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M Metsäranta

Publications and source records attributed to M Metsäranta.

At least 19 recordsLinked to original sources
PubMed2004

Abnormal craniofacial development and expression patterns of extracellular matrix components in transgenic Del1 mice harboring a deletion mutation in the type II collagen gene.

OBJECTIVE: To analyze the effect of a type II collagen mutation on craniofacial development in transgenic Del1 mice. DESIGN: Samples from homozygous (+/+) and heterozygous (+/-) transgenic Del1 mice harboring mutations in the type II collagen gene as well as non-transgenic (-/-) littermates were collected at days 12.5, 14.5, 16.5 and 18.5 of gestation. The cartilaginous and bony elements of the craniofacial skeleton were analyzed after staining with alcian blue, alizarin red S and von Kossa. The expression patterns of type II, IX and X collagens and aggrecan were analyzed by immunohistochemistry and in situ hybridization. RESULTS: Several abnormalities were observed in the craniofacial skeleton of transgenic Del1 mice. These include an overall retardation of chondrogenesis and osteogenesis in Del1 +/+ mice, and to a lesser extent also in Del1+/- mice. Characteristic findings in Del1 +/+ mice included a reduced anterioposterior length, a smaller size of the mandible, a palatal cleft and a downward bending snout. We also detected retarded ossification of calvarial bones in Del1 +/+ and +/- mice when compared with Del1 -/- mice. A surprising finding was the presence of both type II and X collagens and their mRNAs in the periosteum of the cranial base. CONCLUSION: The present study confirms the important role of type II collagen mutation in craniofacial development and growth. In addition to affecting endochondral ossification, the type II collagen mutation also disturbs intramembranous ossification in the developing craniofacial skeleton.

Animals↗
PubMed1998

Expression of type II and IX collagen isoforms during normal and pathological cartilage and eye development.

Cartilage collagens type II and type IX exist in two alternative forms which arise from alternative splicing and alternative use of promoters, respectively. In the present study we analyzed temporal and spatial expression patterns of the two isoforms of type II and type IX collagen transcripts as well as those of alpha2(IX) and alpha3(IX) collagen mRNAs in limb cartilages and eyes during mouse embryonic development. Northern and RNase protection assays revealed temporal coregulation of the two alternative isoforms in limbs, but not in the eye where no long form of alpha1(IX) collagen mRNA was detected. Although in situ hybridization of limbs revealed identical expression patterns of the long form of type II collagen and the short form of alpha1(IX) collagen mRNA in the perichondrium and periosteum of 14.5-18.5-day embryos, the patterns were distinctly different at day 12.5 of development: the long form of type II collagen mRNA was expressed throughout the developing cartilaginous anlage whereas the short form of alpha1(IX) collagen mRNA was expressed in the surrounding mesenchyme. Some differences were also detected in the temporal and spatial expression patterns between the alpha1(IX), alpha2(IX), and alpha3(IX) collagen mRNAs. In the eyes, alpha2(IX) collagen mRNA had highest expression levels at day 12.5, whereas alpha1(IX) and alpha3(IX) collagen mRNAs peaked later, at day 16.5. In the limbs, alpha1(IX) and alpha3(IX), but not alpha2(IX), collagen mRNAs were detected in periosteal cells after 16.5 days of development. In transgenic Dell mice, harboring type II collagen transgenes with a small deletion mutation, expression of mutant mRNA affected neither the alternative splicing of wild-type or mutant transcripts nor the ratio of the two alternative forms of the alpha1(IX) collagen mRNA. Despite some distinct similarities, the two alternative forms of type II and type IX collagen must, therefore, be under differential control during mouse development.

Animals↗
PubMed1998

Incorporation of cortical bone allografts and autografts in rats: expression patterns of mRNAs for the TGF-betas.

Healing of bone grafts is dependent on the rate of new bone formation. To understand better the regulation of new bone formation in the graft we have studied local production of TGF-beta1, 2 and 3, and of the small proteoglycans by determining their mRNA levels in a rat bone graft model. These mRNA levels were compared to the healing rates of autografts and allografts, as determined by histology, UV-microscopic evaluation of tetracycline-labeled new bone formation, microradiography and mechanical testing at 1, 2, 4 and 8 weeks of healing. Analyses showed that, analogous to slower bone formation in allografts, the induction of TGF-beta1 gene expression was slower than in allografts, when compared with autografts. A similar delay was seen in decorin gene expression. The results agree with the suggested role of TGF-beta1 in induction of type I collagen and osteonectin production. Our findings thus support the view that locally produced TGF-beta1 plays a role in normal graft incorporation, while local production of TGF-beta3, and particularly TGF-beta2, may be less important in this respect.

