Collagen II is essential for the removal of the notochord and the formation of intervertebral discs - PubMed (original) (raw)
Collagen II is essential for the removal of the notochord and the formation of intervertebral discs
A Aszódi et al. J Cell Biol. 1998.
Abstract
Collagen II is a fibril-forming collagen that is mainly expressed in cartilage. Collagen II-deficient mice produce structurally abnormal cartilage that lacks growth plates in long bones, and as a result these mice develop a skeleton without endochondral bone formation. Here, we report that Col2a1-null mice are unable to dismantle the notochord. This defect is associated with the inability to develop intervertebral discs (IVDs). During normal embryogenesis, the nucleus pulposus of future IVDs forms from regional expansion of the notochord, which is simultaneously dismantled in the region of the developing vertebral bodies. However, in Col2a1-null mice, the notochord is not removed in the vertebral bodies and persists as a rod-like structure until birth. It has been suggested that this regional notochordal degeneration results from changes in cell death and proliferation. Our experiments with wild-type mice showed that differential proliferation and apoptosis play no role in notochordal reorganization. An alternative hypothesis is that the cartilage matrix exerts mechanical forces that induce notochord removal. Several of our findings support this hypothesis. Immunohistological analyses, in situ hybridization, and biochemical analyses demonstrate that collagens I and III are ectopically expressed in Col2a1-null cartilage. Assembly of the abnormal collagens into a mature insoluble matrix is retarded and collagen fibrils are sparse, disorganized, and irregular. We propose that this disorganized abnormal cartilage collagen matrix is structurally weakened and is unable to constrain proteoglycan-induced osmotic swelling pressure. The accumulation of fluid leads to tissue enlargement and a reduction in the internal swelling pressure. These changes may be responsible for the abnormal notochord removal in Col2a1-null mice. Our studies also show that chondrocytes do not need a collagen II environment to express cartilage-specific matrix components and to hypertrophy. Furthermore, biochemical analysis of collagen XI in mutant cartilage showed that alpha1(XI) and alpha2 (XI) chains form unstable collagen XI molecules, demonstrating that the alpha3(XI) chain, which is an alternative, posttranslationally modified form of the Col2a1 gene, is essential for assembly and stability of triple helical collagen XI.
Figures
Figure 1
Skeletal staining of newborn vertebral columns derived from wild-type and _Col2a1_-null mice. Veterbral columns from wild-type (+/+) (A and C) and _Col2a1_-null (−/−) mice (B and C) were stained with alizarin red and alcian blue. In the wild type, a ventral view of the thoracic regions (A) showed adjacent vertebral bodies (vb) were separated by well-formed intervertebral discs (ivd) that contained a gelatinous nucleus pulposus (np) in the center and a cartilaginous inner annulus (ia) at the periphery. In contrast, the intervertebral discs of the _Col2a1_-null mice (B) lacked the nucleus pulposus. The neighboring vertebral bodies (vb) were connected via blue-stained cartilaginous bridges (arrows). C and D show lateral views of the fifth thoracic vertebra from wild-type and _Col2a1_-null mice, respectively. Vertebral arches of mutant vertebrae failed to fuse. Note that the _Col2a1_-null vertebral body is composed of a blue, cartilaginous tissue that is surrounded by a thin, red, bony sheath.
Figure 2
Comparative histological analysis of the developing vertebral column from wild-type and _Col2a1_-null mice. Sagittal sections of the cervical vertebral column (E12.5–17.5 and newborn [_NB_]) from wild-type (+/+; A, C, E, G, and I) and _Col2a1_-null mice (−/−; B, D, F, H, and J) were stained with hematoxylin and eosin. The sections are presented at same scale to show the size differences between normal and mutant vertebral bodies. The orientation of I and J are at right angles to the others. The segmental differentiation of prevertebrae (pv) and intervertebral mesenchymes (im) were similar at E12.5 between wild-type (A) and _Col2a1_-null embryos (B). In wild-type mice, the notochord gradually expanded at the intervertebral disc areas to form a nucleus pulposus (np) and vanished within the vertebral bodies (vb) (C, E, G, and I). In _Col2a1_-null mice, the rod-like structure of the notochord remained relatively unchanged, without any notable differential expansion or regression (D, F, H, and J). Vertebral bodies of _Col2a1_-null mice became double in size. Other landmarks, such as prevertebrae (pv), intervertebral mesenchyme (im), notochord (n), inner annulus (ia), and outer annulus (oa), are indicated. The arrows identify the notochordal sheath, and asterisks indicate the acellular notochordal tube. Bar, 100 μm.
Figure 3
Immunohistochem-ical localization of collagens in wild-type and _Col2a1_-null vertebral columns. Sagittal and parasagittal sections of the vertebrae from wild-type (+/+; A, C, E, G, I, and K) and _Col2a1_-null (−/−; B, D, F, H, J, and L) embryos were stained with specific antibodies against collagens I (Col1), II (Col2), III (Col3), IX (Col9), X (Col10), and XI (Col11). In the wild type (A), collagen II was present in the cartilage of vertebral bodies (vb), inner annulus (ia), and notochordal sheath (arrow), but absent in the outer annulus (oa) and the nucleus pulposus (np). _Col2a1_-null cartilage lacked collagen II (B), while collagens IX (C and D) and XI (E and F) showed a tissue distribution similar to that of wild type. The immunostaining for collagen XI was significantly weaker in _Col2a1_-null cartilage (F). _Col2a1_-null cartilage showed ectopic deposition of collagens I (J) and III (L). The distribution of collagen X was in the center of normal and in the periphery of _Col2a1_-null vertebral bodies (compare G and H). A–F, I, and J are sections from E14.5 embryos, and G, H, K, and L are sections from E15.5 embryos. Bar: (+/+) 200 μm; (−/−) 100 μm.
