To examine the regulation of collagen types EX and X during the hypertrophic phase of endochondral cartilage development, we have employed in situ hybridization and immunofluorescence histochemistry on selected stages of embryonic chick tibiotarsi. The data show that mRNA for type X collagen appears at or about the time that we detect the first appearance of the protein. This result is incompatible with translational regulation, which would require accumulation of the mRNA to occur at an appreciably earlier time. Data on later-stage embryos demonstrate that once hypertrophic chondrocytes initiate synthesis of type X collagen, they sustain high levels of its mRNA during the remainder of the hypertrophic program. This suggests that these cells maintain their integrity until close to the time that they are removed at the advancing marrow cavity. Type X collagen protein in the hypertrophic matrix also extends to the marrow cavity. Type EX collagen is found throughout the hypertrophic matrix, as well as throughout the younger cartilaginous matrices. But the mRNA for this molecule is largely or completely absent from the oldest hypertrophic cells. These data are consistent with a model that we have previously proposed in which newly synthesized type X collagen within the hypertrophic zone can become associated with type II/IX collagen fibrils synthesized and deposited earlier in development (Schmid and Linsenmayer, 1990; Chen et al. 1990).

During endochondral bone development, individual chondrocytes follow a progression from young cells undergoing rapid division, through mature cells having the greatest capacity for synthesizing matrix components, to hypertrophic cells, which become progressively enlarged and along with their surrounding matrix are eventually removed. In prehypertrophic cartilage, the chondrocytes synthesize a mixture of collagen types, including II, IX and XI, all of which become co-associated into fibrils (van-der Rest and Mayne, 1987; Mendier et al. 1989). After the cells have initiated hypertrophy, they add type X collagen to their biosynthetic program (for review see Schmid and Linsenmayer, 1987), and this molecule also becomes associated with these fibrils (Schmid and Linsenmayer, 1990).

Several types of evidence suggest that as hypertrophy progresses, the synthesis of type X collagen increases, possibly with a concomitant decrease in synthesis of the other collagen types. By biochemical analyses of cultured chondrocytes, both we (Schmid and Conrad, 1982; Schmid and Linsenmayer, 1983) and others (Capasso et al. 1984; Gibson et al. 1984; Thomas et al. 1990) have observed that as these cells age, there is a progressive increase in the proportion of collagen synthesized as type X. In vivo, as hypertrophy progresses, an increased proportion of the collagen is type X (Capasso et al. 1984; Gibson et al. 1984; Reginato et al. 1986a).

During their progression through hypertrophy, it also seems that, for at least part of the time, individual chondrocytes are capable of maintaining the synthesis of the other ‘cartilage-specific’ collagen types, as well as type X. Double-label immunofluorescence analyses of permeabilized chondrocytes in vitro (Solursh et al. 1986), as well as in vivo observations (unpublished data), show cells containing both collagen types II and X.

Morphologically, in the extracellular matrix, both collagen types IX (Vaughan et al. 1988) and X (Schmid and Linsenmayer, 1990; Chen et al. 1990) are found along the surface of type II collagen fibrils - an association that undoubtedly alters fibrillar properties. In the case of type X, we (Schmid and Linsenmayer, 1990) hypothesized that this results from the secondary association of type X molecules, newly synthesized in the hypertrophic zone, with preexisting type II/IX fibrils, synthesized and assembled at earlier stages of cartilage matrix assembly. Consistent with this possibility, we (Chen et al. 1990) have observed, using an in vitro sternal-cartilage model system, that type X collagen can rapidly and extensively move through cartilage matrix and subsequently become associated with preexisting fibrils.

In the present study, we have investigated the production and matrix deposition of these two ‘fibril-associated’ collagens in vivo. We have examined the mRNAs for both collagen types by in situ hybridization and the deposition of their matrix proteins by immunofluorescence histochemistry. We have employed selected stages of the well-characterized (Schmid and Linsenmayer, 1985a; Schmid and Linsenmayer, 19856) embryonic chick tibiotarsus. In this developing long bone rudiment, the phases of endochondral development occur in a precisely defined sequence, allowing for easy evaluation of both temporal and spatial events during hypertrophy.