Animals↗
PubMed1997

Developmental regulation of mRNA species for types II, IX and XI collagens during mouse embryogenesis.

Several techniques were used to study the co-ordination of mRNA levels for five constituent chains of cartilage collagen fibrils during mouse development. Short cDNA clones were first constructed for mouse and human alpha3(IX) and for mouse proalpha1(XI) collagen mRNA species. Northern analysis of developing mouse embryos revealed that the mRNA species for alpha1, alpha2 and alpha3 chains of type IX collagen peaked earlier than those for proalpha1(II) and proalpha1(XI) collagen chains. Quantification of these mRNA species by slot-blot hybridization confirmed this developmental regulation: the mRNA ratios for type II/type IX/type XI collagens changed from 5.7:1:0.6 (at embryonic day 12.5) to 10.6:1:0.9 (in newborn mice). However, the genes coding for the three chains of type IX collagen seemed to be under more co-ordinated regulation during mouse development. In addition to high mRNA levels in cartilages and the eye, low levels of type IX collagen transcripts were identified in brain and skin of newborn mouse using RNase protection and reverse transcriptase-PCR assays. Finally, hybridization in situ revealed identical tissue distributions of the three type IX collagen mRNA species during early chondrogenesis but somewhat more widespread expression of the alpha1(IX) and alpha3(IX) mRNA species during endochondral ossification at day 16.5 of embryonic development. These results suggest a relatively tight co-ordination of the alpha1(IX), alpha2(IX), and alpha3(IX) collagen mRNA species in chondrocytes, but a lack of co-ordination in several non-cartilaginous tissues.

Amino Acid Sequence↗
PubMed1997

Abnormal craniofacial growth and early mandibular osteoarthritis in mice harbouring a mutant type II collagen transgene.

Skull morphology and histology in the heterozygous offspring of a transgenic founder mouse Del1, harbouring 6 copies of deletion mutation in Col2a1 gene, were compared with those in normal siblings. On visual observation and roentgenocephalometric examination the heads of heterozygous Del1 mice were smaller than normal. Histologically the sizes of cartilaginous structures of the cranial base were reduced. Severe defects were seen in the temporomandibular joint as progressive osteoarthritic lesions. These observations elucidate the relationship between the genotype and phenotype and demonstrate that heterozygous Del1 mice are a useful model for studies on a genetic disturbance where 'clinical' manifestations are not evident until adult age.

Animals↗
PubMed1997

Localization of type II collagen mRNA isoforms in the developing eyes of normal and transgenic mice with a mutation in type II collagen gene.

PURPOSE: To elucidate the function of type II collagen in the development and diseases of the eye by analyzing the temporospatial expression of the long (IIA) and short (IIB) isoforms of type II collagen in the normal and transgenic Dell mice. METHODS: Normal and Dell transgenic embryos harboring a deletion mutation in the pro alpha 1 (II) collagen chain were studied from day 10.5 of embryonic development up to day 10 postpartum. Northern and in situ hybridizations and RNase protection assays were used to study the developmental and temporospatial expression of type II collagen isoforms. RESULTS: Expression of type II collagen mRNAs was observed at all developmental stages with maximum expression at 16.5 days of embryonic development. RNase protection analyses confirmed that both wild type and transgene-derived mRNAs underwent similar alternative splicing of exon 2 in the eye. By in situ hybridization, both isoforms were observed in the cornea, sclera, vitreous, ganglion cell layer of retina, developing ciliary body-iris, and in the retinal pigment epithelium-Bruch's membrane as well as in the lens and conjunctiva. Differences were observed between eyes of Dell mice and of control subjects in the levels and temporal expression patterns of type II collagen mRNA, which resulted in structural abnormalities in histologic analysis. CONCLUSIONS: Widespread expression of type II collagen mRNAs in ocular structures suggests an important role for type II collagen in structural development of the eye. As the expression patterns observed correspond to structural abnormalities in the eyes of Dell mice, the current results offer a promising basis for further development of mouse models for arthroophthalmopathies.

Abnormalities, Multiple↗
PubMed1997

Mutation in type II collagen gene disturbs spinal development and gene expression patterns in transgenic Del1 mice.

