Gene expression during hard tissue formation

Abstract

The purpose of this thesis was to study the formation of the extracellular matrix (ECM) of bone and dentin during development. Molecular techniques, such as in situ hybridization and RT-PCR, were used to investigate the occurrence of specific gene products in formative cells. The fibrillar collagen type I is the major extracellular component in both bone and dentin. According to Northern blots, the [alpha]1(I) mRNA chain is composed of two variants. The complete sequences of the [alpha]1(I) chains were established and it was found that these differ in the untranslated regions only.

The expression patterns of the two variants were compared in the hard tissue forming cells of growing rat maxilla and they were found to be expressed at all developmental stages and in all different cell types studied. The mRNA expression of the fibril collagens, types I, II, III, V and XI was compared during formation of the premaxillary-maxillary suture region, an area of intramembranous bone formation. A specific temporospacial expression pattern was found, which could be correlated to collagen fiber construction and tissue segregation. Type III mRNA was expressed in the soft tissues of the suture and periosteum, types V and XI mRNA occurred only transiently in young osteoblasts at the border between soft and hard tissue, while type I transcript was detected in all osteoblasts, but with different signal strength depending on the localization of the cell. Type II mRNA was not detected in the suture region. In contrast, no sutures were formed and no collagen type III mRNA expression took place during the growth of the alveolar bone. Instead, collagen H mRNA was expressed in islands of cells, which later developed into cartilage domains, and which were interspersed between osteoblasts synthesizing collagen type I mRNA, and bone trabeculae. It is proposed that collagen type II and cartilage are instrumental for bone growth and allow bone expansion, functionally replacing collagen type III and sutures.

One of the major non-collagenous extracellular matrix components in both bone and teeth is osteonectin (ON). The sequence of the rat ON mRNA was established and the expression pattern of its mRNA was mapped and found to be similar to the pattern obtained with collagen [alpha]1(I). It seems likely that ON is synthesized concomitantly with the fibrillar collagen proteins and is a component of the ECM. Ameloblasts, associated with mineralizing enamel did not express ON mRNA, thus presence of ON mRNA and the deposition of mineralizing enamel are not correlated. Fibronectin (FN) is also produced by osteoblasts and odontoblasts and is known to be involved in cell attachment but can also bind to e.g. collagen. At three different positions, the transcript can e alternatively spliced, presumably to allow for variable functions of FN. The splicing pattern in bone and teeth cells was investigated. Osteoblasts omitted and odontoblasts used exon EM, a difference that could be related to specific modes of cell-matrix organization.