Depletion of cartilage collagen fibrils in mice carrying a dominant negative Col2a1 transgene affects chondrocyte differentiation

Ottavia Barbieri,1,2 Simonetta Astigiano,1 Monica Morini,1,2 Sara Tavella,2 Anna Schito,2 Alessandro Corsi,3,4 Davide Di Martino,2 Paolo Bianco,4 Ranieri Cancedda,1,2 and Silvio Garofalo1,2

1Istituto Nazionale per la Ricerca sul Cancro, 16132 Genoa; 2Dipartimento di Oncologia, Biologia e Genetica, Università di Genova, 16132 Genoa; 3Dipartimento di Medicina Sperimentale, Università dell'Aquila, 67010 Cappito di L'Aquila; 4Dipartimento di Medicina Sperimentale e Patologia, Università di Roma La Sapienza, and Parco Scientifico Biomedico San Raffaele, 00161 Rome, Italy

Submitted 12 December 2002 ; accepted in final form 8 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We have generated transgenic mice harboring the deletion of exon 48 in the mouse {alpha}1(II) procollagen gene (Col2a1). This was the first dominant negative mutation identified in the human {alpha}1(II) procollagen gene (COL2A1). Patients carrying a single allele with this mutation suffer from a severe skeletal disorder called spondyloepiphyseal dysplasia congenita (SED). Transgenic mice phenotype was neonatally lethal with severe respiratory failure, short bones, and cleft palate. Transgene mRNA was expressed at high levels. Growth plate cartilage of transgenic mice presented morphological abnormalities and reduced number of collagen type II fibrils. Chondrocytes carrying the mutation showed altered expression of several differentiation markers, like fibroblast growth factor receptor 3 (Fgfr3), Indian hedgehog (Ihh), runx2, cyclin-dependent kinase inhibitor P21CIP/WAF (Cdkn1a), and collagen type X (Col10a1), suggesting that a defective extracellular matrix (ECM) depleted of collagen fibrils affects chondrocytes differentiation and that this defect participates in the reduced endochondral bone growth observed in chondrodysplasias caused by mutations in COL2A1.

skeletal dyplasias; growth plate; cartilage extracellular matrix; spondyloepiphyseal dysplasia congenita


ENDOCHONDRAL BONE FORMATION and growth are strictly dependent on chondrocyte differentiation (2). Extracellular matrix (ECM) plays an important role in controlling cell differentiation (6, 25). During endochondral bone formation, the composition of ECM changes at different developmental stages (2). Modifications of ECM composition are associated with the transition from the transient cartilaginous template to the permanent bone scaffold (10). The proliferation of chondrocytes and their capacity to control the expression of the proper ECM genes before terminal differentiation are critical in this process (2).

Genetic defects in ECM components of cartilage are associated with short stature, abnormal bone shape, and reduced bone growth (11). The precise mechanisms involved in these diseases are poorly understood. To better understand how defective cartilage ECM can alter skeletal development, transgenic mice with several dominant negative mutations in type II procollagen, the major collagen of the cartilage matrix, have been generated (4, 21, 27). They have remarkably similar phenotypes. Their bones are short, curved, and, when the mutations are viable, predisposed to osteoarthritis (8). The study of these experimental models was not focused to recognize defective chondrocyte differentiation as a critical step in the genesis of the skeletal problem. However, several mouse mutants with defined defects in chondrocyte differentiation, as PTHrP-(13) and Ihh-(26) null mice, show very similar phenotype and skeletal abnormalities. This similarity raises the question whether the defective ECM can also modify the ability of chondrocytes to follow a correct maturation pattern to sustain bone growth.