Transgenic Del1 mice harboring a deletion mutation in the cartilage-specific type II collagen gene were used for a systematic study on the dose-dependent effects of this dominant mutation on the embryonic development and growth of the vertebral column. Skeletal staining of homozygous and heterozygous Del1 mice and their nontransgenic littermates with Alcian blue/Alizarin red revealed not only a dose-dependent retardation in the appearance of ossification centers in transgene-positive offspring but also abnormal shapes and proportions of their vertebral columns. Histologic analysis confirmed these findings and demonstrated also retarded removal of the notochord, abnormal shapes and sizes of vertebral bodies and intervertebral discs, and the presence of an occult spina bifida in homozygous Del1 mice. In situ hybridization revealed abnormalities in the expression patterns of type I, II, IX, and X collagens and aggrecan, corresponding to the disorganization of the columnar chondrocyte architecture of the growth zones, increased appositional growth activity along the periphery of the vertebrae, increased numbers of hypertrophic chondrocytes, and development of necrotic areas in the central cartilaginous areas of vertebral bodies of homozygous Del1 embryos. Many of these findings parallel those seen in human chondrodysplasias and help us to understand the pathogenetic mechanisms involved in these developmental abnormalities.

Animals↗
PubMed1996

Evidence for insufficient chondrocytic differentiation during repair of full-thickness defects of articular cartilage.

The main objective of this study was to characterize the cellular phenotypes in the repair tissue of full-thickness defects of articular cartilage by histologic and molecular biologic techniques. Healing of the defects in the articular cartilage of the knee joints of 12 rabbits was analyzed at days 3, 7, 14, 28 and 50 using histology and Northern analysis of mRNA levels for type I, II and III collagens and osteonectin. The cellular source of each mRNA was determined by in situ hybridization. Two novel cDNA clones for rabbit type II and III collagen mRNAs were constructed to obtain species-specific hybridization probes. The repair tissue of full-thickness defects consisted of two types of tissue. At the bottom of the defect, bone-derived cells with high levels of type I collagen and osteonectin mRNA were actively producing new osteoid, while superficially a slow transition from a fibrin clot into undifferentiated mesenchyme with cells containing type III collagen mRNA was observed. This tissue subsequently became fibrocartilaginous, with small groups of cells turning on the transcription of the type II collagen gene and acquiring a phenotype typical for hyaline cartilage. The data suggest that small clusters of cells in the repair tissue of full-thickness articular cartilage defects are capable of turning on an apparently correct chondrocytic phenotype. The low transcription level of the type II collagen gene suggests, however, that insufficient amounts of fundamentally important regulatory factors or progenitor cells are present in the repair tissue. In the future, such factors should be administrable into the joint by novel therapeutic means.

Amino Acid Sequence↗
PubMed1995

Conservation of the sizes of 53 introns and over 100 intronic sequences for the binding of common transcription factors in the human and mouse genes for type II procollagen (COL2A1).

Over 11,000 bp of previously undefined sequences of the human COL2A1 gene were defined. The results made it possible to compare the intron structures of a highly complex gene from man and mouse. Surprisingly, the sizes of the 53 introns of the two genes were highly conserved with a mean difference of 13%. After alignment of the sequences, 69% of the intron sequences were identical. The introns contained consensus sequences for the binding of over 100 different transcription factors that were conserved in the introns of the two genes. The first intron of the gene contained 80 conserved consensus sequences and the remaining 52 introns of the gene contained 106 conserved sequences for the binding of transcription factors. The 5'-end of intron 2 in both genes had a potential for forming a stem loop in RNA transcripts.

Animals↗
PubMed1995

Expression of type VI, IX and XI collagen genes and alternative splicing of type II collagen transcripts in fracture callus tissue in mice.

The levels of six mRNAs coding for constituent alpha-chains of three minor collagens of cartilage were analyzed in an experimental fracture model in normal and transgenic Del1 mice harboring a deletion mutation of exon 7 in the type II collagen gene. Reduced and retarded chondrogenesis in Del1 mice was evident in callus samples as reduced mRNA levels for the cartilage specific type IX and XI collagens at days 7 and 9 of fracture healing. Analysis of the calluses for alternative splicing of pro alpha 1(II) collagen mRNA also suggested retarded chondrogenesis in Del1 calluses. Another developmentally regulated step in limb development, a switch between alternative promoters of the alpha 1(IX) collagen gene, was also seen during fracture healing but was less obvious in Del1 calluses. Finally, the current data suggest that the abnormality in bone remodelling in Del1 mice involves activation of the genes coding for alpha 1(XI) and alpha 2(VI) collagens.

