Abnormal compartmentalization of cartilage matrix components in mice lacking collagen X: implications for function

Abstract

There are conflicting views on whether collagen X is a purely structural molecule, or regulates bone mineralization during endochondral ossification. Mutations in the human collagen alpha1 (X) gene (COL10A1) in Schmid metaphyseal chondrodysplasia (SMCD) suggest a supportive role. But mouse collagen alpha1 (X) gene (Col10a1) null mutants were previously reported to show no obvious phenotypic change. We have generated collagen X deficient mice, which shows that deficiency does have phenotypic consequences which partly resemble SMCD, such as abnormal trabecular bone architecture. In particular, the mutant mice develop coxa vara, a phenotypic change common in human SMCD. Other consequences of the mutation are reduction in thickness of growth plate resting zone and articular cartilage, altered bone content, and atypical distribution of matrix components within growth plate cartilage. We propose that collagen X plays a role in the normal distribution of matrix vesicles and proteoglycans within the growth plate matrix. Collagen X deficiency impacts on the supporting properties of the growth plate and the mineralization process, resulting in abnormal trabecular bone. This hypothesis would accommodate the previously conflicting views of the function of collagen X and of the molecular pathogenesis of SMCD.

Figure 1

Targeted disruption of the Col10a1 gene. (A) Targeting strategy and recombinant mutant allele of Col10a-1. Neomycin resistance (neo) and HSV-thymidine kinase (tk) genes (spotted boxes) with arrows indicating the direction of transcription. Restriction sites: B, BglII; E, EcoRI; H, HindIII. Solid boxes, exons of the gene; numerals above those boxes are exon numbers; crossed box, internal probe; hatched box, 3′ external probe; gray box, probe for in-situ hybridization; arrowheads A, B, and C show positions of primers used for RT-PCR analyses which revealed low levels of mutant transcripts in heterozygotes and homozygotes but no wild-type mRNAs in −/− mice. (B) Left, Southern analysis of representative ES cell clones by using 3′ external probe showing the 2.8 kb BglII fragment diagnostic of the mutant allele in targeted ES cell clones (clones 1, 2, 4, 5 ; +/−) and 6.0 kb BglII fragment being characteristic of wild-type allele (clone 3; +/+ and CCE) while the 3.2 kb BglII fragment is common for both wild-type and mutant alleles. Right, Southern analysis of tail biopsies of F2 mice from heterozygous cross by using internal probe showing the 4.9-kb HindIII fragment which is diagnostic of the mutant allele present in heterozygous (+/−) and homozygous (−/−) mutants but absent in wild-type (+/+) mice.

Figure 2

Col10a1 mutant mice lacking collagen X. (A) RT-PCR analyses revealed no wild-type Col10a1 mRNA transcript in the mutant mice (−/−) by using primers A & C (left panel); mutant Col10a1 transcript was detected from heterozygous (+/−) and homozygous (−/−) by using primers B & C (right panel) but not from wildtype (+/+). +RT, with reverse transcriptase; −RT, without reverse transcriptase. TC, targeted ES cell clone DNA; CCE, wild-type ES cells DNA for PCR only as control for size. (B and C) Dark-field views of in situ hybridization on 2-d wild-type (B) and mutant (C) mice proximal tibia growth plate using α1(X) probe (see Fig. 1). A very low level of mutant Col10a1 transcript was detected in mutant. Abbreviations: pro, proliferating chondrocytes; uhy, upper hypertrophic chondrocytes; lhy, lower hypertrophic chondrocytes. Bar, 200 μm. (D and F) Immunostaining of the growth plate cartilage of the proximal tibia of wild-type (D) and null mutant (F) show no detectable collagen X in the mutants but presence of the protein in the hypertrophic zone and cartilaginous remnants in bony trabeculae of wild type. (E and G) Phase contrast pictures of a region shown in D and F, respectively. Abbreviations: pro, proliferating zone; hy, hypertrophic zone; bm, bone matrix. Bar, 200 μm.

