated cells at scattered locations throughout the bone (Fig. 4Go, a and d), similar to those observed in the previous experiment using 35S-labeled probe (see Fig. 3Go). Subsequent detection using the radiolabeled c-fms cRNA probe on the same section (Fig. 4Go, b and c), revealed that transcripts for c-fms and TRAP colocalized inside the skeleton. Next, we performed hybridization of an adjacent section with a cocktail of DIG-labeled TRAP and 35S-labeled type IV collagenase cRNAs (Fig. 4Go, d–f). Although a few of the cells expressing type IV collagenase mRNA also expressed TRAP mRNA, the former was intensely expressed in a distinct cell population located at the chondro-osseous margin, between growth plate chondrocytes and bone. Reciprocal combination of the labeling methods was used to confirm that these different sites of expression were not caused by different sensitivity of the DIG- or 35S-labeled probes (Fig. 4Go, g–i). Among the relatively few cells that coexpressed type IV collagenase and TRAP mRNAs at this stage, most were bi- or multinucleated cells. The majority were found close to the surface of trabecular bones, mostly because relatively few cells at the chondro-osseous border expressed TRAP mRNA. Few, if any, cells expressed TRAP mRNA but not type IV collagenase mRNA throughout the sample.



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  		Figure 4. Dual labeling in situ cRNA hybridization of fetal (ED17) mouse hindlimbs. a–c, Colocalization of TRAP and c-fms mRNAs. The same hindlimb section was hybridized with a cocktail of DIG-labeled TRAP and 35S-labeled c-fms cRNAs. Hybridization signals were detected sequentially, first for DIG-labeled TRAP (a) and then for 35S-labeled c-fms [b, brightfield view (BF); c, darkfield view (DF)]. d–i, Distinct localization of transcripts for type IV collagenase and TRAP at early stages of skeletal development. Reciprocal combination of the labeling methods was used to confirm that the different localization patterns were not the result of differing sensitivity for DIG- or 35S-labeled probes. The section was hybridized with a cocktail of DIG-labeled TRAP and 35S-labeled type IV collagenase cRNAs, and the probes were detected sequentially, first for DIG (d) and then for 35S (e, brightfield view; f, darkfield view). A higher magnification of the area indicated by the rectangle in d shows two multinucleated cells (inset) that are readily identified by their pale nuclear staining relative to the more intense staining of the cytoplasm. Detection of the probes for DIG-labeled type IV collagenase (g, brightfield view) and 35S-labeled TRAP (h, brightfield; i, darkfield) shows that cells expressing type IV collagenase mRNA are predominantly located at the chondro-osseous border, whereas cells expressing TRAP mRNA are uniformly distributed in the ossification center. Bar = 100 µm

 
By ED18 (Fig. 5Go) and thereafter, in bones from 5- and 15 day-old mice (Figs. 6Go and 7Go), signals for type IV collagenase were noted in all cells showing positivity for TRAP/c-fms, but the former also continued to be uniquely and intensely expressed in cells at the chondro-osseous junction. In 15-day-old postnatal bones (Fig. 7Go), all three osteoclast-associated mRNAs also were intensely expressed in cells lining cortical bone surfaces.



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  		Figure 5. Dual labeling in situ cRNA hybridization of fetal (ED18) mouse hindlimbs. a–c, Sequential detection of DIG-labeled TRAP and 35S-labeled type IV collagenase mRNAs on the same section of fetal mouse hindlimbs. At the metaphysis, cells expressing TRAP mRNA also express type IV collagenase mRNA. Type IV collagenase mRNA, however, has a distinct distribution, being highest in cells at the chondro-osseous border. Cells at the chondro-osseous margin that express transcripts for type IV collagenase but no detectable levels of TRAP mRNA are depicted by arrows. d–f, Sequential detection of DIG-labeled TRAP (d) and 35S-labeled c-fms (e, brightfield view; f, darkfield view) mRNAs revealed that these transcripts are similarly distributed in the primary spongiosa. Bar = 85 µm.

 


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  		Figure 6. Dual labeling in situ cRNA hybridization. Expression of osteoclast-associated mRNAs in 5-day-old postnatal endochondral bone. Sections from hindlimbs were hybridized with a cocktail of DIG-labeled TRAP and 35S-labeled c-fms (a–c), DIG-labeled TRAP and 35S-labeled type IV collagenase (d–f), or DIG-labeled type IV collagenase and 35S-labeled TRAP (g–i) cRNA probes. Both brightfield (a, b, d, e, g, and h) and darkfield views (c, f, and i) are shown. Cells at the chondro-osseous margin that uniquely express type IV collagenase mRNA are depicted by arrows. Bar = 120 µm.