To investigate how a defective extracellular environment could affect chondrocyte differentiation in vivo, in an entire transgenic organism, we have used a transgenic mouse strain overexpressing a dominant negative collagen type II, lacking 36 amino acids at the carboxyl terminal end of the triple helical region. The mutation consisted of the deletion of exon 48, corresponding to the last triple helical coding exon of Col2a1, and was designed from the first reported human mutation identified in spondyloepiphyseal dysplasia congenita (SED) (15).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Generation and identification of transgenic mice. The entire mouse Col2a1 gene was cloned from a cosmid library (22) and engineered with the exon 48 mutation. The deletion was created by joining the BspHI and NcoI restriction sites flanking exon 48 in a XbaI-BamHI genomic fragment containing the 3' end of Col2a1 (Fig. 1A). Therefore, the deletion also removed 85 bp in intron 47 and 99 bp in intron 48. Including the exon sequence, the deletion consisted of 292 bp. Using the XbaI-BamHI fragment with the deletion, the entire Col2a1 genomic locus was reconstituted in a 42-kb-long cosmid clone, as described in Fig. 1A. The NotI-ClaI fragment with 5' end of the gene, from the promoter to exon 14, was also engineered with a silent mutation that abolishes the NcoI site in exon 7 and allows transgenic identification (Fig. 1B). The transgene DNA was released from the cosmid vector pWE15 by NotI digestion and separated in agarose gel. After purification by electroelution, it was microinjected into the pronuclei of one-cell B6D2F1 mouse embryos that were then implanted into CD1 pseudopregnant foster mothers (9). Transgenic founder mice could be recognized by NcoI digestion of tail genomic DNA and Southern blot (Fig. 1B) (4). Transgene copy number was estimated by the intensity of transgene specific band compared with those generated by the endogenous Col2a1 alleles. It was calculated as the half ratio of the intensities of transgene over endogenous bands.



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Fig. 1. Construction, identification, and expression of Del48 transgene. A: construction of the Del48 transgene. The XbaI-BamHI fragment carrying exon 48 deletion was used to reconstitute the 40-kb-long Col2a1 locus in a cosmid vector. B, top: Southern blot of NcoI-digested tail genomic DNA. The 1,168-bp fragment is diagnostic of transgenic mice. Ratio of the intensities of wild-type (WT) and transgene (TG) specific bands allows the determination of transgene copy number. Bottom: schematic representation of the silent mutation in exon 7 that abolishes the NcoI site and specifically marks the transgene. The presence of the transgene is detected by the 1,168-bp NcoI fragment. C: schematic of RT-PCR with exon 48 flanking primers that amplify WT (left) and transgenic (middle) cDNA. Right: [32P]dCTP-labeled RT-PCR of limb RNA shows the WT (280 bp) and exon 48-deleted (172-bp) cDNA fragments. The ratio of the intensities of WT(280 bp)- and transgene (172 bp)-specific fragments was used to estimate the level of transgene expression.

 

Transgene RNA expression. The amount of transgene mRNA was measured by reverse transcriptase-PCR (RTPCR) from whole limb RNA. The primers for the reaction were in exon 46 (O9: ACACCGAGGTTTCACTGGA) and in exon 49 (O10: GCAAAGGCTGACATGTCGA) and amplified a 280-bp-long fragment. In transgenic mice with exon 48 deletion, two fragments were generated: one was 280-bp long, corresponding to endogenous wild-type Col2a1 mRNA, and the other was 172-bp long diagnostic of the transgene mRNA with exon 48 deletion. To precisely measure transgene mRNA, [32P]dCTP was incorporated in the PCR reaction. The radiolabeled fragments were separated by PAGE (Fig. 1C) and the intensity of the bands was measured. Transgene RNA was expressed as a percentage of wild-type Col2a1 RNA. This ratio was constant in different transgenic mice after collection of aliquots of the same PCR reaction from cycle 10 to 20 (linear amplification).

Gross examination, histology, and electron microscopy. The fetuses were removed by Cesarean section from day 14.5 throughout day 18.5 of gestation. For visualization of the entire skeletal system, the cleared skeleton of Del48 transgenic mice and wild-type littermates was stained with Alcian blue and/or Alizarin red (20). For histological examination, embryos were sagittally sectioned and their limbs were dissected. All samples were fixed in 4% paraformaldehyde (PAF) in 0.1 M phosphate buffer, pH 7.2, at 4°C for 2 to 4 h. Half head and body and single limbs were dehydrated in graded ethanol series and separately embedded in paraffin; the sections were stained with hematoxylin-eosin and 1% toluidine blue. For electron microscopy, growth plates from fixed long bones of the limbs were carefully dissected under the microscope, postfixed in 1% osmium tetroxide, dehydrated in graded ethanol solutions, and embedded in epoxy resin (Araldite). Semithin sections were stained with Azur II-methylene blue to select appropriate fields for thin sectioning. Ultrathin sections were cut with diamond knives, placed on uncoated grids, contrasted with uranyl acetate and lead citrate, and observed under a Zeiss transmission electron microscope.