Figure 3

Unaltered transcriptional activity of other genes in the growth plate of collagen X mutants. In situ hybridization of proximal tibia growth plates of 129/SvJ 2-d-old wild-type (+/+, A–F) and mutant (−/−, G–L) mice showing expression of collagens α1(II) (A and G); α2(VI) (B and H); α1(VIII) (C and I); α2(VIII) (D and J); α1(IX) (E and K); and link protein (F and L). All pictures are dark-field views showing sections hybridized with corresponding matrix molecules antisense RNA probes. There is no alteration in the expression of these genes in the mutant mice compared with the wild type. Abbreviations: res, resting chondrocytes; pro, proliferating chondrocytes; hy, hypertrophic chondrocytes. Bar, 200 μm.

Figure 4

Altered trabecular morphology and organization in collagen X mutants. The proximal metaphyseal growth plate of the tibia was sectioned longitudinally and the matrix materials were stained with toluidine blue. (A and B) 2-d; (C and D) 4-wk and (E and F) 8-wk wild-type (+/+, A, C, and E) and mutant (−/−, B, D, and F) female mice of 129/SvJ background are shown. In mutants, the trabecular structure (arrowheads) appeared abnormal. Abbreviations: hy, hypertrophic chondrocytes; bm, bone marrow. Bar, 100 μm.

Figure 5

Altered distribution of proteoglycans and matrix vesicles in the growth plates of collagen X mutants. (A) Ultrastructure of the cartilage matrix of RHT-fixed samples from wild-type (+/+) and collagen X deficient (−/ −) mice. Matrix ultrastructure of resting (res, a–d), proliferating (pro, e and f), upper hypertrophic (uhy, g–j), lower hypertrophic (lhy, k and l) zones of the growth plate cartilage and trabeculae (tb, m and n) of wild-type (+/+) and collagen X deficient (−/−) 2-d-old 129/SvJ mice are shown. In the four mutants studied, vesicles (outlined arrowheads), of average 100-nm-diam and containing electron-dense inclusions, characteristic of matrix vesicles, are more abundant in the matrix of the resting and proliferating zones, but are fewer in the upper hypertrophic zone (see Table II). Granular material (filled arrowheads), with the typical appearance of proteoglycans, some of which adhering to the surface of the collagen fibrils (arrows), are found dispersed in the matrix in all zones in the wild-type. The amount of these granular materials are increased in the mutant resting and proliferative zones (d and f) but reduced in the upper hypertrophic zone (j). Collagen fibrils in the proliferating and upper hypertrophic zones of the mutant are abnormal but are normal in the resting zone. Signs of mineralization in the form of calcified spicules can be seen in the lower hypertrophic zone (k and l) and in the trabeculae at the chondrosseous junction (m and n). Distribution pattern of mineral deposits in trabeculae of mutant is patchy compared to wild type. Bar, 200 nm. (B). Ultrastructure of the cartilage matrix of tannic acid–fixed samples from wild-type (+/+) and collagen X deficient (−/−) 2-d 129/SvJ mice. The specimens were fixed in a fixative, containing tannic acid, which in contrast to RHT fixative, would leach out proteoglycans. In the cartilage of the mutant mice, the membrane vesicles (outlined arrowheads) are found in the resting (res) and proliferating (pro) zones (b and d), in addition to the upper hypertrophic zone (uhy) where the membrane vesicles are normally compartmentalized (a, c, and e). The population of membrane vesicles in the upper hypertrophic zone of the mutant cartilage is also significantly reduced (f). Removal of the proteoglycan content in the cartilage revealed more clearly the fibrillar organization. In the resting zone, the fibrils (arrows) in the mutant and wild-type specimens display similar morphology (a and b), but the fibrils in the proliferating and upper hypertrophic zones of the mutant are more contorted and shorter in appearance (d and f). The ultrastructural appearance of the matrix in the lower hypertrophic zone is similar between the wild type (g) and the mutant cartilage (h). Bar, 200 nm.

