The skeleton is able to grow and adapt to mechanical loads by calling on the osteoprogenitor lineage to produce osteoblasts capable of making a mineralizing matrix. Following the paradigm of the hematopoietic field in which anemia is viewed as a defect in the lineage producing mature red ceils, we have developed a series of promoter-GFP reporter transgenic mice which can be used to assess lineage progression both in primary culture and intact mouse bone in response to a perturbation by a hormone/growth factor or a mutation in a genetic pathway controlling bone mass. This renewal builds of the success of this approach by expanding on the number and breadth of differentiation-related GFP reporter constructs. A new type of reporter that is responsive to the transcriptional environment of the cell will also be produced. A combination of 3-4 distinguishable reporter colors will allow simultaneous recognition of multiple levels of differentiation. Quantitative measurement of lineage performance in primary culture and an greatly improve method for visualizing GFP reporters in section of adult mouse bone provide the opportunity to relate studies performed in vitro to the ability of the lineage to maintain bone mass in vivo. The utility of these new reagents to develop molecular pathways controlling lineage maturation will be explored. First, cells at defined levels of differentiation will be isolated by FACS sorting and subjected to cDNA microarray analysis using a bone-focused gene collection. Second, the consequences of an under-active canonical wnt pathway to lineage progression will be analyzed in the LRP5 knock out mouse. Microarray studies will be focused on the cell population within the developing lineage that first manifests the effect of the mutation so that genes immediately downstream of the pathway can be discerned from differences resulting from a failure to acquire full osteoblast maturation. Third, two congenic inbred lines of mice developed at the Jackson Laboratory as a quantitative trait affecting cortical bone thickness will be analyzed. A trait that is associated with an intrinsic alteration in lineage performance will undergo microarray studies to identify genes within the genetic interval with altered expression in the cells that reflect the influence of the congenic interval. To support the possibility that altered expressions of a specific RNA might be responsible for the change in lineage progression, the level of the candidate RNA will be experimentally altered in control cells by delivery of an RNAi or cDNA construct using a lentiviral vector system to determine if the change in lineage progression can be reproduced or rescued. The concepts that will be developed and evaluated in this proposal should be effective in the study of a genetic influence controlling bone mass and its response to the environment.
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