Previous studies on type X collagen mRNA synthesis have implicated both transcriptional (LuValle et al. 1989; Castagnola et al. 1988) and translational controls (Reginato et al. 19866; Thomas et al. 1990) as being of primary importance in the initiation of type X collagen synthesis. Our data clearly show that temporally and spatially mRNA for type X collagen first appears at or about the time that the protein becomes detectable. This result is incompatible with translational regulation, which would require accumulation of the mRNA to occur at an appreciably earlier time. Also, once hypertrophic chondrocytes initiate synthesis of type X collagen, they maintain high levels of its mRNA throughout the remainder of the hypertrophic program. Thus, these cells appear to maintain their integrity until close to the time that they are removed at the advancing marrow cavity. Type IX collagen is found throughout the hypertrophic matrix, as well as in the younger cartilages matrices. However, the mRNA for this molecule in the oldest hypertrophic cells, is largely or completely absent.

In situ hybridization

In situ hybridizations were performed essentially according to Hayashi et al. (Hayashi et al. 1986), with minor modifications.

Preparation of tissues

Embryos were staged according to Hamburger and Hamilton (Hamburger and Hamilton, 1951). Tibiotarsi or whole limbs were removed and immediately fixed for 3h in 4% paraformaldehyde (J.T. Baker, Phillipsburg, NJ) in phosphate-buffered saline, pH 7.4 (PBS). The tissue was washed 3 × 5 min in PBS, dehydrated in a graded series of ethanol, infiltrated with xylene, and embedded overnight in paraffin (Paraplast Plus, Monoject, Sherwood Medical, St. Louis, MO). 6 μ m sections were cut and mounted on subbed slides [prepared by incubating clean microscope slides overnight at 68° in a solution of l × Denhardt’s mixture (Sigma Chemical Co., St. Louis, MO) in 3 × standard saline citrate buffer (SSC) (1 × SSC consists of 0.15 M NaCl and 0.015 M trisodium citrate, pH 7.0) and fixing the drained slides for 20 min in ethanol/ acetic acid (3:1)). The sections were deparaffinized by heating for 1 h at 60°C and adding xylene to the warm jar. After 30min in xylene (with occasional agitation), the slides were washed 2 × 15 min in xylene and 2 × 5 min in 100% ethanol, air dried, and fixed in 4% paraformaldehyde in PBS for 10min. They were then washed 3 × 5min in PBS, 2 × 5 min in 70% ethanol, and 5 min in 95% ethanol, and allowed to air dry.

cDNA probes

32P-labeled fragments of cDNAs corresponding to collagen types II, IX and X were used as probes. 100 ng of each fragment was nick translated to a specific activity of greater than l ×108ctsmin−1 μ g−1. The αl(II) collagen probe was a 1200 bp Pstl/Pvull fragment of pYN1124 (Ninomiya et al. 1984). This fragment encodes the C-propeptide region of the α T(TI) collagen chain, as well as 620bp of the 3’ untranslated sequence. The αl(IX) collagen probe was the 1020bp Pvull fragment of pYN1738 (Ninomiya and Olsen, 1984), which represents only 3’ untranslated sequence. The αl(X) collagen probe was a Pstl PvuII fragment of pYN3116 (Ninomiya et al. 1986), which contains 175 bp of the 3’ untranslated sequence.

In situ hybridization

Before hybridization, sections were incubated in a freshly prepared solution of predigested pronase in 50 mM Tris/5mM EDTA, pH 7.5, for 10 min at room temperature, then immediately immersed in PBS containing 2 mg ml−1 glycine for 30s and’two changes of PBS for 30s each. The sections were fixed in the 4% paraformaldehyde buffer for 20 min, washed twice in PBS containing 2 mg ml−1 glycine for 5 min each, washed once in 0.1 M triethanolamine buffer, pH 8.0, for 5 min, and treated for 10 min in a freshly prepared solution of 0.25 % acetic anhydride in the triethanolamine buffer. They were then washed for 5 min each in two changes of 2 × SSC, dehydrated in two changes of 70% ethanol and one change of 95% ethanol for 5 min each, and air-dried in a sterile hood.

