Journal of Histochemistry and Cytochemistry, Vol. 48, 725-742, June 2000, Copyright © 2000, The Histochemical Society, Inc.


ARTICLE

Developmentally Regulated Alternative Splicing of the {alpha}1(XI) Collagen Chain: Spatial and Temporal Segregation of Isoforms in the Cartilage of Fetal Rat Long Bones

Nicholas P. Morrisa,b, Julia T. Oxfordc, Gillian B.M. Daviesa, Barbara F. Smoodya, and Douglas R. Keenea
a Research Department, Shriners Hospital for Children
b Department of Biochemistry and Molecular Biology, School of Medicine
c Department of Biochemistry, School of Dentistry, Oregon Health Sciences University, Portland, Oregon

Correspondence to: Nicholas P. Morris, Research Dept., Shriners Hospital for Children, 3101 SW Sam Jackson Park Road, Portland, OR 97201. E-mail: npm@shcc.org


  Summary
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Summary
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Materials and Methods
Results
Discussion
Literature Cited

Type XI collagen is a component of the heterotypic collagen fibrils of fetal cartilage and is required to maintain the unusually thin diameter of these fibrils. The mature matrix form of the molecule retains an N-terminal variable region whose structure is modulated by alternative exon splicing that is tissue-specific and developmentally regulated. In the {alpha}1(XI) chain, antibodies to two of the peptides, p6b and p8, encoded by the alternatively spliced exons localized these epitopes to the surface of the collagen fibrils and were used to determine the pattern of isoform expression during the development of rat long bones (humerus). Expression of the p6b isoform was restricted to the periphery of the cartilage underlying the perichondrium of the diaphysis, a pattern that appears de novo at embryonic Day (E) 14. P8 isoforms appeared to be associated with early stages of chondrocyte differentiation and were detected throughout prechondrogenic mesenchyme and immature cartilage. After E16, p8 isoforms gradually disappeared from the diaphysis and then from the epiphysis preceding chondrocyte hypertrophy, but were highly evident at the periarticular joint surface, where ongoing chondrogenesis accompanies the formation of articular cartilage. The spatially restricted and differentiation-specific distribution of {alpha}1(XI) isoforms is evidence that Type XI collagen participates in skeletal development via a mechanism that may be distinct from regulation of fibrillogenesis.

(J Histochem Cytochem 48:725–741, 2000)

Key Words: collagen, cartilage, skeletal development, bone, alternative splicing, type XI, localization, isoforms


  Introduction
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Introduction
Materials and Methods
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Literature Cited

Type xi collagen is a constitutive element of the interstitial collagen fibrils of fetal cartilage (Mendler et al. 1989 ; Eikenberry et al. 1992 ). By resisting the swelling pressure of proteoglycans, these collagen fibrils contribute directly to the biomechanical properties of cartilage. Although present in relatively small amounts, Type XI collagen is necessary to attain a network of thin (<25 nm) collagen fibrils typical of fetal cartilage in the endochondral skeleton. In the absence of Type XI collagen, very large fibrils are formed in the developing cartilage, coincident with the loss of structural integrity, resulting in severe skeletal dysplasia (Li et al. 1995 ).

Type XI collagen is a member of the family of fibrillar collagen genes (Ramirez et al. 1990 ). Although most abundant in cartilage, its expression is not limited to this tissue (Lui et al. 1995a ; Yoshioka et al. 1995 ). It is a heterotrimer composed of three chains, {alpha}1, {alpha}2, and {alpha}3 (Eyre and Wu 1987 ). The MA613 chain is a product of the COL2A1 gene (Furuto and Miller 1983 ; Oxford et al. 1994 ), which produces Type II collagen, the predominant fibrillar collagen in cartilage. Both the {alpha}1 and {alpha}2 chains are distinct gene products and contain large complex amino terminal domains (see Fig 1) (Yoshioka and Ramirez 1990 ; Zhidkova et al. 1993 ). The amino terminal domain of the {alpha}2 chain begins with a disulfide-bonded amino propeptide (Npp) of about 250 amino acids, originally isolated as an independent cartilage protein called PARP (proline–arginine rich protein) (Neame et al. 1990 ). The {alpha}1 chain contains an amino propeptide homologous to the {alpha}2-Npp, followed by a variable region (Zhidkova et al. 1993 ), and finally by the minor triple helix characteristic of all of the fibrillar collagens.



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Figure 1. Structure of the amino-terminal domain of Type XI and isoforms of the {alpha}1(XI) chain arising by alternative splicing. (A) Procollagen XI contains a 300-nm uninterrupted triple-helical domain (TH) flanked by short, non-triple-helical telopeptides (n-tp and c-tp) characteristic of the fibrillar collagens. A carboxyl propeptide (Cpp), contributed to by all three chains, is rapidly removed after secretion. At the N-terminus is a complex amino terminal domain (NTD) containing amino propeptides (Npp), variable regions (VR), and the minor triple helix (mh). The Npp of the {alpha}1 chain is proteolytically processed (vertical arrow) very slowly so that the intact {alpha}1-NTD is incorporated into the fibril with Type XI collagen. The variable region is composed of four peptides, p6a (39aa), p6b (51aa), p7 (31aa), and p8 (85aa) encoded by consecutive exons 6A, 6B, 7, and 8, respectively. Alternative splicing of exons encoding p6a, p6b, and p8 leads to six protein isoforms: p6a+p8, p6b+p8, p8, p6b, p6a, and p0 (lacking all three peptides). The intervening peptide, p7, is constitutively expressed. The {alpha}2-Npp is rapidly processed after secretion. The {alpha}2(XI) NTD also contains a variable region, but none of the alternatively spliced exons is expressed after chondrocyte differentiation. After chondrocyte differentiation, {alpha}3(XI) NTD lacks a region distal to the minor helix and is not proteolytically processed. Antibodies recognizing the p6b and p8 peptides were utilized in this study. Isoform-independent antibodies used as controls included one recognizing the carboxyl telopeptide (c-tp) and a newly prepared antiserum to the p7 peptide. (B) The specificity of the p7 antibody was examined by immunoblotting of an NaCl extract of fetal rat chondrocytes in pellet culture. Lane 1, Coomassie-stained sample; Lane 2, sample blotted with p7 antibody; Lane 3, bacterial collagenase-digested sample blotted with p7 antibody (polyclonal); Lane 4, sample blotted with c-tp antiserum. The p7 antiserum recognizes the same bands as the c-tp antiserum, and these bands are collagenase-sensitive.