Figure 4
Immunohistochemical localization of noncollagenous cartilage proteins in wild-type and _Col2a1_-null vertebral columns. Sagittal sections of the vertebrae of E14.5 embryos from wild-type (+/+; A, C, and E) and _Col2a1_-null (−/−; B, D, and F) were stained with specific antibodies against aggrecan (Agn), fibromodulin (FM), and cartilage oligomeric protein (COMP). Aggrecan and COMP were similarly distributed in wild-type (A and E) and mutant (B and F) vertebral column. Fibromodulin (D) staining was decreased in _Col2a1_-null cartilage. The vertebral body (vb), notochord (n), and inner annulus (ia) of the developing vertebral column are indicated. Bar: (+/+) 200 μm; (−/−) 100 μm.
Figure 5
Localization of Col1a1 and Col3a1 mRNAs in wild-type and _Col2a1_-null cartilage. Paraffin sections from wild-type (+/+; A and C) and _Col2a1_-null (−/−; B and D) E14 vertebral columns were hybridized with 35S-labeled antisense cRNAs specific for collagens I (A and B) and III (C and D). Chondrocytes in _Col2a1_-null vertebral bodies expressed ectopic collagens I or III (arrowheads) or lacked the expression of these collagens (arrows, B and D). No cells expressing collagens I or III were observed in cartilage of normal vertebral bodies (A and C). The location of the vertebral body (vb), annulus fibrosus (a), and the perichondrium (p) are indicated. Bar, 50 μm.
Figure 6
Electrophoretic analysis of collagens from wild-type and mutant cartilage. Collagens extracted from wild-type and homozygous mutant (−/−) limb cartilage were analyzed by 5% SDS/PAGE stained with Coomassie blue (lanes 1–4, and lanes 13–15) or by immunoblot with the appropriate antibody (lanes 5–12). Samples were analyzed with (+) or without (−) limited pepsin digestion. p in lane 12 indicates a less stringent pepsin digestion condition. Collagen extracts from −/− skin (lane 14) and wt bone (lane 15) were included for comparative purposes. Unless otherwise indicated, wild-type cartilage collagen samples were derived by pepsin digestion of the Gu/ HCl insoluble fractions, whereas −/− cartilage collagen samples were derived from the Gu/HCl soluble fraction. The identities of the various collagen bands are indicated. β11, β12, and β22 represent dimers with the following composition: α1(I)2, α1(I)1α2(I)1, and α2(I)2, respectively; β is a general term for these dimers. The arrows in lane 12 indicate degraded fragments of collagen XI chains. All samples were analyzed under nonreducing condition.
Figure 7
Electrophoresis of pepsin-digested collagen produced by wild-type and mutant chondrocytes cultured on alginate beads. After 8 wk of culturing wild-type (wt) and mutant (−/−) limb chondrocytes on alginate beads, cells were labeled with
l
-[2,3-3H]proline, and the collagen from the cell-associated (cell), immediate extracellular matrix (alginate), and medium fractions were subjected to limited pepsin digestion. The resultant collagen chains were analyzed on a 5% SDS–polyacrylamide gel (A). Samples were analyzed without reduction of disulphide bonds, and the protein bands were detected by fluorography. The identities of the various collagen bands are indicated. The migration position of the disulphide bonded trimer of collagen III (α1(III)3) is also indicated. (B) An immunoblot of similar samples hybridized to a collagen II antibody. The position of the α1(II) chain is indicated, and a lane (std) containing purified collagen II is also included for comparison.
Figure 8
Electron microscopy of collagen fibrils in wild-type and _Col2a1_-null cartilage. Ultrastructural anaylsis was performed on wild-type (A and C) and _Col2a1_-null (B and D) cartilage. Longitudinal sections showed a reduced number of collagen fibers and no well-organized fibrillar network in the mutant cartilage (B and D). The abnormal fibrils had rough surfaces, and their diameters and banding pattern were irregular (D). Bars: (A and B) 0.4 μm; (C and D) 0.2 μm.
Figure 9
Analysis of cell death, cell proliferation, and metabolic activity during normal notochord dismantling. Sagittal sections through the lumbar part of vertebral column at E 13.5 (A–C, E, and F) and E14.5 (D). Cellular death was analyzed by TUNEL assay (A and B). No apoptosis was detected either in the notochord (n) or in the vertebral body (vb) (A). Positive control section treated with DNase I stain all cells (B). Cell proliferation was monitored by BrdU incorporation assay (C and D). Proliferation rate of cells was similar in the intravertebral and intervertebral segments of notochord E13.5 (C). Dividing cells were detectable in the intravertebral segment even at E14.5 (D). Radioactive sulfate incorporations assay (E, dark field image; F, bright field image). Both intra- and intervertebral notochordal cells were metabolically active. High metabolic activity was also obvious in the cartilage of vertebral bodies. ia, inner annulus. Bars: (A–C and F) 200 μm; (D and E) 100 μm.
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