Alternative Splicing↗
PubMed1995

Developmental expression of a type II collagen/beta-galactosidase fusion gene in transgenic mice.

The correct temporal and spatial expression of the type II collagen gene is believed to be important for normal development and growth of the skeleton and the eye, i.e., tissues where the protein product is predominantly found. To study transcriptional activation of type II collagen gene in skeletal and nonskeletal tissues we produced transgenic mice carrying murine proalpha1(II) collagen/beta-galactosidase fusion gene constructs. The expression of the fusion gene was found to depend on the presence of intron 1 deleted failed to reveal any beta-galactosidase activity confirming the important role of regulatory sequences within intron 1 of the gene. High-level expression of the functional construct was clearly confined to cartilaginous tissues but transient low-level expression was also observed in extraskeletal locations, such as the developing brain and the notochord. The results demonstrate that the regulatory elements in the proalpha1(II) collagen/beta-galactosidase fusion gene construct confer both temporal and spatial specificity indistinguishable from that of the endogenous proalpha1(II) collagen gene as determined by the presence of the corresponding mRNA by in situ hybridization. Furthermore the beta-galactosidase activity correlated well with the progression of chondrogenesis as seen by staining of whole mouse embryos with Alizarin red S and Alcian blue in the hybrid mouse strain used for microinjections. The transgenic mouse line produced should prove useful for studies on various aspects of chondrogenesis. Furthermore, the data shows that the regulatory elements present in the construct are sufficient for targetting the expression of other genes in cartilage.

Animals↗
PubMed1995

Coordinate patterns of expression of type I and III collagens during mouse development.

The extracellular proteins types I and III collagen are abundantly expressed during development. Here, the patterns of the pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen mRNAs are systematically examined from 7.5 to 17.5 days of development (E7.5 to E17.5) in the mouse using in situ hybridization with specific riboprobes. Coordinated expression of pro alpha 1(I) and pro alpha 2(I) collagen mRNA was found throughout development in all regions examined. Widespread type I collagen expression starting at E8.5 occurred in embryonic mesoderm, sclerotomes, dermatomes, and in the forming connective tissues. After E14.5, regions of ossification showed highest levels of type I collagen expression. Pro alpha 1(III) collagen expression was specific to and coordinated with patterns of type I collagen expression in many fibroblast-containing tissues. No expression of type III collagen occurred in osteoblasts. This comprehensive study of the transcripts of abundantly expressed structural proteins should provide a basis for comparison of other key extracellular matrix molecules and serve as a reference for studies on the patterns of activities of various promoter/enhancer-reporter gene constructions of type I and III collagen genes in transgenic mice.

Animals↗
PubMed1995

Transgenic mice with deficiencies in cartilage collagens: possible models for gene therapy.

We address three issues that are important when considering somatic gene therapy approaches to osteoarthritis (OA) and related syndromes. First, only those diseases for which a precise molecular etiology has been established should be contemplated for somatic gene therapy. Second, DNA sequences should be identified that restrict expression of correcting genes to chondrocytes; we discuss the use of transgenic mice to identify such sequences. Third, we emphasize the usefulness of establishing animal models that mimic human OA syndromes by genetic manipulations. These transgenic models should be essential for testing gene therapy approaches in vivo.

Animals↗
PubMed1994

The exon structure of the mouse alpha 2(IX) collagen gene shows unexpected divergence from the chick gene.

One cosmid and two overlapping phage clones covering the entire mouse alpha 2(IX) collagen gene including 12 kilobase pairs (kb) of 5'- and 8 kb of 3'-flanking sequences were isolated from two genomic libraries. The overall gene structure was determined by restriction mapping and nucleotide sequencing. The gene spans 16 kb from the start of transcription to the polyadenylation site and contains 32 exons. It codes for a mRNA of 3 kb that translates into a polypeptide of 688 amino acids. The intron-exon junctions and mRNA structure were confirmed by amplification of cDNA made for mouse cartilage RNA. The coding sequence of the mouse alpha 2(IX) collagen gene shows marked similarities to those for other type IX collagen chains. Although the overall exon-intron organization of the mouse gene is very similar to the chick alpha 2(IX) gene, some unexpected differences were observed at the splice junctions. Split codons characteristic for the central triple helical domain of the chick were not found in the mouse gene that thus exhibited a long stretch of exons with sizes that are multiples of 9 base pairs in this domain. The promoter of the mouse alpha 2(IX) collagen gene contains some G + C-rich elements including three Sp1 consensus recognition sites and a far upstream CCAAT box but no TATAA box. Both primer extension and RNase protection assays revealed several transcription start sites within 418 base pairs of the promoter. The present study reports the first complete nucleotide sequence of any type IX collagen gene and forms the basis for comparative structural studies on this collagen type and for experiments involving transgenic mice.

