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Overlapping genomic clones covering the 7.2 kb mouse alpha 1(X) collagen gene, 0.86 kb of promoter and 1.25 kb of 3′-flanking sequences were isolated from two genomic libraries and characterized by nucleotide sequencing. Typical features of the gene include a unique three-exon structure, similar to that in the chick gene, with the entire triple-helical domain of 463 amino acids coded by a single large exon. The highest degree of amino acid and nucleotide sequence conservation was seen in the coding region for the collagenous and C-terminal non-collagenous domains between the mouse and known chick, bovine and human collagen type X sequences. More divergence between the sequences occurred in the N-terminal non-collagenous domain. Similarity between the mammalian collagen X sequences extended into the 3′-untranslated sequence, particularly near the polyadenylation site. The promoter of the mouse collagen X gene was found to contain two TATAA boxes 159 bp apart; primer extension analyses of the transcription start site revealed that both were functional. The promoter has an unusual structure with a very low G + C content of 28% between positions -220 and -1 of the upstream transcription start site. Northern and in situ hybridization analyses confirmed that the expression of the alpha 1(X) collagen gene is restricted to hypertrophic chondrocytes in tissues undergoing endochondral calcification. The detailed sequence information of the gene is useful for studies on the promoter activity of the gene and for generation of transgenic mice.

Transcription of the type-II procollagen gene (COL2A1) is very specifically restricted to a limited number of tissues, particularly cartilages. In order to identify transcription-control motifs we have sequenced the promoter region and the first intron of the human and mouse COL2A1 genes. With the assumption that these motifs should be well conserved during evolution, we have searched for potential elements important for the tissue-specific transcription of the COL2A1 gene by aligning the two sequences with each other and with the available rat type-II procollagen sequence for the promoter. With this approach we could identify specific evolutionarily well-conserved motifs in the promoter area. On the other hand, several suggested regulatory elements in the promoter region did not show evolutionary conservation. In the middle of the first intron we found a cluster of well-conserved transcription-control elements and we conclude that these conserved motifs most probably possess a significant function in the control of the tissue-specific transcription of the COL2A1 gene. We also describe locations of additional, highly conserved nucleotide stretches, which are good candidate regions in the search for binding sites of yet-uncharacterized cartilage-specific transcription regulators of the COL2A1 gene.

Cartilage collagen fibrils, which are characterized by their thin, uniform diameters, are formed of a multicomponent system of three collagen types (II, IX, and XI) and interacting proteoglycans. We have used a genetic approach to test whether the proper assembly of this multiprotein structure was altered by overexpression of one of its normal components. Here we show that in transgenic mice in which the normal mouse alpha 1(II) collagen is overexpressed, thick abnormal collagen fibrils are generated. Mice that showed the highest expression of the transgene also displayed a larger proportion of abnormal fibrils and died at birth. We propose that an imbalance among the constituents of the cartilage collagen fibrils disrupts the mechanism that controls their assembly. The results show the applicability of the transgenic mice system to studies of complex multicomponent protein assemblies in intact animals.

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.

Purpose. To characterize the morphological changes in the eyes of transgenic mice harboring different mutations in type II collagen gene to elucidate the function of this collagen in the eye, and to find out whether these animals could function as models for the human arthro-ophthalmopathies of the Kniest, Stickler and Wagner types.Methods. Three genetically engineered mouse lines representing two types of mutations in the triple-helical domain of type II collagen and their nontransgenic littermates used as controls were analyzed on day 18.5 of embryonic development. After genotyping by polymerase chain reaction (PCR) and Southern hybridization the embryos were prepared for routine histology. Polarization microscopy was done on hyaluronidase-treated sections.Results. Histological analysis revealed several genotype-dependent abnormalities in the eyes of the transgenic mice. Most striking changes were observed in the vitreous architecture; in one line of mice the vitreous was tightly packed in the posterior region of the vitreous space with thick fibrils, empty cavities and dense membrane-like material. The other mutation resulted in reduced filament density of the vitreous. In the most severely affected phenotype the internal limiting membrane was detached from the retinal layers and was markedly thickened, and the posterior lens capsule was thickened. The anterior chamber was shallow or absent in all transgenic lines but was well formed in the normal animals. Changes were also observed in the lens, corneal and scleral structures.Conclusions. The ocular changes observed in transgenic mice harboring mutations in type II collagen gene show similarities to the human ocular findings in Kniest dysplasia, and in Stickler and Wagner syndromes. We therefore propose that these animals could serve as models for systematic analysis of vitreoretinal degeneration and other abnormalities, as seen in these syndromes.

The extracellular proteins types I and III collagen are abundantly expressed during development. Here, the patterns of the proα1(I), proα2(I), and proα1(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 proα1(I) and proα2(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. Proα1(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.

We have generated transgenic mice by microinjection of a 39-kb mouse pro alpha 1(II) collagen gene construct containing a deletion of exon 7 and intron 7. This mutation was expected to disturb the assembly and processing of the homotrimeric type II collagen molecule in cartilage. Expression of transgene mRNA at levels equivalent or higher than the endogenous mRNA in the offspring of two founder animals resulted in a severe chondrodysplastic phenotype with short limbs, hypoplastic thorax, abnormal craniofacial development, and other skeletal deformities. The affected pups died at birth due to respiratory distress. Light microscopy of epiphyseal growth plates of transgenic pups demonstrated a marked reduction in cartilaginous extracellular matrix and disruption of the normal organization of the growth plate. The zone of proliferating chondrocytes was greatly reduced whereas the zone of hypertrophic chondrocytes was markedly increased extending deep into the diaphysis suggestive of a defect in endochondral ossification. Electron microscopic examination revealed chondrocytes with extended RER, a very severe reduction in the amount of cartilage collagen fibrils, and abnormalities in their structure. We postulate that the deletion in the alpha 1(II) collagen acts as a dominant negative mutation disrupting the assembly and secretion of type II collagen molecules. The consequences of the mutation include interference with normal endochondral ossification. These mice constitute a valuable model to study the mechanisms underlying human chondrodysplasias and normal bone formation.