Chondromodulin I is dispensable during enchondral ossification and eye development - PubMed (original) (raw)
Chondromodulin I is dispensable during enchondral ossification and eye development
Oliver Brandau et al. Mol Cell Biol. 2002 Sep.
Abstract
Chondromodulin I (chm-I), a type II transmembrane protein, is highly expressed in the avascular zones of cartilage but is downregulated in the hypertrophic region, which is invaded by blood vessels during enchondral ossification. In vitro and in vivo assays with the purified protein have shown chondrocyte-modulating and angiogenesis-inhibiting functions. To investigate chm-I function in vivo, we generated transgenic mice lacking chm-I mRNA and protein. Null mice are viable and fertile and show no morphological changes. No abnormalities in vascular invasion and cartilage development were detectable. No evidence was found for a compensating function of tendin, a recently published homologue highly expressed in tendons and also, at low levels, in cartilage. Furthermore, no differences in the expression of other angiogenic or antiangiogenic factors such as transforming growth factor beta1 (TGF-beta1), TGF-beta2, TGF-beta3, fibroblast growth factor 2, and vascular endothelial growth factor were found. The surprising lack of phenotype in the chm-I-deficient mice suggests either a different function for chm-I in vivo than has been proposed or compensatory changes in uninvestigated angiogenic or angiogenesis-inhibiting factors. Further analysis using double-knockout technology will be necessary to analyze the function of chm-I in the complex process of enchondral ossification.
Figures
FIG. 1.
Targeted disruption of mouse chm-I. (A) Structure of wild-type allele, targeting construct, and recombinant locus. Boxes, exons. The expected fragment sizes after _Bgl_II digestion are 11 kb for the wild-type allele and 4.5 kb for the recombinant allele. (B) (Top) Southern blot analysis of mouse tail DNA isolated from the progeny of a mating between heterozygous parents. DNAs were digested with _Bgl_II and hybridized with the probe indicated in panel A. (Bottom) PCR genotyping with primers for wild-type and recombinant alleles. +/+, wild-type mouse; +/−, heterozygous mouse; −/−, homozygous mutant mouse. (C) (Left) Northern blot analysis of total RNA from limb cartilage derived from newborn wild-type and homozygous mutant mice. The filter was hybridized with a cDNA probe specific for chm-I. No RNA is detectable in the mutant mice. (Right) RT-PCR with _chm-I_-specific primers, cartilage, thymuses, and whole eyes of homozygous mutant and homozygous wild-type mice gives no evidence for chm-I mRNA in the chm-I-deficient mouse. (D) Immunohistochemistry of newborn tibial growth plate with a chm-I-specific antibody confirms complete deletion of the chm-I protein in the mutant mouse.
FIG. 2.
Analysis of the skeleton in wild-type and chm-I-deficient mice. (A) No gross abnormalities are detectable in alcian blue and alizarin red skeletal staining of newborn mice. The relation of the bony shafts of E16 forelimbs to the complete bone length is unchanged in chm-I mutant mice. (B) X-ray examination of 6-month-old mutant and wild-type mice shows no differences in bone morphology and bone density.
FIG. 3.
Immunostaining of E15 and newborn cartilage. (A) Consecutive sections of tibias from wild-type and chm-I-deficient E15 littermates were stained with specific antibodies against type II and type X collagens and endomucin. Collagen distributions and levels of vascular invasion were comparable. (B) HE and safranin O-van Kossa staining of the tibias from newborn littermates of heterozygous crossings show no obvious morphological differences or altered proteoglycan content. (C) Consecutive sections of the tibias from wild-type and chm-I-deficient 6-month-old littermates were stained with safranin O. The articular cartilage and the articular surface are normal and give no indications for degeneration.
FIG. 4.
Analysis of chondrocyte proliferation and differentiation. (A) Consecutive sections of newborn knee joints were stained with BrdU (left). BrdU-positive cells in the femoral and tibial growth plates and epiphysis were counted. No significant differences in the number of positive cells per square millimeter section could be observed (right). (B) In situ hybridization with probes specific for Col2a1, Col X, PTHrPR, and Ihh probes showed similar distributions of these markers in the proliferative, early hypertrophic, and hypertrophic zones in wild-type and mutant newborn mouse tibial sections. (C) Electron microscopy revealed no changes in cell morphology or extracellular matrix in the hypertrophic or proliferative zone.
FIG. 5.
Analysis of epiphyseal cartilage by RT-PCR and immunoblotting. (A) RT-PCR experiments gave no evidence for changed expression of genes for FGF-1, FGF-2, TGF-β1, TGF-β2, TGF-β3, tendin, or VEGF. (B) Immunoblotting for FGF-2, TGF-β2, TGF-β3, and VEGF with total protein extracts from cartilage also gave no evidence for a changed expression of these growth factors.
References
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