The final hybridization solution consisted of 0.5 –1 gg ml−1 of nick-translated probe, 150 – 300gg ml−1 yeast tRNA, 25 – 50ggml−1 salmon sperm DNA, 50% formamide, 10mM Tris-HCl, pH7.0, 0.15M NaCl, ImM EDTA, pH7.0, l × Denhardt’s solution, and 10% dextran sulfate. The lyophilized probe plus yeast tRNA and salmon sperm DNA was dissolved in the aqueous components; then the nonaqueous components were added, and finally the dextran sulfate. The solution was heated for 3 min to 95°C to denature the probe and rapidly cooled in an ice/water bath. 20 μ l of the hybridization mix was spread on the sections and covered with a 22 mm × 22 mm sterile coverslip. The edges of the coverslip were sealed with a 1:1 mixture of rubber cement and petroleum ether. The slides were incubated for 18 – 20 h on a preheated metal pan floating in a 45°C water bath.

After hybridization, the rubber cement was peeled off and the slides were transferred to a slide holder and incubated in 2 × SSC at 45°C for 1h to remove the cover slips. They were then washed for 10 min each in three changes of 45°C 2xSSC, three changes of 45°C I × SSC containing 10% formamide, and three changes of 45°C 0.1 × SSC, allowed to cool for a few minutes, dehydrated in two changes of 70% ethanol and two changes of 95 % ethanol for 5 min each, and allowed to dry.

The hybridized slides were dipped into Kodak NTB-2 emulsion diluted 1:1 with 0.6M ammonium acetate, pH7.0, maintained at 43°C. The slides were allowed to stand vertically for a few minutes, then were laid horizontally to dry at room temperature for at least one hour. They were then exposed for 2 to 3 days in a partially evacuated desiccator over Drierite desiccant. The exposed slides were developed in Kodak D-19 developer at 18°C for 2.5 – 3 min. After drying, a coverslip was mounted using Permount.

The slides were viewed by dark-field and Hoffman Interference optics, and photographed with Kodak Technical Pan film.

Immunofluorescence histochemistry

Immunofluorescence analyses for collagen types IX and X were performed employing monoclonal antibodies on sections cut from the same blocks of tissue used for the in situ hybridizations. After being deparaffinized, the sections were rehydrated in PBS. Further processing for immunofluorescence visualization with monoclonal antibodies was performed as previously described (Linsenmayer et al. 1988). The monoclonal antibodies employed were IX-4D6, which is specific for type IX collagen (Irwin et al. 1985), and X-AC9, which is specific for type X (Schmid and Linsenmayer, 19856).

Stages 30 – 33

We (Schmid and Linsenmayer, 1985a) previously observed that in the tibiotarsus, immunohistochemically detectable type X collagen first appeared at stage 32 (7.5 days). To determine whether the appearance of type X mRNA correlates with this event, we performed in situ hybridizations using type X collagen cDNA on sections of tibiotarsi from a somewhat earlier stage (30) (6.5 – 7 days), and from a somewhat later stage (33) (7.5 – 8 days). We also employed a cDNA probe for collagen type IX.

In stage 30 limbs, there was no detectable hybridization with the cDNA for type X collagen (Fig. 1A). At stage 33 there was clear hybridization of cells in the mid-diaphyseal region of the tibiotarsus, but nowhere else (Fig. IB). cDNAs for collagen type IX (Fig. 1C) hybridized with cells throughout these cartilaginous anlagen.

Fig. 1.

Dark-field micrographs of in-situ hybridizations of sections of tibiotarsi from embryonic stages 30 (A); and 33 (B and C). ‘A’ and ‘B’ were hybridized with the type X collagen cDNA. ‘C’ was hybridized with the type IX collagen cDNA. In addition, an immunofluorescence micrograph of type X collagen in the mid-diaphysis of the same stage 33 embryo is shown in the insert in ‘B’. Bar, 200 μ m.