Growth plate morphometric analysis. The proximal epiphysis of the second metatarsal bone was chosen for quantitative analysis of cartilage. The analysis was performed with a semiautomatic image analyzer (IAS2000; Delta System, Rome, Italy). The following parameters were established: chondrocyte density (number of chondrocytes/104 µm2 of cartilage matrix), the amount of cartilage matrix for chondrocyte (µm2 of cartilage matrix/chondrocyte), and the thickness of reserve, proliferative, prehypertrophic, and hypertrophic cartilage. Chondrocyte density and the amount of cartilage matrix for chondrocyte were established after measurement of the area of the epiphyseal cartilage and the number of chondrocytes within the same area.

Immunohistochemistry. Five-micrometer-thick sections were cut, deparaffinized, and treated with hyaluronidase. Endogenous peroxidase was inhibited with methanol and H2O2, and nonspecific binding was reduced by incubation with goat serum. Primary antibodies were mouse monoclonal antibodies anti-human type X collagen X53 (5), mouse monoclonal antibody anti-type II collagen CIIE8 (a generous gift from K. Von der Mark), and commercial antibodies against Cdkn1a, Ihh, and Fgfr3 (Santa Cruz Biotechnology). After incubation with the primary antibody, the sections were treated with a biotinylated goat anti-mouse and peroxidaseconjugated streptavidin complex (Jackson). The peroxidase activity was visualized with 3-amino-9-ethylcarbazole sub-stratum (Merck). The sections were counterstained with Harris' hematoxylin and mounted in Dako (Santa Barbara, CA).

Proliferation and apoptosis assays. Pregnant mice were injected intraperitoneally with 50 µg of bromodeoxyuridine (BrdU) (Sigma, St Louis, MO) per g/body wt 1 h before death. Chondrocyte nuclei that had incorporated BrdU were detected by incubating the sections with a monoclonal antibody against BrdU (Clone BU-33, Sigma) diluted 1:100 for 2 h at room temperature. The color reaction was developed by using diaminobenzidine (DAB; Sigma) as substrate. Apoptotic chondrocytes were detected by using the ApopTag Plus terminal TdT-mediated dUTP nick end labeling (TUNEL) kit (Oncor, Gaithersburg, MD) according to the manufacturer's instructions. Proliferation and apoptotic indices were calculated as the number of labeled nuclei per 100 chondrocyte within the reserve and proliferative zones.

In situ hybridization. Paraffin sections on (3-aminopropyl)triethoxysilane-coated glass slides were defatted, rehydrated, treated with 0.2 M HCl, and washed in phosphate-buffered saline (PBS). After being incubated with proteinase K and washed with glycine/PBS, sections were postfixed in PAF, treated with 0.25% acetic acid/0.1 M triethanolamine, and incubated 1 h in a moist chamber with a prehybridization buffer (10% dextran sulfate, salmon sperm DNA 0.1 mg/ml, yeast tRNA 0.1 mg/ml, poly A-poly C 0.01 mg/ml, Na-pyrophosphate inorganic 1 mg/ml, and 40% deionized formamide). Hybridization to digoxigenin (DIG-UTP)-labeled RNA probes (0.5 ng/µl) was carried out overnight in the same hybridization mixture in a moist chamber. After this step, sections were treated with RNAse and washed in sodium chloride-sodium citrate (SSC). The probes were subsequently visualized by staining with anti-DIG-alkaline phosphatase-conjugated antibody, washed in 0.1 M Tris, pH 9.5, 0.1 M NaCl, 0.01 M MgCl2, and developed by immersion in a Boehringer Mannheim developing reagent. The probes were as follows: Col10a1 (3) and Runx2 (12).

