Osteocytes are terminally differentiated cells derived from osteoblasts that become entrapped in their own matrix. Osteocytes survive by establishing a mutually connected canalicular network of dendritic processes by which they communicate with other osteocytes and other osteoblast lineage cells. It is well known that bone tissue has the capacity to alter its mass and structure in response to mechanical strain using osteocytes as mechanosensory cells. Identification of genes involved in these processes requires isolation of a pure population of osteocytes and a comparison to the gene expression profile to cells at other maturation stages of the osteoblast lineage. In this project we propose to evaluate the gene expression profile of an isolated osteocyte population and contrast its gene expression to osteoblasts as they transition into the osteocyte stage. A defined model of mouse calvarial osteoblast differentiation during the process of intramembranous ossification will be utilized. Cells of all maturation stages will be isolated using a sequential enzymatic digestion of parietal bones derived from mice harboring GFPtopaz driven by the dentin matrix protein 1 (DMP-1) promoter. This promoter has been shown to drive the expression of GFP to cells that are becoming entrapped in the matrix (preosteocytes) and in the osteocytes. To separate osteocytes from osteoblasts, DMP1-tpz transgenic mice (osteocyte specific marker) will be crossed with pOBCol2.3GFP-saphirre mice (transgene active in osteoblasts and osteocytes). Enzymatically released cells from calvaria will be fluorescence-sorted to obtain isolated populations of osteoblasts and osteocytes on the basis of the two-color system. In addition to gene expression analysis, this study will address the role of genes that are highly expressed at the osteocyte stage and have the potential to play an important role in osteocyte biology. The analysis of their gene expression profile will provide important information for a better understanding of the mechanisms that regulate bone mass. We will evaluate the effects of retroviral overexpression or downregulation of genes of interest by siRNA. The results of this study will generate the basis for a larger proposal that will include testing the in vivo effects of deficiency or overexpression of particular genes identified by this analysis.