The variable regions of the {alpha}1 and {alpha}2 chains are not homologous. In both cases, considerable sequence and presumably structural complexity results from alternative splicing of three exons encoding peptides in this region (Oxford et al. 1995 ; Tsumaki and Kimura 1995 ; Zhidkova et al. 1995 ). During cartilage formation and chondrocyte differentiation, complex splicing of the {alpha}2 chain rapidly converges to the predominance of a single form lacking all three alternatively spliced exons (Tsumaki and Kimura 1995 ; Sugimoto et al. 1998 ) and a highly truncated variable region. In the {alpha}1 chain, the variable region is encoded by consecutive exons 6A, 6B, 7, and 8, formerly designated I, II, III, and IV (Zhidkova et al. 1995 ) or v1a, v1b, c2, and v2 (Oxford et al. 1995 ). Alternative splicing of exon 6A (peptide p6a), exon 6B (peptide p6b), and exon 8 (peptide p8) generates six possible protein isoforms, p6a+p8, p6b+p8, p8, p6a, p6b, and p0 (containing p7, but lacking p6a, p6b, and p8) (Fig 1A). Mesenchymal cells express the p6a+p8 isoform typical of noncartilaginous tissues. Differentiation of mesenchymal cells to chondrocytes in micromass culture leads to the expression of five of the isoforms (the p6a isoform is in very low amounts), and p0 and p6b are the most abundant. These isoform-specific peptides can be detected immunochemically both in cell culture and in fetal cartilage tissue (Davies et al. 1998 ). P6a and p8 are acidic peptides (pI 3.4), whereas p6b is a very basic peptide (pI 11.9) containing clusters of three, four, and five basic residues, mostly lysine (Oxford et al. 1995 ; Zhidkova et al. 1995 ).

It is likely that the amino terminal domain, or parts of it, are responsible for the effect of Type XI collagen on limiting fibril diameter (Eikenberry et al. 1992 ), presumably by steric hindrance of the close packing of Type II collagen molecules in the fibril. The p6a, p6b, and p8 peptides encoded by the alternatively spliced exons may contribute to this function, directly or indirectly, or they may contribute additional functionality to the collagen molecule. The removal of the {alpha}2-Npp is relatively rapid (t1/2 1 hr), but less than 50% of the {alpha}21-Npp is processed after 20 hr of organ culture of embryonic chick sterna (Thom and Morris 1991 ). This is extraordinarily slow relative to the rate of matrix production, collagen fibril formation, and tissue growth. Proteolytic processing occurs between the amino propeptide region and the variable region (Zhidkova et al. 1993 ; Rousseau et al. 1996 ), such that all of the variable region and minor helix are retained in the mature matrix form of the molecule. The retained variable regions are incorporated into the fibril and the isoforms show distinct patterns of expression in cartilage. For example, the p6a isoform is found predominantly in the proximal rib, the portion that becomes bone, and is scarce or is absent from the distal portion which will become permanent cartilage, even though Type XI collagen is generally distributed in the fetal rat rib cartilage. This suggests a linkage between the expression of the p6b isoform and the complex process of endochondral ossification (Davies et al. 1998 ). The p6a+p8 and p8 isoforms are prevalent in prechondrogenic mesenchyme and immature cartilage and may be involved in the process of chondrocyte differentiation (Davies et al. 1998 ). To further examine the relationship between isoform expression and skeletal development, we have investigated the pattern of expression of the p6b- and p8-containing isoforms during the development of fetal rat long bones, primarily the humerus. As in the rib, p6b showed a restricted distribution in the cartilage of the developing humerus. Before primary ossification, p6b was detected only in the diaphysis, primarily adjacent to the perichondrium, and not in the epiphysis. This pattern arose de novo after differentiation of the cartilaginous rudiment and concomitant with the formation of an organized perichondrium as reflected by the staining pattern of Type XII collagen. The p8-containing isoforms were most strongly expressed in areas of newly forming cartilage and disappeared as chondrocyte maturation proceeded. The distinct distribution of isoforms of the {alpha}1(XI) chain in the cartilage of developing limbs suggests the potential for unique functional capability beyond the regulation of collagen fibril formation.


  Materials and Methods
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Materials and Methods
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Antibodies
Antibodies recognizing various epitopes on the collagen {alpha}1(XI) chain as well as collagens X and XII have been previously described. These include monoclonal and rabbit polyclonal antibodies raised against a 20-amino-acid sequence within the p8 peptide, a monoclonal antibody raised against the intact p6b peptide (Davies et al. 1998 ), rabbit polyclonal antibodies raised against the {alpha}1(XI) carboxyl telopeptide (c-tp) (Li et al. 1995 ; Davies et al. 1998 ), a rabbit polyclonal antibody raised against Type X collagen (Davies et al. 1998 ), and a rabbit polyclonal antiserum raised against Type XII collagen (Keene et al. 1991 ). A monoclonal antibody to Type II collagen was purchased from NeoMarkers (Union City, CA). Secondary antibodies linked to FITC or CY3 were obtained from Sigma (St Louis, MO). A rabbit polyclonal antiserum recognizing the p7 peptide encoded by exon 7 was prepared as previously described (Oxford et al. 1994 ). The full-length p7 peptide, ANIVDDFQDYNYGTMETYQTESPRRVSGSNE(C), was prepared by peptide synthesis in the analytical core facility of Shriners Hospital (Portland, OR). The carboxy terminal cysteine residue was included for coupling to carrier and was not part of the original sequence. Analysis of antibody specificity using immunoblotting of an SDS-PAGE gel was performed as previously described (Davies et al. 1998 ).

Animals
Timed pregnant rats and pups were purchased from Simonsen (Gilroy, CA). Animals were sacrificed according to the guidelines of the Institutional Review Board of the Oregon Health Sciences University.