Amino Acid Sequence↗
PubMed1994

Retarded chondrogenesis in transgenic mice with a type II collagen defect results in fracture healing abnormalities.

We have examined the biological and biomechanical consequences of defective type II collagen production for fracture repair employing a genetically engineered mouse line Del1 which was generated by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7 (Metsäranta et al. [1992] J. Cell Biol. 118:203-212). Standardized tibial fractures were produced in transgenic Del1 mice and their nontransgenic littermates were used as controls. The fracture callus tissues were analyzed at days 7, 9, 14, 28, and 42 using radiography, histomorphometry, biomechanical testing, and Northern analysis of mRNAs for several tissue-specific matrix components. Deficient production of cartilage in Del1 mice resulted in reduced radiographic callus size, smaller cross-sectional area, and impaired biomechanical properties when compared with fractures of nontransgenic control mice. The differences were most evident in 14-day fracture calluses. Consequently mRNAs for cartilage-specific type IX and X collagens and aggrecan were also reduced in Del1 calluses. Levels of type II collagen mRNAs were unaffected since the mutated transgene produced additional type II collagen mRNA molecules. Further abnormalities in the fracture repair process of Del1 mice were observed in callus remodeling. In the control animals a typical feature of external callus remodeling was reduction of callus size during endochondral ossification between days 14 and 28. Such reduction was not observed in the transgenic mice. Histological examination of fracture calluses suggested also a reduction in trabecular surface area, which was found to be even more pronounced in metaphyseal bone of Del1 mice. Despite these differences the biomechanical properties of the calluses in the two groups became similar by day 28 of fracture healing. The results thus suggest that reduced chondrogenesis due to the presence of mutated transgenes in Del1 mice not only causes a temporary impairment in biomechanical properties of healing fractures but also affects later stages of callus remodeling.

Animals↗
PubMed1994

Characterization of primary cultures of chondrocytes from type II collagen/beta-galactosidase transgenic mice.

Studies on the function of extracellular matrix components of cartilages and on chondrocyte-specific regulatory mechanisms will benefit from approaches in which transgenic mice and cell cultures will complement each other. We therefore established and extensively characterized primary cultures of mouse chondrocytes isolated from rib growth plates of newborn mice harboring a transgene in which type II collagen gene regulatory sequences were driving expression of an E. coli beta-galactosidase reporter gene. Primary chondrocytes expressed a fully differentiated phenotype in monolayer culture, producing mRNAs for the collagen types II, IX and X, and for the transgene. Transgenic cells also synthesized high levels of E. coli beta-galactosidase, easily quantifiable and also detectable in individual cells by X-gal staining. When chondrocytes were isolated from transgenic mice in which beta-galactosidase was fused to the product of the neomycin resistance gene, they displayed resistance to G418. After one to two weeks in culture, chondrocytes progressively lost expression of the transgenes, in parallel with that of cartilage-specific genes, and started expressing high levels of type I collagen RNA. The use of transgenic chondrocytes allowed us to easily score phenotypic changes by assaying beta-galactosidase activity and neomycin resistance. Cultures of mouse chondrocytes, such as those reported here, should also help characterize biochemically the phenotypes of other transgenic mice in studies of genetic diseases of cartilages and of mechanisms involved in chondrogenesis.

Animals↗
PubMed1994

Type II collagen mutations in rare and common cartilage diseases.

Cartilage diseases include a wide variety of clinical phenotypes from common osteoarthrosis to several different types of chondrodysplasias, i.e. 'disorders of cartilage', of which more than 100 different have been described. Patients frequently suffer from various symptoms affecting their joints and/or the growth of their long bones. The amount of hyaline cartilage at articular surfaces is often diminished and structurally abnormal. The surface of the cartilage may have an irregular appearance with defects extending into the subchondral bone. The major constituents of this hyaline cartilage are collagens and proteoglycans, the most abundant protein being type II collagen. It is a homotrimer of three identical alpha-chains, which are encoded by a single gene on human chromosome 12. The gene for type II collagen therefore became a likely candidate for some forms of chondrodysplasias and cartilage degeneration. Recently, both linkages and exclusions between this gene and various cartilage diseases have been reported and a growing number of mutations within the gene have also been identified.

Animals↗