Individual osteoblasts in the developing calvaria express different gene repertoires

Several studies in vitro and a few in vivo have suggested that mature osteoblasts heterogeneously express osteoblast markers. In one recent study of the osteoblasts associated with bone nodules formed in vitro in rat calvaria cell populations, extensive diversity was documented in the overall gene repertoires expressed. To address whether comparable heterogeneity is evident in vivo, we investigated the expression of nine osteoblast lineage markers by both in situ hybridization and immunohistochemistry. At 21 days of fetal rat development, the calvaria is a rapidly growing bone with distinct maturational zones that are readily observed in coronal sections; that is, an osteogenic front emerging at sagittal and coronal sutures is adjacent to areas of growing trabeculae of bone, followed by more mature areas of remodeling bone. Based on expression patterns, markers can be divided into two categories. One category comprises markers that are globally expressed by all osteoblasts irrespective of their position in the calvaria. Of those tested, only two, alkaline phosphatase and the pth/pthrp receptor, fit into this category. All other markers analyzed, including transcription factors (c-fos and msx-2), matrix molecules (bone sialoprotein, osteopontin, and osteocalcin), and a hormone (pthrp), were differentially expressed only in subpopulations of osteoblasts, based on cell maturational status, environment (ectocranial vs. endocranial surfaces), and microenvironment (adjacent osteoblasts). Preosteoblasts and osteocytes in different regions of the calvaria also expressed different subsets of the lineage markers. Mechanisms responsible for generating differential gene expression profiles appear to be both transcriptional and posttranscriptional. These results indicate that postproliferative, morphologically indistinguishable osteoblasts are not a homogeneous class of cells, but instead are molecularly diverse. The present results also raise the possibility that lineage progression and/or maintenance of the differentiated state may be adaptable in the calvaria.