Immunohistochemistry
Frozen sections were prepared and analyzed as previously described (Sakai et al. 1986 ). Acetone-fixed, paraffin-embedded sections (AmeX procedure) were prepared according to the protocol of Sato et al. 1986 . Briefly, freshly obtained tissues were rinsed in PBS and placed in acetone at room temperature (RT). After at least 4 hr, the samples were moved to -20C overnight. Samples were then placed successively into fresh solutions of acetone, 4C, 15 min; acetone at RT 15 min; and methyl benzoate, RT, 30 min. Tissue samples were then placed in cassettes and into an automated tissue processor (Citadel 1000; Shandon, Cheshire, UK) for cycling through two changes of xylenes, 30 min each, and two changes of melted paraffin, 1 hr each, the last under vacuum. Six-µm sections were cut on a Reichert–Jung model 820 II microtome (Nussloch, Germany). Sections were deparaffinized in xylene and rehydrated through graded ethanol series before immunohistochemistry.

Unmasking
Sections (frozen or paraffin) were routinely treated with chondroitinase ABC (Sigma), 0.25 ul/ml in PBS for 30 min at RT to remove proteoglycans. As a control, additional unmasking was attempted utilizing 6 M guanidine-HCl buffered in 0.05 M Tris to pH 7.5, or 0.5 M acetic acid, or digestion with the protease ficin (Zymed; San Francisco, CA).

Immunofluorescence Micrographs
Some immunofluorescence images were obtained with a Zeiss Axiophot fluorescence microscope as color slides, which were then digitized. Other images were obtained using a Nikon E800 microscope equipped with a Sensys digital camera (Photometrics; Tucson, AZ) and utilizing Metamorph software (Universal Imaging; West Chester, PA).

Histology
Paraffin sections were rehydrated as above and stained with silver nitrate (von Kossa method) to visualize calcified areas of the tissue and were counterstained with safranin-O to visualize proteoglycans (Clark 1980 ).

Ultrastructural Localization
Localization of p6b and p8 epitopes by immunoelectron microscopy was performed as previously described (Keene et al. 1995 ). A solution of 6-nm colloidal gold was obtained from Aurion (Wageningen, The Netherlands) and was coupled to the p6b and p8 MAb according to the manufacturer's instructions.


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Materials and Methods
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Isoform-specific Antibodies
Characterization of the expression of the {alpha}1(XI) protein isoforms during the cartilaginous phase of long bone development required the use of isoform-specific antibodies. It was not possible to generate antibodies specific to each of the six isoforms because the three peptides modulated by alternative splicing occur in more than one isoform (Fig 1a). The monoclonal antibody specific to the p6b peptide recognizes both the p6b+p8 and the p6b isoform. It is likely that p6b antibody detects primarily the p6b isoform, because the patterns obtained with the p6b and p8 antibody did not significantly overlap at later times of development (see below) and the p6b+p8 isoform is expressed at low levels (Oxford et al. 1995 ; Davies et al. 1998 ). The p8 monoclonal and polyclonal antibodies recognize both p6a+p8 and p8 isoforms, which predominate very early in chondrocyte differentiation (Davies et al. 1998 ). An antibody specific for p6a was not obtained, but the p6a isoform is minimally expressed in cartilage (Davies et al. 1998 ). It is not possible to make an antibody specific to the p0 isoform. The absence of peptides at two different sites in p0 precludes the use of a junctional peptide. However, this is the most abundant isoform in cartilage (Rousseau et al. 1996 ; Davies et al. 1998 ) and is likely to be well represented by the signal obtained with the splicing-independent antibody recognizing the carboxyl telopeptide (c-tp) of the {alpha}1(XI) chain. As an additional control for the isoform-independent distribution of {alpha}1(IX) chains, a polyclonal antibody specific for the p7 peptide in the variable region was prepared (see Materials and Methods). The p7 peptide, encoded by exon 7, is constitutively expressed and is therefore present in all six isoforms, and is topologically adjacent to both the p6b and p8 peptides. The p7 antiserum specifically recognized {alpha}1(XI) chains in an extract of cartilage (Fig 1B), yielding a pattern of immunoblotting identical to that of the previously characterized c-tp antibody. Bands relating to {alpha}1(II) or {alpha}2(XI) were not recognized, and all blotted bands were collagenase-sensitive.

Localization of the {alpha}1(XI) Collagen Chain and Its Isoforms in Fetal Cartilage
E17 and E18 fetal rat humerus were initially examined for the distribution of {alpha}1(XI ) isoforms. At this stage (Fig 2), the central diaphysis had been invaded by vascular tissue and endochondral ossification was under way. The cartilage (stained with safranin-O) adjacent to the ossification front was hypertrophic and mineralized (stained black with von Kossa stain), while the mineralized bony collar in the periosteum surrounded much of the remaining diaphyseal cartilage (Fig 2C). Well-differentiated perichondrium/periosteum covered the diaphysis. At the epiphysis, the joint cavity was formed and the cartilage appeared relatively uniform. The articular surfaces were covered by immature chondrocytes and mesenchymal cells in a layer defining the developing articular cartilage (Fig 2B).



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Figure 2. Histology of E18 fetal rat elbow. (A) Section was stained with safranin-O (orange-pink) for proteoglycans and von Kossa for mineralized tissue (black). (B) Enlargement of the larger boxed area containing the developing articular cartilage and joint space. (C) Enlargement of the smaller boxed area containing the lateral region of the diaphysis in the region of the hypertrophic cartilage. as, articular surface; bc, bony collar: D, diaphysis; E, epiphysis; H, humerus; hc, hypertrophic cartilage; js, joint space; pc, perichondrium; poc, primary ossification center; U, ulna. Original magnifications x 100.

