The osteoprogenitor lineage provides a renewing source of osteoblast for embryogenesis, somatic growth, bone fracture repair and states of estrogen deficiency. Many diseases of bone can be viewed as a failure of the lineage to provide sufficient numbers of functional to meet the mechanical load place on the skeleton. This resource development application is designed to implement a comprehensive strategy for recognizing stages of differentiation within the lineage and to appreciate the RNA expression profile in relatively homogeneous population of cells at defined levels of differentiation. There are nine elements of the grant submitted by a complementary group of cell and molecular, mouse geneticists, computational engineers and computer scientists. The project is based on the use of promoter-GFP transgenes that active at defined stages within the osteoblast lineage. This allows the lineage to be characterized in vitro (project 1) or in vivo (project 2). Both approaches use a computer controlled microscopic work station that allows repetitive images to be recorded in tissue culture or histological section. Digital analysis of the images will allow the lineage to be defined in quantitative terms. A bone focused microarray library will be assembled (project 3) so that informative expression studies can be performed of GFP positive population of cells isolated by FAC sorting (project 1). This library will be annotated with particular reference to molecular pathway and its relationship to bone biology (project 4). Data from a microarray study will be tested for statistical significance prior to being place into standard and newly developed clustering algorithms (project 5). A second feature of the strategy is that all the murine models will be assessed in a uniformly heterozygous environment found in F1 mice of C57/B16 and C3H mice. Project 6 provides the discipline to build the congenic inbred lines from which the testor F1 mice will be produced. The applicability of the reagents and protocols for general use by bone biologists will be evaluated by 3 investigators at the Health Center using murine models that perturb the osteoprogenitor pathway. The data that is generated from control and mutant mouse models will be organized in a cartoon based query database (project 8). The strategies, reagents and protocols that prove to be valuable to defining lineage performance and molecular pathways will be accessible from a web interface.