Fig. 1.

Dark-field micrographs of in-situ hybridizations of sections of tibiotarsi from embryonic stages 30 (A); and 33 (B and C). ‘A’ and ‘B’ were hybridized with the type X collagen cDNA. ‘C’ was hybridized with the type IX collagen cDNA. In addition, an immunofluorescence micrograph of type X collagen in the mid-diaphysis of the same stage 33 embryo is shown in the insert in ‘B’. Bar, 200 μ m.

We also probed for the presence of the protein products of these mRNAs by immunofluorescence histochemistry performed on sections from the same tissue preparations used for the in situ hybridizations. Type X collagen was not detectable in the stage 30 limb (not shown) but was present at stage 33 in the same mid-diaphyseal position (Fig. IB, insert) as its mRNA. Type IX collagen (not shown) was found throughout the cartilaginous anlagen at both stages of development.

Stage 40

To determine whether, during the progression through hypertrophy, chondrocytes lose the ability to synthesize any of these collagens, we performed in situ hybridizations on sections of the epiphyseal growth region from stage 40 (14 days) embryos. Figs 2A and 2A’ are, respectively, Hoffman Interference and dark-field micrographs of the same section of the hypertrophic region hybridized with the cDNA for type IX collagen. Figs 2B and 2B’ are the same types of micrographs of a sister section hybridized with the cDNA for type X collagen. That these tissue sections are entirely within the hypertrophic zone can be determined by the presence of periosteal bone (see figure legend and Discussion) and the proximity of the advancing marrow cavity (see ref. Schmid and Linsenmayer, 1985b). Both types of micrographs are shown because the autoradiographic data are more easily evaluated by dark field, whereas the cells are visible by Hoffman Interference.

Fig. 2.

Micrographs of in situ hybridizations of sections from the hypertrophic region of a tibiotarsus from a stage 40 embryo. Most of the periosteal bone was stripped off during removal of the cartilage, but a small piece remains in the upper left of ‘A’. ‘A’ and ‘B’ utilize Hoffman Interference optics, ‘A’ and ‘B’ are dark field. ‘A’ and ‘A’ were hybridized with the cDNA for type IX collagen. *B’ and ‘B’’ were hybridized with the cDNA for type X. Regions designated ‘a’ to ‘c’ and the arrows in ‘B’ are described in the text. Bar, 200μm.

Fig. 2.

Micrographs of in situ hybridizations of sections from the hypertrophic region of a tibiotarsus from a stage 40 embryo. Most of the periosteal bone was stripped off during removal of the cartilage, but a small piece remains in the upper left of ‘A’. ‘A’ and ‘B’ utilize Hoffman Interference optics, ‘A’ and ‘B’ are dark field. ‘A’ and ‘A’ were hybridized with the cDNA for type IX collagen. *B’ and ‘B’’ were hybridized with the cDNA for type X. Regions designated ‘a’ to ‘c’ and the arrows in ‘B’ are described in the text. Bar, 200μm.

Within the hypertrophic zone, the progression from younger to older chondrocytes resulted in three regions in which different patterns of hybridization were found. In the frames in Fig. 2, these regions are designated ‘a’ to ‘c’. The chondrocytes in ‘a’, the younger ones, showed hybridization with the cDNA for collagen type IX but not that for type X. Those in ‘b’ showed hybridization with the cDNAs for both collagen types.

In region ‘c’ the cells showed hybridization with the cDNA for collagen type X, but little if any with that for type IX. Such cells were found all the way to the margin of the advancing marrow cavity, the site where the oldest chondrocytes reside. Only a small number of the cells (in Fig. 2B indicated with arrows) did not show hybridization with the type X collagen cDNA.