In the distal humerus of E17/E18 rat fetuses, antibodies to Type II collagen defined the cartilaginous zones (Fig 3A). The general distribution of the {alpha}1(XI) chain, as determined by the c-tp antibody, was found to overlap that of Type II, except that staining was less intense at the distal third of the epiphysis and was somewhat less in the diaphysis (Fig 3B). The general distribution of the {alpha}1(XI) chain was also examined by labeling with the antibody to the p7 peptide (Fig 3C). In this case, the diminished staining of the distal epiphysis was less pronounced, being primarily restricted to the margin adjacent to the joint space. This suggests that the c-tp epitope of the {alpha}1(XI) chain is not uniformly available to the antibody. The p8 antibody preferentially labeled the epiphysis (Fig 3D–3F). The staining was intense out to the extreme edge of the joint surface in both the humerus and the ulna, as observed with Type II collagen. Towards the diaphysis, overall staining became weaker and discontinuous; strong, even staining was limited to the lateral margin adjacent to the perichondrium. Staining for p8 was very weak in the hypertrophic zone (compare to Fig 3G). Double labeling with c-tp and p8 antibodies (Fig 3E and Fig 3F) showed that these epitopes co-distributed in the proximal epiphysis and the adjacent diaphysis. However, p8 predominated at the most distal epiphysis and joint surface, whereas c-tp staining predominated in the diaphysis towards the middle of the bone. Higher magnification of this double stain (Fig 3F) showed that p8 surrounded small clusters of cells, whereas c-tp labeled individual cells. This change in p8 staining from the epiphysis to the diaphysis is shown in more detail in Fig 4, comparing in this case p8 and p7 antibodies. Staining with p8 antibodies was general in the epiphysis (Fig 4B), although a few septa between cells labeled much more intensely with p7 than with p8 antibodies (Fig 4C). In the diaphysis, there was incomplete and discontinuous staining with the p8 antibody (Fig 4E and Fig 4F). This suggests that more recently synthesized Type XI collagen did not include p8 isoforms of {alpha}1(XI) and that this new matrix and subsequent cell division displaced the existing p8 isoform-containing matrix.



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Figure 3. Distribution of p6b- and p8-containing isoforms of the {alpha}1(XI) collagen chain in the distal humerus of E18 fetal rat. (A) Stained with Type II collagen antibody; edges of ulna and radius are visible. (B) Stained with c-tp polyclonal antibody (general {alpha}1(XI)); note weaker staining in the distal epiphysis. (C) Stained with p7 antibody. (D) Stained with monoclonal p8 antibody; note diminished and non-uniform staining of the diaphysis. (E) Stained with polyclonal c-tp (red) and monoclonal p8 (green); note lack of overlap in the distal epiphysis and proximal diaphysis. (F) High magnification of E18 epiphysis stained with c-tp (red) and p8 (green). P8 surrounds groups of cells; c-tp surrounds individual cells. (G) Staining with p6b antibody (green) and collagen Type X antibody (red); compare with A–D. (H) Cross-section of humerus through the distal diaphysis, stained with p7 (red) and p6b (green) antibodies. (I) Ulna (lower left) radius (lower right), and humerus stained with p6b antibody. (J) Staining with polyclonal c-tp (red) and monoclonal p8(green) antibodies after treatment of the section with 6 M guanidine. (K) Staining with p6b antibody after treatment of the section with 6 M guanidine. (L) Frozen section stained with polyclonal p8 antibody. (M) Frozen section stained with p6b antibody. (N) PBS instead of primary antibody with anti-mouse IgG–FITC secondary antibody. (O) Same as N, with anti-rabbit IgG–Cy3 secondary antibody. All sections are from E18 embryos except for H, which is from E17. Original magnifications: A–E, G–O x 100; F x 400.



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Figure 4. Gradient of p8 isoform distribution from the epiphysis to the diaphysis. E18 humerus labeled with antibody to p8 (green) and isoform-independent antibody to peptide p7 (red). (A–C) Epiphyseal region, including developing articular surface. The p8 and p7 epitopes generally co-distribute in the epiphysis. Occasionally the matrix between adjacent cells does not stain for p8, suggesting that this isoform was no longer secreted. (D–F) Diaphyseal region distal to the hypertrophic zone, similar to Fig 3F. Secretion of p8-negative isoforms of {alpha}1(XI) after cell division leads to the open lace-like appearance of p8 staining in the diaphysis. Original magnification x 400.

More extreme asymmetric labeling of cartilage in the humerus was observed with antibodies to p6b isoforms (Fig 3G–3I). Staining for p6b was most intense at the lateral boundary of the diaphyseal cartilage and became weaker and difficult to detect in the central region farthest away from the perichondrium. Staining was completely absent from the epiphysis. Staining for p6b was not restricted to hypertrophic cartilage, identified by staining with antibodies to Type X collagen, but did overlap slightly at the margin (Fig 3G). Examination of cross-sections of the diaphysis (Fig 3H) confirmed the peripheral staining pattern for p6b isoforms (green) in contrast to the uniform staining observed with the p7 antibody (red). This asymmetric distribution of p6b labeling was also apparent in the ulna and radius (Fig 3I).

The pattern of labeling of p8- and p6b-containing isoforms of the {alpha}1(XI) chain at this stage of development, as well as the nonuniform labeling of {alpha}1(XI) with the c-tp antibody, was not altered by pretreatment of sections with 6 M guanidine (Fig 2J and Fig 2K) or acetic acid, mild pepsin digestion, or proteolysis with ficin (not shown). Utilization of frozen sections (Fig 2K and Fig 2M) also did not fundamentally alter the staining pattern with antibodies to p6b or p8. Repeated experiments showed that the broader p6b staining reaching the central diaphysis in Fig 2K and Fig 2M probably resulted from the location of the plane of section closer to the perichondrial surface of the humerus. Other contributing factors, such as section thickness (15 µm for frozen, 6 µm for AmeX), better preservation of epitopes in frozen sections, or unmasking of some epitopes by treatment with guanidine may also be involved.

The localizations of p8 and p6b isoforms were refined by comparison to the distribution of Type XII collagen (Fig 5), used here as a marker for noncartilaginous connective tissues containing primarily Type I collagen. Antibodies to collagen XII labeled the perichondrium. The epiphyseal cartilage underlying the joint surface was also labeled. The p8 staining overlapped with Type XII collagen staining in this distal epiphyseal region, but the matrix corresponding to the layers of cells at the extreme articular surface stained only with collagen XII antibodies (Fig 5A). The p6b and collagen XII antibodies recognized adjacent but almost mutually exclusive domains in the diaphysis (Fig 5B and Fig 5C). Collagen XII antibodies stained the perichondrium, whereas p6b antibodies labeled the underlying cartilage. Nearer to the epiphysis, there appeared to be a very narrow region of overlap at the cartilage–perichondrial interface, but closer to the hypertrophic zone there was virtually no overlap in staining (Fig 5C). This indicated a rather abrupt transition between perichondrium and cartilage in the developing cartilaginous template.