Statistical analysis. A statistical comparison between transgenic and wild-type mice was performed for thickness of reserve, proliferative, prehypertrophic, and hypertrophic cartilage and BrdU and TUNEL labeling index. Data were reported as means ± SD. Comparisons between groups were made by one-way ANOVA, and post hoc comparison was carried out with Scheffe's test. A probability level of 5% was used to establish a significant difference.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lethal short-limbed dwarfism in transgenic mice with a 36-amino acid deletion in collagen II. Three transgenic lines carrying the Del48 transgene were generated and exhibited the same phenotype, two of which were studied in great detail. Over 100 transgenic embryos, carrying several copies of transgene, were generated and analyzed. The two founders were mosaic and phenotypically normal, whereas the entire progeny carrying the same transgene copy number revealed the severe phenotype of a lethal chondrodysplasia (Fig. 2A). This phenotype was fully penetrant, with none of the transgenic mice surviving beyond birth because of acute neonatal respiratory distress. The respiratory failure was due to simultaneous defects in the volume of the thorax from reduced growth of the ribs and in the airway diameter from structural defects in tracheal cartilages. Newborn transgenic mice were unable to breathe, became cyanotic, and died within minutes after birth with severe heart and liver congestion. Gross and histopathological examination of lungs revealed no sign of inflation.



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Fig. 2. Phenotype of Del48 transgenic mice. A: Del48 transgenic embryos from 18.5 to 16.5 days post coitum (pc) (left to right). Alizarin red (bone) and Alcian blue (cartilage) staining of the cleared skeleton. B: control WT embryos from 18.5 to 16.5 days pc. Alizarin red and Alcian blue staining of the cleared skeleton. C: hard palate of WT (left) and Del48 transgenic mouse (right) after removal of lower jaw, showing the complete clefting in transgenic (black arrow). D: base of the skull of WT and Del48 transgenic embryos 18.5 days pc stained with Alizarin red showing the size of palate splitting (blue arrow). E: Alizarin red staining of WT and Del48 mandiboles prepared from embryos 18.5 days pc showing the reduction in size. F: Alizarin staining of forelimbs (top) and hindlimbs (bottom) of WT (left) compared with Del48 transgenic (right) embryo 18.5 days pc. The staining reveals that transgenic long bones are 50% shorter than wild type.

 

A short snout with a protruding tongue was also observed (Fig. 2A). This was due to reduced growth of the mandible and upper jaw bones (Fig. 2E). Incomplete closure of the hard palate due to defective cartilage growth was found in all transgenic mice (Fig. 2, C and D). Severe shortening (about 50%) of limbs was present due to reduced growth and mineralization of long bones (Fig. 2F). Membranous and periosteal bone formations, which do not require the formation of an intermediate cartilage template, were unaffected. These distinctive anomalies made it easy distinguish transgenic from normal embryos since day 16.5 post coitum (pc); however, the presence of transgene was always confirmed by a diagnostic 1,168-bp long NcoI fragment in tail genomic DNA Southern blot (Fig. 1B).

During embryo development, the first recognizable alteration of the normal pattern of ossification was observed at 15.5 days pc. At this stage, embryo size was comparable, but vertebral bodies of transgenic embryos were not stained by the bone-specific dye Alizarin red, whereas normal vertebrae were stained. Transgenic ribs and long bones were also faintly colored and shorter than normal. By 16.5 days pc, transgenic embryos displayed a marked difference in skeletal growth (Fig. 2A). They were smaller due to reduced length of vertebral column, limbs, and ribs. At this developmental stage, the vertebral bodies of transgenic mice were not yet mineralized and the intervertebral distance was strongly reduced due to the absence of intervertebral disks. Shortening of ribs and other long bones was evident, as was impaired ossification of metacarpal and metatarsal bones. At later developmental stages, the delay in ossification was even more pronounced (Fig. 2A).

The level of transgenic mRNA expression was estimated by radiolabeled RT-PCR (Fig. 1C). The intensity of the 172-bp long, transgenic fragment with the deletion ranged from three to five times the intensity of the undeleted, wild-type fragment, at a different PCR cycle number of the linear phase of amplification, in transgenic embryos at different developmental stages and from different transgenic strains. It was impossible to distinguish between the normal and the deleted proteins because of both the minimal protein amount in cartilage (see electron microscopy) and the small size of the deletion.

