Age-dependent changes in the expression of matrix components in the mouse eye.

Although the presence of 'cartilage-specific' collagens in the eye has been documented earlier, very little is known about their synthesis rates during ocular development, growth and aging. The purpose of the present study was to follow changes in the mRNA levels and distribution of key components of the extracellular matrix in the eyes of normal and transgenic Del1 mice, harboring a short deletion mutation in the type II collagen gene, during ocular growth and aging. Total RNAs extracted from mouse eyes were studied by Northern analysis for mRNA levels of type I, II, III, VI, IX and XI collagens, biglycan, fibromodulin and decorin. A predominant finding of the present study was the marked reduction in the mRNA levels of type I and II collagens in the eye upon aging. The changes in the mRNA levels of type III and VI collagen and proteoglycans were smaller. Localization of type II and IX collagen in the eye was performed by immunohistochemistry. Despite the reduction in the type II collagen mRNA levels, immunohistochemistry confirmed widespread distribution of the protein also in aging mouse eyes, suggesting its slow turnover. Although the Del1 mutation caused gradual degenerative lesions in the eyes, the distribution of the protein remained essentially unchanged. The widespread distribution and marked downregulation of type II collagen production in the mouse eye upon aging probably explain the gradual development of degenerative lesions, particularly in the eyes of transgenic Del1 mice, where production of mutant type II collagen chains also contributes to the process.

Type XIII collagen is widely expressed in the adult and developing human eye and accentuated in the ciliary muscle, the optic nerve and the neural retina.

The distribution of mRNAs coding for type XIII collagen, a novel nonfibril-forming collagen, was studied by Northern and in situ hybridizations of adult and fetal human eyes and the corresponding protein was localized by indirect immunofluorescence in frozen sections of 12 and 17 week human fetal eyes using a polyclonal antipeptide antibody to type XIII collagen. Type XIII collagen was found to be widely expressed in ocular tissues when studied at both the mRNA and protein levels in fetal and adult human tissues. No major differences were observed in the expression patterns between fetal and adult tissues. Surprisingly, the strongest signals seen in in situ hybridizations and immunofluorescence stainings occurred in the optic nerve bundles and in the ganglion cell layer of the retina. Other notable locations containing type XIII collagen included the developing ciliary smooth muscle, the posterior two-thirds of the corneal stroma and the striated extraocular muscles. Low level signals were also detected in the blood vessel walls and mesenchymal cells of the other ocular tissues. All immunosignals detected were adherent to cells, and the extracellular matrices appeared to be devoid of type XIII collagen. Our results are in concert with the presumed plasma membrane location of type XIII collagen, and it is hypothesized that this molecule could be involved in cell-matrix and perhaps cell-cell interactions. The wide expression of type XIII collagen in the eye, and especially in the neural structures, warrants future studies on type XIII collagen in other nerve structures and in pathological conditions affecting the eye. Due to its wide expression, type XIII collagen is likely to be an important factor for the normal development and functioning of the eye.

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.

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.

Ocular abnormalities in transgenic mice harboring mutations in the type II collagen gene.

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 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.