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Figure 5. Distribution of p6b and p8 isoforms of {alpha}1(XI) relative to collagen XII in E17/E18 fetal rat distal humerus. (A) E18, p8 (green) and collagen XII (red). The p8-containing isoforms co-distribute with Type XII collagen in the periarticular chondrogenic region, but collagen XII is much less abundant in the interior of the epiphysis. Note that p8 staining is almost absent in the diaphysis. (B) E18, p6b (green) and collagen XII (red). (C) E17, p6b (green) and collagen XII (red). The p6b antibody stains only the cartilage and not the perichondrium, as defined by collagen XII staining. There is a very narrow region of overlap at the interface of these tissues, which is less pronounced where perichondrial/periosteal bone invasion is taking place (C). There appears to be some staining of interior hypertrophic cartilage with the collagen XII antibodies. Original magnifications: A,B x 100 and C x 200.

Ultrastructural Localization of p8 and p6b Isoforms
The site of proteolytic processing (Fig 1A) and the matrix staining obtained above with the p6b and p8 antibodies suggested that these peptides in the variable region should be part of the collagen fibrils and available at the fibril surface. This possibility was examined by immunoelectron microscopy using primary antibody-colloidal gold conjugates (Fig 6). The area of p6b labeling shown in Fig 6a was near the end of the bony collar where mineralization was discontinuous (between the hypertrophic zone and the epiphysis; see Fig 2C). The thin collagen fibrils characteristic of fetal cartilage were extensively labeled with p6b antibody. At higher magnification (Fig 6B), the labeling was periodic, with a minimum spacing of about 54 nm corresponding to a periodicity characteristic of the quarter stagger arrangement of collagen molecules within the fibril. The large Type I collagen-containing fibrils and dispersed foci of mineralization of the bony collar were not labeled with p6b antibody. Consistent with the observations by light microcopy, there was no transition or structural gradation between the extracellular matrix of the perichondrium and the underlying cartilage. The thin fibrils of cartilage were juxtaposed to the thick, banded fibrils of perichondrium. Examination of the distal epiphyses with antibodies to p8 showed that these collagen fibrils were also extensively labeled, but it was more difficult to demonstrate periodic labeling (Fig 6C). At the level of light and electron microscopy, the p6b and p8 peptides in their respective {alpha}1(XI) isoforms were accessible without need for unmasking, except for the removal of proteoglycans with chondroitinase.




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Figure 6. Ultrastructural localization of p6b and p8 peptides to the surface collagen fibrils. Five-nm colloidal gold particles directly conjugated to p6b and p8 primary antibodies were used to localize these isoforms and epitopes in the tissue. (A) Colloidal gold–p6b labeling of the cartilage–perichondrial interface in the diaphysis where the bony collar is newly forming and extending towards the epiphysis (Fig 2C). Bar = 200 nm. The forming bony collar is represented by the large electron-dense (black) crystals of hydroxyapatite scattered within the large-diameter collagen fibrils of the perichondrium, which are not labeled with p6b antibody. The thin fibrils of cartilage between the perichondrium and the chondrocyte are extensively labeled with antibody to p6b. Note the very sharp transition between the fibril architectures of the perichondrium and the cartilage. (B) Higher magnification of the p6b labeling, showing periodic distribution of gold particles along the fibril. Bar = 100 nm. Corrected for shrinkage, the interval is about 67 nm, characteristic of the staggered alignment of fibrillar collagens. (C) Labeling of collagen fibrils in the epiphysis with antibodies to p8. Bar = 100 nm.

Developmental Expression of p6b- and p8-containing Isoforms
It is possible that the asymmetric and almost mutually exclusive distributions of p6b and p8 arose at the time of cartilage formation. Alternatively, both sets of isoforms could have been uniformly expressed initially and then have become restricted as development and chondrocyte differentiation proceeded. To distinguish between these two possibilities, the distribution of p6b- and p8-containing isoforms was examined at the initial stages of cartilage formation in the rat humerus, E13.5–14.5 (Fig 7). At E13.5 (Fig 7A–7C), the condensing mesenchyme/immature cartilage of the humerus was generally labeled with the p7 antibody (Fig 7A), stronger in the middle and weaker at the ends where differentiation (or chondrogenesis) was still occurring. At this developmental stage, the tissue was highly cellular, with minimal extracellular matrix, and there was no staining for p6b. Diffuse staining for Type XII collagen around the condensation indicated a poorly differentiated perichondrium (Fig 7B). Staining for the p8 isoform was general and resembled that of the p7 antibody (Fig 7C). At E14 (Fig 7D–7F), the extended region of the anlage (diaphysis) contained more differentiated chondrocytes surrounded by matrix that labeled well with the p7 antibody (Fig 7D). The end of the anlage (epiphysis) was stained less intensely and resembled the E13.5 tissue. The perichondrium was well labeled with collagen XII antibodies and had become striated or fibrillar in appearance (Fig 7E), indicating a more advanced stage of differentiation. Staining with the p6b antibody became apparent in a highly focal zone of chondrocytes adjacent to the perichondrium, but not deeper into the cartilage. The staining pattern with the p8 antibody was similar to that of p7, although not as intense (Fig 7F). By E14.5, the region of strong labeling of the p7 epitope in the diaphysis was more extended (Fig 7G). Staining for p6b reached more distally, along with the collagen XII staining of the perichondrium, but was still limited to the region adjacent to the perichondrium (Fig 7H). The collagen XII localization appeared more fibrillar and less diffuse, indicating differentiation of the perichondrium. The staining with the p8 antibody was similar to that on E14 but, in comparison to p7, a more open network was apparent (Fig 7I). This pattern is reminiscent of the p8 staining in the later diaphysis (Fig 3F), although more subtle. The p6b staining at higher magnification is shown in Fig 7J and Fig 7K. Differential interference microscopy revealed that the cells to the left and far right were flattened perpendicularly to the long axis of the forming bone. The cells in the central region appeared rounder, with more matrix between cells, and it was around these cells, adjacent to the perichondrium, that p6b was first detected up to a depth of about five cells.



