1 Department of Anatomy and Cellular Biology, Tufts University Health Sciences Schools, 136 Harrison ... al. 1990). Key words: collagen types IX and X, chick tibiotarsus,. mRNA .... Interference optics, and photographed with Kodak Technical.
Development 111, 191-196(1991) Printed in Great Britain © The Company of Biologists Limited 1991
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Collagen types IX and X in the developing chick tibiotarsus: analyses of mRNAs and proteins THOMAS F. LINSENMAYER1*, QIAN CHEN1, EILEEN GIBNEY1, MARION K. GORDON 1 , JEFFREY K. MARCHANT1, RICHARD MAYNE2 and THOMAS M. SCHMID3 1 Department of Anatomy and Cellular Biology, Tufts University Health Sciences Schools, 136 Harrison Ave, Boston, MA 02111, USA 'Department of Cell Biology and Anatomy, University of Alabama at Birmingham, Birmingham, AL, USA 3 Department of Biochemistry, Rush-Presbyterian-St Luke's Medical Center, Chicago, 1L, USA
* Author for correspondence
Summary 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 translations! 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 El/EX collagen fibrils synthesized and deposited earlier in development (Schmid and Linsenmayer, 1990; Chen et al. 1990).
Introduction
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
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 coassociated into fibrils (van-der Rest and Mayne, 1987; Mendler 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
Key words: collagen types IX and X, chick tibiotarsus, mRNA, protein.
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T. F. Linsenmayer and others
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 'fibrilassociated' 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. Materials and methods
In situ hybridization In situ hybridizations were performed essentially according to Hayashiera/. (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, pH7.4 (PBS). The tissue was washed
3x5 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/zm sections were cut and mounted on subbed slides [prepared by incubating clean microscope slides overnight at 68° in a solution of lxDenhardt's mixture (Sigma Chemical Co., St. Louis, MO) in 3x standard saline citrate buffer (SSC) (lxSSC consists of 0.15 M NaCl and 0.015 M trisodium citrate, pH7.0) 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 2x15 min in xylene and 2x5 min in 100% ethanol, air dried, and fixed in 4% paraformaldehyde in PBS for 10min. They were then washed 3x5min in PBS, 2x5 min in 70% ethanol, and 5 min in 95% ethanol, and allowed to air dry.
cDNA probes 32
P-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 lxK^ctsmin"' jug"'. The ol(II) collagen probe was a 1200 bp Pstl/PvuW fragment of pYN1124 (Ninomiya et al. 1984). This fragment encodes the C-propeptide region of the 0-1(11) collagen chain, as well as 620bp of the 3' untranslated sequence. The o-l(IX) collagen probe was the 1020bp Pvull fragment of pYN1738 (Ninomiya and Olsen, 1984), which represents only 3' untranslated sequence. The