 


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  		Figure 7. Dual labeling in situ cRNA hybridization. Expression of osteoclast-associated mRNAs in 15-day-old postnatal endochondral bone. A, Sections from mouse hindlimbs were hybridized with 35S-labeled c-fms (a and d), 35S-labeled TRAP (b and e), or 35S-labeled type IV collagenase (c and f) cRNA probes. Brightfield (a–c) and darkfield (d–f) views are shown. Whereas the intensity of TRAP and c-fms mRNA expression is uniform throughout the bone, the level of type IV collagenase expression is not; the latter is more intensely expressed at the junction between cartilage and bone than in the diaphysis. B, Sequential detection of DIG-labeled type IV collagenase (a and d) and 35S-labeled TRAP (b, c, e, and f) mRNA on the same section is shown. a–c, Transcripts for type IV collagenase and TRAP colocalize in cells throughout bone, including numerous cells at the chondro-osseous border. d–f, A higher magnification of the area indicated by rectangles in a–c shows that some cells at the chondro-osseous border continue to uniquely express type IV collagenase. Arrowheads depict cells at the chondro-osseous border expressing both mRNAs; the remaining cells expressing type IV collagenase mRNA do not express TRAP mRNA. Bars = 120 µm (A) and 75 µm (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate a highly distinct pattern of osteoclast-associated gene expression in endochondral bones, especially early in their development (ED15 to ED17). At later stages (ED18 to 15-day-old postnatal bone), this distinction is blurred as transcripts for TRAP, type IV collagenase, and c-fms are progressively expressed by the same cells.

These observations raise questions regarding the ontogeny of the cells expressing genes that are characteristic of osteoclasts. Previous work showed that type IV collagenase is a relatively early marker for cells of the osteoclast lineage (19). Experimental deletion of the protooncogene c-fos in mice led to osteopetrosis due to a loss of functional osteoclasts. In contrast to wild-type bone, in which cells expressing transcripts for type IV collagenase colocalized with TRAP-positive mono- and multinucleated osteoclasts, expression of the type IV collagenase gene in the mutant skeleton was restricted to mononucleated cells at the invading metaphyseal front of 10-day-old postnatal bone. No TRAP-positive cells were detected. This suggested that although committed osteoclast progenitors are able to progress through their initial developmental stages, which are characterized by expression of type IV collagenase mRNA, their subsequent differentiation to postmitotic TRAP-positive cells does not occur in the absence of c-fos. Our data showing that expression of the type IV collagenase gene precedes and then coincides with the expression of genes encoding TRAP and c-fms at various skeletal sites during endochondral bone formation are consistent with, but do not prove, the hypothesis that expression of type IV collagenase mRNA identifies the same population of osteoclasts that later express all three genes. Without methods for simultaneously detecting these transcripts, however, we could not exclude the alternative possibility that type IV collagenase-positive and TRAP/c-fms-positive cells represent distinct cell populations that migrate simultaneously into the same area. Dual labeling in situ cRNA hybridization allowed us to address this issue in more detail. Whereas cells expressing TRAP and c-fms mRNAs always colocalized in the skeleton of late fetal as well as postnatal bone, cells that expressed type IV collagenase mRNA were largely distinct from TRAP/c-fms-positive cells before ED18. From ED18 onward, however, the number of cells coexpressing type IV collagenase and TRAP/c-fms progressively increased in primary and secondary spongiosa (data not shown) and also in cells at the chondro-osseous border. Thus, osteoclasts and/or osteoclast-like cells that accumulate at the chondro-osseous border first express type IV collagenase mRNA; TRAP/c-fms-positive cells that presumably migrate to this site acquire this property at a later time. These data add to the work by others (5, 26, 27), showing that cells of the osteoclast lineage need not express detectable levels of c-fms to migrate to the chondro-osseous border.

This study also suggests that concomitant with the morphological changes that occur as bone grows in width, osteoclasts migrating from the outside to the inside of the forming bone collar gradually acquire a more mature phenotype: small (mononucleated) osteoclast precursors first appear along the outer periosteal surface on ED15; 1 day later, cells that express osteoclast-associated genes, some of which are large, multinucleated osteoclasts, are seen along the inner surface of the bone collar; and by ED17, numerous multinucleated osteoclasts have invaded the mineralized cartilage core. In vitro experiments performed with cultured metatarsal bone explants support this migration pattern (28, 29, 30), which is inhibited by transforming growth factor-ß1 (28).

The unique and highly restricted spatial pattern of cells expressing type IV collagenase mRNA, especially during early embryonic bone development, suggests that this enzyme has a unique role in both invasion of bone by osteoclast precursors and modification of the cartilage matrix for replacement by bone. Several lines of evidence suggest that there is a close spatial relationship between osteoclasts and vascular elements in the subepiphyseal region of the growth plate (31, 32, 33). Interestingly, this is also the site where type IV collagenase-positive cells are most abundant. Type IV collagenase is the predominant MMP secreted by osteoclast-like multinucleated giant cells of human giant tumor of bone, and it has been associated with vascular invasion (34, 35). Type IV collagenase (MMP-9) digests type IV collagen, which is a major structural protein of blood vessel basement membranes (36, 37). Our findings and those of others suggest that type IV collagenase may have a unique role in the invasion of bone by osteoclast-like cells, possibly by facilitating transit of these precursor cells from blood to resorptive sites. Such a role for type IV collagenase is consistent with the observation that the migration of TRAP-positive (pre)osteoclasts from the periosteum to the developing marrow cavity is completely prevented by MMP inhibitors in fetal metatarsal bone explants (29). However, inhibitors of metalloproteinases did not affect bone resorption in metatarsal explants, nor did they affect isolated osteoclasts (38, 39), suggesting that type IV collagenase may have a more important role in osteoclast migration than in matrix resorption, although its intense expression at the chondro-osseous border also is consistent with a unique role in the degradation of cartilage matrix in this specialized microenvironment.

Received May 30, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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