Regulation of alkaline phosphatase and alpha 2(I) procollagen synthesis during early intramembranous bone formation in the rat mandible.

We have studied intramembranous bone formation in the developing rat mandible. In this system discrete developmental stages can be readily distinguished: mesenchymal condensation, osteoid deposition, and mineralization. In mandibles of 14-day rat embryos avascular condensed mesenchymal cells can be discerned in a region lateral to Meckel's cartilage and anterior to the first molar bud. In 18-day embryos primary bone structures with mineral deposition are evident, and at 2 days postnatally the mandible is extensively mineralized. In the developing mandible we investigated the pattern of bone/liver/kidney/placenta (BLKP) alkaline phosphatase (ALP) and alpha 2(I) procollagen expression in the differentiating osteoblasts. The level of ALP activity in loose mesenchymal tissue is close to background levels. In contrast, the condensed mesenchymal cells in 14-day embryos, which will subsequently form bone, display intense ALP activity prior to discernible osteoid or mineral deposition. ALP activity in the condensed mesenchymal cells can be inhibited by levamisole, indicating activity of the BLKP gene product. We could not detect a corresponding increase in transcript level for either ALP or alpha 2(I) in the condensed mesenchyme in 14-day embryo using in situ hybridization, probably due to low message abundance. At 18 days, cells throughout the developing mandible express ALP activity, and intense in situ hybridization to BLKP ALP probes is evident in cells lining the developing bone trabeculae. Alpha 2(I) procollagen transcripts have accumulated in cells of the developing mandibular bone, but are not specifically localized to osteoblastic cells. Our results demonstrate that ALP activity is a very early marker of differentiation of cells of the osteogenic lineage, since a marked increase in ALP enzyme activity is clearly detectable in condensed mesenchymal cells prior to osteoid or mineral deposition. In contrast, Wright and Leblond, using the same model system and immunohistochemistry, could not localize type I collagen to preosteoblastic cells surrounding the developing bone trabeculae, and demonstrated localization of type I collagen to osteoblasts bordering developing trabeculae, indicating a substantial increase in type I collagen expression (at least at the protein level) during preosteoblast to osteoblast differentiation. These results indicate a discrete pattern of regulation for both the ALP and alpha 2(I) genes during osteogenic differentiation, which may involve both transcriptional and posttranscriptional regulation.