Structural and ultrastructural changes in long bones. Structural abnormalities of growth plate cartilage were found in all examined transgenic mice. They consisted of an irregular organization of the proliferating chondrocytes that were unable to form distinct columns of cells along the bone longitudinal axis (Fig. 3E). This resulted in a short and disorganized proliferative zone, and, due to the collapse of the columns, an increased cell number in the prehypertrophic region (Fig. 3E). In the proliferative zone, chondrocytes did not form clusters of dividing cells (Fig. 3F) and appeared larger and isolated in a matrix that was poorly stained with cationic dyes (Fig. 3F). Periodic acid Schiff (PAS)-positive cytoplasm inclusions, a hallmark of human SED, were never found in chondrocytes. In contrast, multiple rounded cytoplasmatic vacuoles were obvious in semithin sections (Fig. 3F).



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Fig. 3. Histology of tibia growth plate cartilage. Growth plate, hypertrophic zone, and proliferating chondrocytes of WT (A-C) and Del48 newborn mice (D-F). In transgenic mice, growth plate is shorter (D), with a higher number of cells lacking organization in vertical columns of chondrocytes (E) and chondrocytes showing cytoplasmatic inclusions (F). Magnification: x1,000 (A and D), x2,000 (B and E), and x4,000 (C and F).

 

Striking changes in the structure of the ECM of epiphyseal cartilage were observed by electron microscopy (EM) in transgenic mice. The typical dense network of thin and straight type II collagen fibrils observed in wild-type littermates (Fig. 4A) was replaced in transgenic mice by an extremely loose, empty-looking matrix, in which abnormally short and curved fibrils were detected (Fig. 4B). Like normal fibrils observed in the epiphyseal cartilage of wild-type litter-mates, these fibrils were associated with proteoglycan granules. An apparent relative excess of such granules could be observed in some areas of the matrix due to reduced number of fibrils. A marked excess of proteoglycans was noted in close association with the chondrocyte plasma membrane, resulting in obvious masses of basophilic material readily distinguishable by light microscopy in the semithin section. Thick, banded fibrils were never observed.



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Fig. 4. Ultrastructure of chondrocyte and cartilage extracellular matrix. A: WT cartilage extracellular matrix (ECM) with normal amount of thin cartilage collagen II fibrils. B: cartilage ECM of Del48 transgenic mouse severely depleted of collagen II fibrils. C: WT mouse chondrocyte. D: Del48 transgenic mouse chondrocyte with distinct alterations. (A and B: original magnification, x18,000, bar 500 nm; C and D: original magnification, x4,900, bar 1 µm).

 

Chondrocytes embedded in this abnormal ECM were characterized by obvious dilation of the rough endoplasmatic reticulum (RER) cisternae (Fig. 4D). The dominant pattern of cisternal dilation observed in most chondrocytes consisted of multiple, small, and rounded cisternal profiles. Occasionally, multiple, nonrounded, more uneven dilations were detected. Single, large distensions were never observed.

Growth plate morphometry, proliferation, and apoptosis assays. Morphometric analysis of the proximal growth plate of the second metatarsal bone revealed significant differences in the thickness of resting, proliferative, prehypertrophic, and hypertrophic cartilages between transgenic and wild-type mice (Fig. 5, A and B). Although reserve, proliferating, and hypertrophic cartilages were thicker in wild-type mice, prehypertrophic cartilage was broader in transgenic mice. Consistent with the depletion of type II collagen fibrils in the cartilaginous matrix, density of chondrocytes and the amount of cartilage matrix for chondrocyte were, respectively, greater and lower in transgenic compared with wild-type mice (Fig. 5, C and D). Thus, to investigate the potential effect of collagen II fibrils reduction on chondrocyte cell cycle, proliferation and apoptosis were assessed by BrdU labeling and TUNEL assay, respectively. No significant difference was detected for both labeling indexes comparing transgenic mice to their wild-type littermates; however, mean values for proliferation and apoptosis were lower and greater, respectively, in transgenic mice (Fig. 5, E and F). These data suggest that in transgenic Del48 mice, shortness of endochondrally formed skeletal segments is not related to an abnormal rate of chondrocyte proliferation and apoptosis.



