Spatio-temporal expression and distribution of collagen VI during zebrafish development.

Collagen VI (ColVI) is an extracellular matrix (ECM) protein involved in a range of physiological and pathological conditions. Zebrafish (Danio rerio) is a powerful model organism for studying vertebrate development and for in vivo analysis of tissue patterning. Here, we performed a thorough characterization of ColVI gene and protein expression in zebrafish during development and adult life. Bioinformatics analyses confirmed that zebrafish genome contains single genes encoding for α1(VI), α2(VI) and α3(VI) ColVI chains and duplicated genes encoding for α4(VI) chains. At 1 day post-fertilization (dpf) ColVI transcripts are expressed in myotomes, pectoral fin buds and developing epidermis, while from 2 dpf abundant transcript levels are present in myosepta, pectoral fins, axial vasculature, gut and craniofacial cartilage elements. Using newly generated polyclonal antibodies against zebrafish α1(VI) protein, we found that ColVI deposition in adult fish delineates distinct domains in the ECM of several organs, including cartilage, eye, skin, spleen and skeletal muscle. Altogether, these data provide the first detailed characterization of ColVI expression and ECM deposition in zebrafish, thus paving the way for further functional studies in this species.

Three novel collagen VI chains, alpha4(VI), alpha5(VI), and alpha6(VI).

We report the identification of three new collagen VI genes at a single locus on human chromosome 3q22.1. The three new genes are COL6A4, COL6A5, and COL6A6 that encode the alpha4(VI), alpha5(VI), and alpha6(VI) chains. In humans, the COL6A4 gene has been disrupted by a chromosome break. Each of the three new collagen chains contains a 336-amino acid triple helix flanked by seven N-terminal von Willebrand factor A-like domains and two (alpha4 and alpha6 chains) or three (alpha5 chain) C-terminal von Willebrand factor A-like domains. In humans, mRNA expression of COL6A5 is restricted to a few tissues, including lung, testis, and colon. In contrast, the COL6A6 gene is expressed in a wide range of fetal and adult tissues, including lung, kidney, liver, spleen, thymus, heart, and skeletal muscle. Antibodies to the alpha6(VI) chain stained the extracellular matrix of human skeletal and cardiac muscle, lung, and the territorial matrix of articular cartilage. In cell transfection and immunoprecipitation experiments, mouse alpha4(VI)N6-C2 chain co-assembled with endogenous alpha1(VI) and alpha2(VI) chains to form trimeric collagen VI molecules that were secreted from the cell. In contrast, alpha5(VI)N5-C1 and alpha6(VI)N6-C2 chains did not assemble with alpha1(VI) and alpha2(VI) chains and accumulated intracellularly. We conclude that the alpha4(VI)N6-C2 chain contains all the elements necessary for trimerization with alpha1(VI) and alpha2(VI). In summary, the discovery of three additional collagen VI chains doubles the collagen VI family and adds a layer of complexity to collagen VI assembly and function in the extracellular matrix.