Francesca V. Mariani - US grants

The ability to manipulate the mouse genome via ES cell technology has had a tremendous impact on our ability to identify the genes required for the development of mammalian embryos. Much is still to be understood about the cell-cell interactions necessary to establish cell lineages and to organize tissues. We propose to develop a binary transgenic system based on Cre/lox technology for ablating specific cell populations in mice. Using this system it will be possible to produce embryos with virtually any cell type ablated by mating two phenotypically normal mouse lines. One line (the "universal ablator") will have the potential to express a cytotoxin in eves cell, but cytotoxin will not be produced unless Cre enzyme is expressed. The other mouse line will express the cre gene in the intermediate mesoderm (IM). By mating these two lines, we will eliminate the IM. This will enable us to investigate the function of the IM in the early mouse embryo, and specifically to test the hypothesis that the IM produces the limb inducer. This system will potentially be useful for analyzing the functions of many different tissues in the regulation of normal and abnormal organ growth and development.

DESCRIPTION (provided by applicant): The skeleton provides a rigid, protective frame for the rest of the body and large injuries or defects can be incapacitating. In particular, the treatment f segmental defects remains an unresolved problem today. Autogenous bone graft is limited in supply and the biological activity of bone graft substitutes requires further refinement. Thus, new strategies for more physiological healing of critical sized bone defects are much needed. Studying models for bone regeneration in small mammals is a vital first step to developing treatments for severe skeletal injuries. Although humans do not repair as well as amphibians, human adults demonstrate a remarkable ability to regenerate large portions of the rib (>6 inches). We have developed a mouse model for rib repair that is amenable to genetic and surgical manipulation. Our preliminary studies have shown that 1) complete repair occurs within 1-2 months, 2) regrowth occurs in the middle of the resection site as well as from the ends, and 3), that the presence of the surrounding connective tissue sheath, or periosteum, is required for complete healing to occur. Based on our preliminary findings we now aim 1) to determine what steps occur to build new bone with the expectation that a rapidly produced cartilage intermediate is key and 2) to determine if the cells involved in the repair arise from progenitors in the periosteum. Our ultimate goal is to harness the unique features of rib skeletal repair to develop methods for enhancing bone healing in other locations of the body.

The repair of extensive bone injuries remains an unmet clinical challenge. By developing two new and complementary models of large-scale bone regeneration in zebrafish and mouse, we aim to understand the role of the cartilage callus in generating large segments of full thickness bone. While the periosteum generates osteoblasts during homeostasis, the specific subpopulation that builds the repair callus remains poorly defined. Using transgenic lineage tracing, we provide compelling preliminary evidence that a rare bi-potent Sox9+/Runx2+ periosteal population generates new cartilage and bone during repair. This newfound ability to label, manipulate, and isolate a specific stem cell population allows us to test whether the remarkable regenerative capacity of the rib is due to the unique properties of its periosteal stem cells. In Aim 1, we team up with an orthopaedic surgeon to test that Sox9+ cells from the rib periosteum can be expanded in culture and used to heal a critical-sized femoral defect. The formation of a cartilage callus is a common feature in bone repair, yet how the periosteum generates cartilage only during repair remains a mystery. In preliminary data, we find that the cartilage callus is severely compromised when either the Ihha ligand is deleted in zebrafish or the Hh receptor Smo is deleted from Sox9+ cells in mice. In Aim 2, we test that this reflects a repair-specific role for Ihh, which is markedly different from its developmental role in osteoblast differentiation and chondrocyte proliferation. Further, our preliminary data suggest that these Ihh-induced repair chondrocytes differ in important ways from those in the growth plate since repair chondrocytes co-express osteoblast genes even at pre-hypertrophic stages. This increase in osteogenic character subsequently correlates with a conversion of chondrocytes into osteocytes. In Aim 3, we test whether repair and developmental chondrocytes represent distinct cell types by comparing global gene expression at different stages of maturation. We also use lineage tracing to quantitate the extent to which cartilage-derived osteocytes preferentially contribute to full thickness bone during repair. Lastly, we use powerful new chromatin accessibility assays to test that as Sox9+ periosteal cells become cartilage, their greater osteogenic character results from the maintenance of poised osteoblast enhancers. Our findings will reveal how a rare population of periosteal stem cells can be induced to make cartilage during injury, and how this specialized repair cartilage can be used to regenerate full thickness bone. A better understanding of these important stem cells will form the basis of future pre- clinical trials aimed at healing large-scale skeletal lesions in other parts of the body.