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Fig. 5. Growth plates morphometry, proliferation, and apoptosis assays. A: comparative histology of the proximal epiphysis of metatarsal bone from transgenic (left) and WT (right) mice. B: statistical comparison revealed significant differences in the thickness (µm) of the reserve, proliferative, prehypertophic, and hypertrophic zones between transgenic and WT mice (*P < 0.05). Of note, whereas the mean values are greater for WT in reserve (RC), proliferative (PC), and hypertophic cartilage (HC), the prehypertrophic zone (preHC) is thicker in transgenic mice. C: chondrocytes from transgenic mice are more crowded compared with WT, as proved by statistically significant mean values for chondrocyte density (n = number of chondrocytes/104 µm2 of cartilage). D: amount of cartilage matrix per chondrocyte (µm2/cell, µm2 of cartilage matrix/chondrocyte) is significantly reduced in transgenic cartilage. E: no significant statistical difference of chondrocyte bromodeoxyuridine (BrdU)-labeling index was found between transgenic and WT mice. F: TUNEL assay did not show a significant difference in chondrocyte apoptosis between transgenic and WT mice.

 

Chondrocyte differentiation is altered in Del48 transgenic mice. Because chondrocyte proliferation and apoptosis were minimally affected by transgene expression and ECM depletion of collagen fibrils, we have determined whether this defect could affect chondrocyte maturation in growth plate cartilage. Differentiation in transgenic mice cartilages was studied following the protein and mRNA expression pattern of molecules specific to different stages of chondrocyte maturation. The expression of Col2a1, Fgfr3, Ihh, and Cdkn1a was studied using highly specific antibodies. Runx2 expression was studied by in situ hybridization using specific probes. Expression of Col10a1, a collagen specifically produced and secreted in the ECM by hypertrophic chondrocytes, was studied by both immunolocalization and in situ hybridization.

Consistent with the EM finding, we observed in all cartilages of transgenic mice a substantial reduction of the amount of collagen II protein (Fig. 6B) that was undetectable. Normal control mice had a very high level of collagen II expression throughout epiphyseal cartilage. (Fig. 6A).



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Fig. 6. Immunohistochemistry and in situ hybridization of Del48 cartilage. Expression of Col2a1 (A, B), Cdkn1a (C, D), and Col10a1 (E, F) in 18.5 days p.c. WT (left) and Del48 transgenic (right) femur. Specific anti-collagen II antibody shows that it is expressed in epiphyseal cartilage of WT femur (A), but it is completely absent in Del48 transgenic cartilage (B). Cdkn1a expression is detected by a specific antibody into the nuclei of prehypretrophic chondrocytes of WT mice (C), but it is missing in Del48 femur chondrocytes (D). By immunohistochemistry, collagen 10a1 is found in WT hypertrophic zone (E) but cannot be found in Del48 mice (F). In situ hybridization with Col10a1-specific probe (G) shows the absence of detectable Col10a1 in Del48 hypertrophic chondrocytes (H). x100 (A, B), x400 (C-H). Expression of Ihh (I, J), Fgfr3 (K, L), and Runx2 (M, N) in WT (left) and Del48 transgenic (right) humerus. Immunohistochemistry with anti-Ihh-(I, J) and anti-Fgfr3-(K, L) specific antibodies shows that in WT mouse, Ihh (I) and Fgfr3 (K) are expressed in prehypertrophic chondrocytes, whereas in Del48 mice Ihh (J) and Fgfr3 (L) are undetectable. In situ hybridization with specific probe for Runx2 (M, N) shows that the high expression level of Runx2 mRNA in WT hypertrophic chondrocytes (M) is missing in Del48 transgenic cartilage (N). x200 (I-N).