Immunofluorescence analyses of sections taken from the same blocks of stage 40 tissue used for the in situ hybridizations, showed type IX collagen (Fig. 3A) to be distributed throughout both the non-hypertrophic and hypertrophic regions, at most showing a slight decrease in the latter. The distribution of type X collagen (Fig. 3B) in the matrix of the hypertrophic zone corresponded to that of its mRNA. The fluorescence signal for this collagen remained at approximately the same intensity all the way to the front of the advancing marrow cavity.

Fig. 3.

Fluorescence micrographs of sections from the same embryo used in Fig. 2 reacted with monoclonal antibodies for collagen type IX (A) and collagen type X (B). Bar, 200 μm.

Fig. 3.

Fluorescence micrographs of sections from the same embryo used in Fig. 2 reacted with monoclonal antibodies for collagen type IX (A) and collagen type X (B). Bar, 200 μm.

The data that we have obtained on the temporal and spatial distribution of the mRNA for type X collagen argue against translational regulation being the major form of control involved in the appearance of type X collagen in developing embryos in vivo. Type X mRNA was not detectable in limbs about half a day younger than those in which we had observed the first immunohistochemically detectable type X collagen (Schmid and Linsenmayer, 1985a). When the mRNA did become detectable, it corresponded both temporally and spatially to the appearance of the protein. Outside of the mid-diaphyseal region, the chondrocytes, which themselves will subsequently become hypertrophic and synthesize type X collagen, had no detectable type X collagen mRNA. Thus, if translational regulation is involved at all, it must occur during a very brief period of time.

In a previous immunohistochemical analysis of the epiphyseal growth region of 13-day embryos, we (Schmid and Linsenmayer, 1985b) observed that the advancing front of immunohistochemically detectable type X collagen lagged considerably behind that of the surrounding sleeve of periosteal bone, generally considered to demarcate the level of a developing limb at which hypertrophy is initiated. Those data allowed us to conclude that the initiation of type X collagen synthesis occurred subsequent to the onset of hypertrophy; it did not precede hypertrophy.

The current in situ hybridization data allow us to extend this conclusion to the level of mRNA. As is the case for the protein, during early hypertrophy the cells contain mRNAs for collagen type IX, but not for type X which does not appear in detectable amounts until later. Thus, the accumulation of type X collagen mRNA is a result of a previously initiated hypertrophic program, and not the cause of it.

We have used the term ‘hypertrophic program’ to suggest that hypertrophy may involve a regulated cascade of events. Consistent with this, we have observed that the progression of a chondrocyte through hypertrophy does not simply involve the acquisition of mRNA for type X collagen. It also involves the extensive diminution, and possibly the complete loss of the mRNA for collagen type IX. Similar results have been reported for type II collagen mRNA in the hypertrophic cartilage of juvenile chickens (Oshima et al. 1989), and for chondrocytes in suspension culture (Castagnola et al. 1988).

Type X collagen mRNA, conversely, continues to be sustained at high levels within the chondrocytes nearest the advancing marrow cavity. This suggests that even the most hypertrophic cells maintain their integrity and metabolic capabilities until about the time of their removal (see also Farnum and Wilsman, 1987; Hun-ziker et al. 1985). Thus, if the removal of chondrocytes and matrix during marrow cavity formation involves cell death, it probably occurs rapidly and is confined to the cells immediately bordering the cavity, as is suggested also by the morphological observations of Farnum and Wilsman (1987).

The observations on the distributions of mRNAs for these collagens, when coupled with the immunofluorescence data on the distribution of their proteins, are consistent with our hypothesis that newly synthesized type X collagen molecules become added onto preexisting type II/IX collagen fibrils. The absence of detectable type IX collagen mRNA throughout the portion of the hypertrophic zone nearest the marrow cavity, with the continued presence of the protein, suggests that previously synthesized type IX is retained within the matrix. Conversely, the maintainance of type X collagen mRNA by the terminal hypertrophic chondrocytes, demonstrates that these cells remain capable to the very end of using this molecule to modify the matrix.

We thank Yoshi Ninomiya for providing cDNAs and John Fitch for critically reading the manuscript. Supported by NIH Grant HD23681 to T.F.L. and AM30481 to R.M., and an Arthritis Investigator Award to T.M.S.

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