Figure 5

Altered distribution of proteoglycans and matrix vesicles in the growth plates of collagen X mutants. (A) Ultrastructure of the cartilage matrix of RHT-fixed samples from wild-type (+/+) and collagen X deficient (−/ −) mice. Matrix ultrastructure of resting (res, a–d), proliferating (pro, e and f), upper hypertrophic (uhy, g–j), lower hypertrophic (lhy, k and l) zones of the growth plate cartilage and trabeculae (tb, m and n) of wild-type (+/+) and collagen X deficient (−/−) 2-d-old 129/SvJ mice are shown. In the four mutants studied, vesicles (outlined arrowheads), of average 100-nm-diam and containing electron-dense inclusions, characteristic of matrix vesicles, are more abundant in the matrix of the resting and proliferating zones, but are fewer in the upper hypertrophic zone (see Table II). Granular material (filled arrowheads), with the typical appearance of proteoglycans, some of which adhering to the surface of the collagen fibrils (arrows), are found dispersed in the matrix in all zones in the wild-type. The amount of these granular materials are increased in the mutant resting and proliferative zones (d and f) but reduced in the upper hypertrophic zone (j). Collagen fibrils in the proliferating and upper hypertrophic zones of the mutant are abnormal but are normal in the resting zone. Signs of mineralization in the form of calcified spicules can be seen in the lower hypertrophic zone (k and l) and in the trabeculae at the chondrosseous junction (m and n). Distribution pattern of mineral deposits in trabeculae of mutant is patchy compared to wild type. Bar, 200 nm. (B). Ultrastructure of the cartilage matrix of tannic acid–fixed samples from wild-type (+/+) and collagen X deficient (−/−) 2-d 129/SvJ mice. The specimens were fixed in a fixative, containing tannic acid, which in contrast to RHT fixative, would leach out proteoglycans. In the cartilage of the mutant mice, the membrane vesicles (outlined arrowheads) are found in the resting (res) and proliferating (pro) zones (b and d), in addition to the upper hypertrophic zone (uhy) where the membrane vesicles are normally compartmentalized (a, c, and e). The population of membrane vesicles in the upper hypertrophic zone of the mutant cartilage is also significantly reduced (f). Removal of the proteoglycan content in the cartilage revealed more clearly the fibrillar organization. In the resting zone, the fibrils (arrows) in the mutant and wild-type specimens display similar morphology (a and b), but the fibrils in the proliferating and upper hypertrophic zones of the mutant are more contorted and shorter in appearance (d and f). The ultrastructural appearance of the matrix in the lower hypertrophic zone is similar between the wild type (g) and the mutant cartilage (h). Bar, 200 nm.

Figure 6

Altered bone content and incidence of coxa vara in collagen X mutants. (A–D) Microradiographs of the femurs of 2-d (A and B) and 4-wk (C and D) wild-type (+/+, A and C) and mutant (−/−, B and D) female mice of 129/SvJ background. The opacity of the bony region (white in X-ray image) correlates with the amount of radiographically opaque bone materials. The bone content was measured by densitometry at the sites indicated on the wild-type radiographs and the data are summarized in the Table III. Single-headed arrows in B and D indicate orientation proximal-distal. Abbreviations for bony landmarks: h, distal head (articular condyle); j, knee joint, m, marrow cavity; n, neck; t, tibia; s, shaft. Bar, 0.5 mm. (E–H) Radiographic study of the angle of the femur head of wild-type (+/+, E and G) and mutant (−/−, F and H) mice at 13–14 months (E and F) and 20–22 months (G and H). A decrease in the angle between the neck (fn) and the shaft (fs) of the femur (coxa vara) is observed at both ages. There is always a significant foreshortening of the length of the neck of the femur of the mutant mice shown in F and H. Bar, 1 mm. (I) Measurements of the angle of the head of the left and right femur of the wild-type and mutant mice at various postnatal ages. Significant unilateral (right) coxa vara, showing varus angulation was found in some collagen X deficient mutants (see Results).