 

To recognize modifications of chondrocyte maturation, we then studied the expression of the cdk inhibitors Cdkn1a, of Ihh, and Fgfr3. The expression of these molecules was detected in normal prehypertrophic chondrocytes (Fig. 6, C, I, and K) (24, 26, 12), but it was almost undetectable in transgenic chondrocytes (Fig. 6, D, J, and L).

Furthermore, in the presumptive hypertrophic zone of transgenic mice, expression of collagen X was not recognizable by specific antibody (Fig. 6, E and F). To assess whether the abnormality was due to a defective protein assembly or degradation in the pathological ECM lacking collagen II fibrils, we also determined whether Col10a1 mRNA was present. Northern blot (data not shown) and in situ hybridization did not show detectable levels of Col10a1 mRNA in the cartilage of transgenic mice (Fig. 6H), whereas normal chondrocytes showed a highly restricted and intense signal in the hypertrophic zone (Fig. 6G).

In situ hybridization using a specific probe for Runx2, which is expressed at a low level in resting and proliferating chondrocytes but at very high levels in hypertrophic chondrocytes and in osteoblasts (14) (Fig. 6M), showed a very weak signal all over Del48 transgenic mice cartilage without the peak of expression that characterizes the transition from cartilage to bone (Fig. 6N).

These experiments show that the reduction of collagen II fibrils in cartilage matrix due to transgene expression of deleted collagen II chains is associated with chondrocyte inability to undergo proper differentiation into hypertrophic cells, to express detectable levels of Col10a1, Cdkn1a, Ihh, and Fgfr3, and to sustain bone formation.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several transgenic mice carrying dominant negative Col2a1 mutations have been generated in the past. Such mutations were randomly distributed along the collagen triple helix and range from single amino acid (glycine) substitutions (4, 18, 19) to large deletions (21, 27) or inactivating insertions (16). However, nobody had previously described a deletion at the COOH-terminal end of the triple helix of Col2a1. Deletions were reported in the middle (27) or at the amino-terminal end of the helix (21) and, in the case of the spontaneous mouse mutant Dominant micromelia (Dmm), in the C-propeptide (23). The phenotype of all these mutant mice was remarkably similar, and apparently the site or the size of deleted residues did not generate phenotype diversity. Other mutant mice with genetic defects in different collagen components of the cartilage fibrillar network, such as chondrodysplasia mouse (cho) (17), also show a very similar phenotype, although the genetic defect does not act in a dominant negative way. Although in humans COL2A1 mutations can generate a spectrum of skeletal dysplasias of different severity, including SED, achondrogenesis type II, hypochondrogenesis, Kniest dysplasia, and Stickler arthro-ophthalmopathy (7), in mice a lethal phenotype is usually observed.

We generated transgenic mice carrying a transgene with the deletion of exon 48 in Col2a1. This mutation was the first identified in humans (15) and determines a nonperinatal or neonatal lethal SED congenita phenotype in which abnormalities in iuxtatruncal epiphysis and spine predominate. In contrast, an invariably lethal short-limbed dwarfism that better overlaps the spectrum of hypochondrogenesis/achondrogenesis was observed in Del48 transgenic mice. Compared with the severity of the disease observed in the human family in which exon 48 deletion of COL2A1 was found (15), the severity of the skeletal phenotype in our murine model appears much more pronounced. This difference can be related to the different level of expression of the transgene. In fact, in Del48 transgenic mice, the amount of transgene mRNA was estimated to be higher than the amount of normal collagen II mRNA expressed by both wild-type alleles. Even though the protein biochemical consequences of the deletion remain to be characterized, it has been clearly established that the level of transgene expression is critical for the severity of phenotype (21); thus we can assume that in the human family, the expression level of procollagen chains from the mutant allele was lower than in Del48 transgenic mice.