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Figure 7. Early developmental expression of Type XI collagen isoforms. Upper forelimbs from E13.5 (A–C), E14.0 (D–F,J,K), and E14.5 (G–I) embryos were sectioned, showing immunofluorescence labeling of the humerus (distal to the left) with antibodies to {alpha}1(XI). Each set represents consecutive sections. Sections in A,D,G were labeled with p7 antibody. Sections in B,E,H,K were double labeled with p6b antibody (green) and collagen XII antibody (red). Sections in C,F,I were labeled with p8 antibody. J is a DIC micrograph of the same section shown in K. P8 staining of chondrogenic tissue is uniform at the start. The restricted distribution of p6b-containing isoforms arises de novo at the interface between the cartilage and perichondrium in a group of cells that appear more mature (rounded) and prehypertrophic. Original magnifications: A–I x 200; J,K x 400.

The distribution of the p6b and p8 isoforms was examined at subsequent stages in the development of the humerus (Fig 8). The {alpha}1(XI) chain was generally distributed in the cartilage at E16, as indicated by staining with p7 antibodies (Fig 8A). This pattern was maintained through the regression of cartilage in the diaphysis due to primary endochondral ossification (Fig 8D), the establishment of a metaphyseal growth plate (postnatal Day 3; Fig 8G), and the secondary ossification center in the epiphysis (postnatal Day 7; (Fig 8J). Labeling of the cartilage with the p8 antibody was widespread at E16 (Fig 8B) but rapidly diminished in the diaphysis by E18–E20 (Fig 8E; see also Fig 3D and Fig 4F). Staining of the epiphysis persisted through postnatal Day 7 (Fig 8K). However, by E20 (Fig 8E) the staining was stronger in the developing articular cartilage. By postnatal Day 3 (Fig 8H), the epiphyseal cartilage was only weakly stained, whereas the developing articular cartilage at the joint surface continued to stain strongly, a pattern that was also observed at postnatal Day 7 (Fig 8K). The disappearance of the p8 isoform from the diaphysis and the epiphysis preceded chondrocyte hypertrophy associated with primary and secondary endochondral ossification, as indicated by staining for Type X collagen (Fig 8C–8L).



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Figure 8. Distribution of p6b and p8 isoforms of {alpha}1(XI) during later embryonic and early postnatal development. Upper forelimbs from E16 (A–C), E20 (D–F), and Day 3 pups (G–I), Day 7 pups (J–L) were sectioned. Indirect immunofluorescent labeling utilized p7 antibody (A,D,G,J); p8 polyclonal (red) and p6b monoclonal (green) antibodies (B,E,H,K); and p6b (green) and anti-Type X collagen (red) antibodies (C,F,I,L). A and C, at x 100 magnification, show the distal two thirds of the humerus. D–I, at x 40 magnification, show the distal humerus and proximal ulna and, in some cases (G–I), a grazing edge of the radius. J–L, also at x 40 magnification show the distal humerus and proximal radius, with just a small fragment of the ulna on the left. After E16 there is little overlap between p6b- and p8-containing isoforms. P6b staining maintains its association with the perichondrium, whereas p8 staining disappears from the diaphysis and epiphysis ahead of chondrocyte hypertrophy (after E16) but remains strongly associated with periarticular chondrogenesis.

In contrast, from E16 to postnatal Day 7, the p6b isoform was detected only at the periphery of the diaphyseal cartilage along its entire length, overlapping with Type X staining as the cartilage became hypertrophic, and disappearing only when cartilage became bone (Fig 8C, Fig 8F, Fig 8I, and Fig 8L). Staining for p6b was not detected in association with the secondary ossification center in the epiphysis at postnatal Day 7 (Fig 8L), although it was detectable at the periphery of the well-defined metaphyseal growth plate. Weak, diffuse, and variable staining with the p6b antibody was observed at times in the epiphysis after birth (near the articular surface in Fig 8J and Fig 8L); however, this was an inconsistent finding. By E20, the patterns of immunohistochemical staining for p6b- and p8-containing isoforms were largely segregated (Fig 8H) and remained so (Fig 8H and Fig 8K). This overall pattern of isoform localization during development was also observed in the radius and the ulna (Fig 8E, Fig 8H, and Fig 8K; and data not shown).

Distribution of p6b and p8 Isoforms in Other Skeletal Systems
To determine if the differential staining pattern of {alpha}1(XI) isoforms was a general phenomenon, other skeletal elements and cartilage systems were analyzed. Examination of the forepaw of E18 animals revealed a pattern of p6b and p8 staining in the metacarpal bones that was very similar to that of the long bones described above (Fig 9A–9C). The development of metacarpals is similar to that of long bones, except that the distal ends ossify directly from the diaphysis rather than via a secondary ossification center. As before, p6b staining was restricted to the diaphysis, although it was not confined to the periphery. This may be due to the small size of these bones. P8 staining was most pronounced at the ends of the bones, although there was still some staining in the diaphysis. The carpal bones, which ossify much later than the other bones in the hand, stained uniformly with p8 antibody, but p6b staining was not detected, either in the frontal plane shown, which emphasizes the articulating surfaces, or in sagittal section, which revealed the perichondrium at the dorsal and ventral surfaces (not shown). A section through the spine at E18 also revealed differential staining for the p6b isoform (Fig 10B). Compared to the staining with the isoform-independent p7 antibody (Fig 10A), p6b localization was absent from the ends of the neural arch and from the center of the vertebral body. Staining for p6b was observed at the periphery of the vertebral body and throughout the narrow shaft of the neural arch adjacent to the perichondrium (Fig 10C). In this section, the p8 epitope was labeled throughout the vertebral body (Fig 10B) but was stained more strongly at the ends of the neural arch, similar to long bones and digits.



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Figure 9. Localization of p6b and p8 isoforms of {alpha}1(XI) in fetal rat paw. Sections were taken through the metacarpals and digits (A–C) and through the carpal bones (D–F) of an E17 fetal rat, in the plane of the paw. Sections were labeled with anti-p8 antibody (A,D), anti-p6b (green) and anti-p8 (red) (B,E), and anti-p6b (green) and anti-collagen XII (red) (C,F). Inset in C is one of the longer metacarpals stained with anti-collagen X antibodies, showing that the region lacking p6b staining in the middle of the metacarpal bone represents hypertrophic chondrocytes. The staining pattern of metacarpals and digits resembled that of long bones, with p6b only in the diaphysis and p8 staining more weakly in the diaphysis and strongly at the ends of the bone. P6b staining was uniform across the diaphysis. P8 strongly labels the carpal bones, and no p6b staining was detected regardless of plane of section.



