Expression of the fibrillin gene family in the development, differentiation and maintenance of mesenchyme cell types
Davis, Margaret Rose
Connective tissue initially arises from embryonic mesenchymal stem cells (MSC) that originate from the mesoderm during embryogenesis and are capable of differentiating into connective tissue lineages such as adipocytes, osteoblasts, chondrocytes and fibroblasts. Connective tissue is composed of cells held together by the extracellular matrix (ECM). The fibrillins and latent transforming growth factor binding proteins form a superfamily of ECM proteins characterised by the presence of a unique domain, the 8- cysteine transforming growth factor beta binding domain (TGFß). The fibrillin proteins (fibrillin-1, fibrillin-2 and fibrillin-3 in most vertebrates, encoded by the FBN1, FBN2 and FBN3 genes respectively), are major components of the 10nM microfibrils found in ECM of many tissue types, for example mesenchyme-derived connective tissues. Fibrillin-1 and fibrillin-2 are also thought to be required for stabilization and storage of latent TGFβ complexes. Mutations in FBN1 cause Marfan syndrome, a connective tissue disorder characterised by abnormalities in the microfibrils resulting in musculoskeletal, ocular, cardiovascular and other complications. FBN2 mutations lead to congenital contractural arachnodactyly, which has a musculoskeletal phenotype similar to Marfan syndrome. There are currently no known diseases associated with FBN3 mutations. In this project, the expression of fibrillins was investigated using human cell lines during early development, mesenchymal stem cell differentiation and in further differentiated mesenchymal cell lines, for example in osteocytes (osteosarcomas), chondrocytes and fibroblast lineage. Immunocytochemistry was used to examine protein expression, real-time PCR and expression microarrays to determine mRNA synthesis and RNAi suppression of gene expression to determine possible functions of fibrillins and associated ECM proteins. In addition, a genome wide bioinformatics evaluation was performed of transcription start sites for the fibrillin gene family utilising the information obtained from the FANTOM5 consortium. The three fibrillin genes showed differing expression patterns in cell lines depending on the stage of development/differentiation. During embryogenesis, expression of FBN3, FBN2 and FBN1 increased sequentially in that order. Expression of FBN3 followed the same pattern as expression of known pluripotency markers, while expression of FBN2 correlated with expression of markers for later stages of mesoderm differentiation. FBN1 expression was associated with mesenchymal markers, and this was supported by a study of mesenchymal stem cells differentiation to the adipose lineage. Fibrillin-1 microfibrils and RNA expression were present early in primary adult human MSC differentiating to adipocytes, suggesting that a fibrillin matrix is required for initial MSC attachment. As differentiation proceeded, fibrillin -1 expression decreased, with rapid degradation of the microfibrils. Fibrillin-2 expression increased following differentiation and fibrillin-3 was not expressed. These results suggest that fibrillin-1 plays an important structural and regulatory role in the early stages of connective tissue development but is not required to maintain the differentiated state. Many genes showed the same expression pattern as FBN1. To better understand the importance of fibrillin-1 and its interaction with these coexpressing genes, fibrillin-1 was knocked down using siRNA in fibroblast, chondrocyte and osteosarcoma cell lines. There were little to no effects identified in chondrocyte and osteosarcoma cell lines, and only a few genes were altered following the reduction of fibrillin-1 mRNA in fibroblasts, suggesting that fibrillin-1 is not a central regulator but an endpoint. This was surprising given its potential role in controlling bioavailability of TGFβ, a key regulator of mesenchymal cells. In addition, the evolution of the fibrillin gene family was studied and it was found that the gene structure, amino acid sequence and genomic positions of each gene are widely conserved across vertebrates, suggesting an important role in vertebrate body structure. However, the differences in gene structure and sequence between the three fibrillin genes suggest divergent function. Fibrillin-1 mutations with the most severe phenotypes are located in regions that are highly conserved. This study shows that there is a clear developmental and evolutionary distinction between the three fibrillins. Fibrillin-3 was associated with pluripotency and its presence in differentiating foetal liver and brain may suggest that there are residual pluripotent cells in these developing tissues. Fibrillin-2 appeared to be a marker for the mesodermal stage and its role in adult cells is currently not clear. Fibrillin-1 was present in cells already predetermined to go to mesenchymal lineages, but it was minimal in the advanced stages of differentiation suggesting that it may be a marker for relatively plastic mesenchymal cells prior to commitment to a specific lineage. These results will assist in the understanding of disorders resulting from fibrillin gene mutations and have identified coexpressed proteins, potential modifiers that could be the targets of gene therapy and candidates for similar connective tissue.