It is reasonable that high transgene expression level can explain not only the phenotypic difference with the patients but also the similarity with the phenotype Col2a1-null mice. Indeed, the skeletal defects of Del48 mice overlap with the most severe phenotype observed in homozygous null mice (16, 1), which also resembles human achondrogenesis type II, because mice die at birth with a cleft palate and gross morphological and histological malformations in their endoskeleton, their long bones shortened, a thickened cortical collar, and absent endochondral bone and epiphyseal growth plates. However, these apparent similarities reflect different pathogenetic mechanisms leading to severe reduction of collagen fibrils in cartilage matrix. Whereas in null mice the production Col2a1 mRNA is impaired due to the insertional mutation in the genomic locus, in Del48 mice both wild-type alleles and the transgene are fully transcribed and translated in collagen II chains that cannot assemble in functional triple helical collagen molecules due to the lack of registry generated by the 36-amino acid deletion at the COOH-terminal end. Because the collagen triple helix is formed by a series of hydrogen bonds that link the Gly-Xaa-Yaa sequences in one procollagen chain to equivalent Gly-Xaa-Yaa sequences in the two other chains, the presence of a shortened chain in a procollagen molecule can prevent folding into a stable triple helix, and degradation of all three chains can occur in a process referred to as procollagen suicide. Such an event occurs in many other fibrillar collagens that are implicated in a variety of human pathologies and without doubt reflects the molecular events leading to ECM defect of collagen fibrils also in Del48 patients and the transgenic model.

The generation of chondrodysplastic transgenic mice harboring exon 48 deletion allowed detailed study on the effects of mutant type II collagen gene on skeletal development. More specifically, because the phenotype was fully penetrant and neonatally lethal, our study was focused on the embryonic development of the skeleton. The first recognizable alteration of the normal pattern of skeletal development was observed at 15.5 days pc. At this stage, although transgenic and wild-type embryos were comparable in size, Alizarin red did not stain or faintly stained vertebral bodies and ribs and long bones of the extremity of the transgenic embryos. By 16.5 days pc, transgenic embryos were smaller due to reduced length of vertebral column, limbs, and ribs. At this developmental stage, the vertebral bodies of transgenic mice were not yet mineralized. At later developmental stages, the delay in ossification and shortness of vertebral column, limbs, and ribs was even more pronounced.

Our morphological study revealed that chondrocyte populations at different maturation stages were found in the growth plates of transgenic mice, but, compared with their wild-type littermates, their distribution was different. In fact, while resting, proliferative and hypertrophic zones were shorter or indistinguishable in transgenic mice compared with the same zones of the wild type, in which the prehypertrophic zone was significantly thicker. Based on this result, we asked whether either an abnormal balance of chondrocyte proliferation and apoptosis or an abnormal chondrocyte differentiation or both contributed to the development of the skeletal phenotype. Although significant differences were not detected in the proliferation and apoptotic indexes, a defective chondrocyte differentiation was proved by the absence or marked reduction in the expression of mRNA and protein of diverse markers of chondrocyte, including Cdkn1a, Ihh, Fgfr3, Col10a1, and Runx2. In addition, as the growing of long bones is also strictly dependent on the appropriate deposition of newly synthesized matrix, we measured chondrocyte density and the amount of cartilage matrix per chondrocyte in resting and proliferating cartilage. Because chondrocyte density and the amount of cartilage matrix per chondrocyte were, respectively, significantly greater and lower in transgenic mice compared with wild type, we conclude that deposition of cartilage matrix is notably impaired in transgenic mice. The greatly extended cisternae of rough endoplasmic reticulum of chondrocytes also suggest that collagen type II secretion may in turn be altered in the transgenic mice, leading to intracellular retention of a proportion of procollagen destined to extracellular export. Thus the inability to assemble procollagen triple helical molecules properly reduces the amount of secreted collagen and results in the depletion of collagen II fibrils in the cartilage matrix, which in turn interferes with the proper interstitial growth of the cartilage and with the normal sequence of chondrocyte maturation and differentiation.


    DISCLOSURES
 
This work was supported by Telethon-Italy Grant D.112, by European Union Grant BIO 2 CT-942002, and by European (ESAERISTO) and Italian (ASI) Space Agencies (to R. Cancedda).


    ACKNOWLEDGMENTS
 
We are grateful to Giuliano Campanile and Elvira Noviello for technical assistance and to Giovanni Levi for advice.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Garofalo, Università di Genova, Centro Biotecnologie Avanzate (Rm. C305), Largo Rosanna Benzi n.10, 16132 Genova, Italy (E-mail: garofalo{at}cba.unige.it).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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