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Figure 10. Differential localization of p6b and p8 isoforms of {alpha}1(XI) in the vertebrae. Transverse sections were taken through the spine of an E17 rat fetus and immunohistochemically labeled as in Fig 9. (A) Antibodies to p7. All of the cartilage in the vertebral body (bottom) and the neural arch (above) are labeled. (B) Antibodies to p6b (green) and p8 (red). The vertebral body is well stained with antibodies to p8, whereas p6b is localized at the rim. In the neural arch, p6b is at the edges and somewhat in the interior everywhere but at the distal end, whereas p8 is more evident proximally and distally. (C) Antibodies to p6b (green) and collagen XII (red). The p6b staining generally coincides with collagen XII staining in the adjacent perichondrial tissue.


  Discussion
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

During chondrogenesis and endochondral ossification, the extracellular matrix of cartilage serves diverse functions and is produced by chondrocytes according to their state of phenotypic differentiation: chondroblast or immature chondrocyte, mature or prehypertrophic chondrocyte, and hypertrophic chondrocyte. It is not surprising, therefore, that regional heterogeneity of extracellular components would also arise from such differences in support of specific functions or the process of differentiation. The developing articular cartilage matrix produced by mesenchymal cells and immature chondrocytes is enriched in fibromodulin (Archer et al. 1996 ), tenascin-C (Koyama et al. 1995 ; Pacifici 1995 ), and collagen XII and XIV (Watt et al. 1992 ) (see Fig 5A). Conversely, Type X collagen, as well as bone proteins such as bone sialoprotein and osteopontin, is produced by hypertrophic chondrocytes in the diaphysis and later in the secondary ossification center of the epiphysis (Schmid and Linsenmayer 1985 ; Bianco et al. 1991 , Bianco et al. 1993 , Bianco et al. 1998 ; Galotto et al. 1994 ; Gerstenfeld and Shapiro 1996 ). However, within the bulk fetal cartilage, the differential distribution of isoforms of the {alpha}1(XI) collagen chain, as presented here, represents a striking example of regional differences of one protein that is linked both to sequential stages of chondrocyte differentiation and to topological location.

As depicted schematically in Fig 1, structural studies indicate that the variable region is a distinct domain with a rather extended conformation for all isoforms (unpublished observations). The highly charged composition of the p6a, p6b, and p8 peptides would alter the chemical nature of the variable region according to the isoform and could modulate potential biological activity as well. Lying adjacent to the proteolytic processing site (Fig 1), the peptides could influence the rate of removal of the large N-propeptide and hence fibril morphology. However, despite the differential localization of the p8- and p6b-containing isoforms shown here, no significant difference in fibril diameter was noted in the different regions of developing cartilage (compare Fig 6B and Fig 6C; and unpublished observations). Ultrastructural analysis confirmed that the variable region was an integral part of the collagen fibril because p6b and p8 epitopes were available and were localized at the fibril surface. The functional significance of the isoforms may therefore reside in an ability to mediate interactions between the fibrils and other components of the extracellular matrix.

p8-containing Isoforms Are Associated with Chondrogenesis
The overall pattern of p8 staining reflects the developmental age and stage of the tissue, oldest in the diaphysis and youngest at the margins of the joint. The p8-containing isoforms were found throughout the early differentiating cartilage starting at E13.5, and by 7 days postnatally they were restricted to the area of chondrogenesis at the articular surface, where ongoing differentiation of prechondrogenic mesenchyme supports the formation of articular cartilage (Bland and Ashhurst 1996 ). In between, p8 staining was observed in a distal to proximal gradient, as p8 disappeared first from the diaphysis and then from the central epiphysis. Removal of p8-containing isoforms from the matrix is not necessarily a prerequisite for chondrocyte hypertrophy because the initial expression of Type X collagen, a well-defined marker for hypertrophic chondrocytes (Schmid and Linsenmayer 1985 ), was detected at E16 in a cartilage matrix that stains strongly for p8. However, the disappearance of p8 staining from the matrix ahead of chondrocyte hypertrophy, both in the advancing front of primary ossification in the diaphysis and in the secondary ossification at postnatal Day 7, suggests a possible relationship.

Detailed examination of the developmental changes in p8 distribution suggested that such a pattern was unlikely to be sustained at the transcriptional level, although this was not examined directly. It is more likely that p8 isoforms predominantly expressed by early immature chondrocytes persist in the matrix. The localization pattern would therefore derive from the timing and location of turnover or modification of epitope than from synthesis. Turnover could involve degradation of the fibril or proteolytic removal of the variable region from the surface of the fibril. Cessation of p8-containing isoform synthesis with the continued synthesis of other isoforms, predominantly p0, would lead to loss of p8 staining near cells. Synthesis of p0 and cell division displace the peripheral p8-containing matrix, which becomes progressively more difficult to detect, presumably due to turnover. This interpretation is supported by the observation that in the later stages of long bone development, p8 isoforms persist at the articular surface, the only area of active chondrogenesis. This interpretation is also consistent with the observation that in micromass culture, p8 isoforms predominate in prechondrogenic mesenchyme and become a minor component in fully differentiated chondrocytes (Davies et al. 1998 ). Finally, the distribution of p8 isoforms is analogous to the distribution of the IIA splice form of collagen Type II produced by prechondrogenic mesenchyme and chondroblasts (Sandell et al. 1991 , Sandell et al. 1994 ; Ng et al. 1993 ; Lui et al. 1995b ). Whereas expression of the IIA splice form ceases on differentiation, the protein isoform persists in the tissue in a pattern similar to the staining for p8 isoforms of {alpha}1(XI) (Oganesian et al. 1997 ; Zhu et al. 1999 ).

