Organization and characterization of fibrillar collagens in fish scales in situ and in vitro

Abstract

The characterization of the fibrillar collagens and the cellular control of their spatial deposition were studied in fish scales using immunofluorescence, electron microscopy, electrophoretic and HPLC analyses, immunoprecipitation and hybridization with cDNA probes. This study was carried out on undisturbed and regenerating scales in situ and in organ and cell cultures from regenerating scales. The hyposquamal scleroblasts forming a pseudoepithelium show an apico-basal polarization and synthesize thick collagen fibrils (100 nm) organized in a plywood pattern as long as the integrity of the cell-cell and cell-collagenous matrix contacts are preserved. In culture, scleroblasts become fibroblastlike and produce an unordered meshwork of thin collagen fibrils (30 nm). Comparison of the synthesized collagens in culture with those extracted from the scales indicates that culture conditions modify fibrillogenesis but do not change the expression of fibrillar collagen genes. Type I collagen, the predominent component, is associated with the minor type V collagen. Type III collagen was not present. In type I collagen, a third chain, α3 chain, was identified. The ratio between the 3 chains suggests the coexistence of two heterotrimers (α(I))2 α2(I) and αl(I) α2(I) α3(I). Analysis by HPLC and electrophoresis of the cyanogen bromide-derived peptides obtained from the purified a3 chain support the hypothesis that α(I) and α3(I) chains are encoded by two different genes. The presence of the two types of heterotrimers in vivo as well as in vitro could correspond to an innate property of the goldfish scleroblasts. Despite the fact that teleost cyanogen bromide-derived peptides differ from those of higher vertebrates, homologies with the mammalian collagen genes (human, for example) are sufficient to allow the detection of mRNA transcripts for αl(I), α2(I) and α2(V) from confluent scleroblast cultures with human probes.