The function of the p8 isoform in the cartilage of the developing bone is unknown. However, the association of the p8 isoform with areas of chondrogenesis suggests that it may provide an extracellular matrix conducive to chondrocyte differentiation. In this regard, it has recently been shown that the cystine-rich peptide in the amino terminal domain of collagen Type IIA has homology to noggin and can bind to BMP-2, suggesting that modulating BMP/GDF activity could be a function of Type II collagen distinct from its structural role in cartilage (Zhu et al. 1999 ). There is no homology between the p8 sequence and known signaling elements in skeletal development. Nevertheless, there remains the potential for interaction between p8, as part of the insoluble extracellular matrix, and the soluble growth and differentiation factors that profoundly influence chondrogenesis, chondrocyte proliferation, and hypertrophy, and the development of the epiphysis and joint (Macias et al. 1997 ; St-Jacques et al. 1999 ; Storm and Kingsley 1999 ).

p6b Isoforms Are Associated with Primary Endochondral Ossification
The distribution of the p6b isoform was observed to be highly restricted in the developing long bone. Strongest immunohistochemical staining was adjacent to the perichondrium and diminished towards the interior, eventually becoming undetectable by this method. This pattern is typical of the humerus, radius, and ulna, as well as the femur and tibia (not shown). Ultrastructural analysis clearly showed that the p6b isoform of {alpha}1(XI) was a component of the thin collagen fibrils typical of fetal cartilaginous matrix that was indistinguishable from that in deeper unlabeled areas of cartilage. Under the forming bony collar, there was a surprisingly sharp transition to the extracellular matrix of the perichondrium, which is characterized by large, banded collagen fibrils.

Although of different geometry, the vertebral bodies also show preferential localization of p6b at the periphery in cross-section. In the smaller bones, such as the metacarpals and digits, p6b is restricted to the diaphysis but staining was not limited to the periphery similar to the pattern observed in proximal rib (Davies et al. 1998 ). This is probably due to the narrow diameter of these bones, although other intrinsic differences cannot be ruled out. These observations suggest that the pattern of p6b expression may be a general property of endochondral skeletal development.

The common feature of the p6b distribution was its association with the perichondrium. In the epiphysis, which lacks a differentiated perichondrium, chondrocytes progress through all stages of differentiation and endochondral ossification without deposition of detectable levels of p6b isoform into their matrix. The proximal end of the metacarpal is not a true epiphysis because there is no secondary ossification center. Ossification of this pseudo-epiphysis is a continuation of the primary ossification of the diaphysis. However, p6b-containing isoforms are excluded from the pseudo-epiphysis as well. In addition, no p6b was detected in the cuboidal bones of the carpus, whose surfaces are largely articulating. This was true even at the dorsal and ventral surfaces and at late stages of development after the onset of endochondral ossification (unpublished observations). The absence of p6b suggests a developmental relationship between the epiphysis of long bones and cuboidal bones that is distinct from the perichondrium-dependent development of the diaphysis (Rooney and Archer 1992 ; Long and Linsenmayer 1998 ).

The restricted distribution of the p6b isoform and its association with the perichondrium arises de novo. At stage E14 in fetal rat (equivalent to about E12.5 in the mouse), p6b was first detected in a group of rounded prehypertrophic chondrocytes at the midpoint of the diaphysis adjacent to the perichondrium, as defined by light microscopy and staining with collagen XII antibodies (Fig 7J and Fig 7K). Before this time the diaphysis contains only flattened proliferative cells oriented perpendicularly to the long axis of the bone (Fig 7A–7C). The expression of p6b also coincides with a change in the appearance of the perichondrium, which shows diffuse staining with collagen XII antibodies at E13.5 and a more striated fibrillar-like staining at E14.

In the development of the long bone, both the cartilage and the perichondrium carry out programs of coordinated differentiation, starting at midpoint of the diaphysis, where the first rounded cells appear, and spreading distally in both directions in a progressive fashion (Rooney and Archer 1992 ; Bianco et al. 1998 ). Therefore, later events, such as chondrocyte hypertrophy, formation of the bony collar between the cartilage and the perichondrium, initiation of vascular invasion, and endochondral ossification, all originate in this zone. It is tempting to speculate from the unique localization of p6b that it is involved in the complex developmental crosstalk between the signals of the perichondrium and associated chondrocytes (Chazaud et al. 1996 ; Lanske et al. 1996 ; Vortkamp et al. 1996 ; Serra et al. 1997 ; von Schroeder and Heersche 1998 ; St-Jacques et al. 1999 ). For example, indian hedgehog is produced by these early prehypertrophic cells and its receptor, patched, and downstream response gene, gli3, are produced in the perichondrium in an Ihh-dependent fashion (St-Jacques et al. 1999 ). It has recently been shown that this Ihh signaling is required for differentiation of the osteoblasts that form the bony collar in the perichondrium. The localization of the p6b peptide to the surface of the collagen fibrils indicates that these peptides would be available for interaction with signaling molecules, such as Ihh, traversing the intercellular space to the perichondrium. Because carpal bones do not express the p6b isoform, it would be interesting to know whether their development is under the control of Ihh.

Alternatively, p6b peptide could mediate interactions between the collagen fibrils and other cartilage matrix macromolecules to strengthen the diaphysis. Early fetal cartilage is highly cellular and lacks the rigidity typically associated with mature cartilage. The highly extended shape of the diaphysis would make it prone to distortion by mechanical stress due to contraction of developing muscle or pressure from position within the uterus. In concert with the mechanical constriction and support of the perichondrium, stabilizing interactions mediated by p6b peptide could enhance the stiffness of the diaphysis. Whether for a structural or a regulatory purpose, p6b and p8 peptides are localized and available at the surface of the collagen fibrils.

The implication of the differential distribution of {alpha}1(XI) isoforms in developing cartilage is that there must be a mechanism for cell-specific alternative splicing. This likely involves cis elements such as exonic splice enhancers and associated splicing machinery (Tian and Kole 1995 ; Coulter et al. 1997 ; Muro et al. 1998 ; Tacke et al. 1998 ), as well as higher-order regulation governing the expression of specific splicing genes. Such possibilities are now under investigation. Furthermore, such regulation is not likely to be limited to the splicing of Type XI collagen. However, direct evidence for specific functions of p6b or p8 splice forms may be approached by targeted exon deletion, studies of which are currently under way.


  Acknowledgments

Supported by grants from the Shriners Hospital for Children (NPM) and the Arthritis Foundation (JTO).

We wish to acknowledge Jay Gambee for the preparation of synthetic peptides, Sara Tufa for technical assistance with electron microscopy, and Dr Kate Gregory for critical reading of the manuscript.

Received for publication December 16, 1999; accepted January 26, 2000.


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Top
Summary
Introduction
Materials and Methods
Results
Discussion
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