The vertebrate skeleton is an intricate system of approximately 200 parts of distinct morphologies that are arranged to ensure fluid motion and to confer unique mechanical properties to the body. The long-term objectives of this research are to determine the molecular mechanisms that regulate morphogenesis of the individual skeletal elements and to elucidate how errors in these pathways result in skeletal defects. Defining the pathways that regulate skeletal morphogenesis will both aid the development of therapies to prevent birth defects and improve our ability to repair defective elements of the skeleton by enhancing methods in tissue engineering. The objectives of this proposal are to define the role of Frizzled (Fzd) signaling in the regulation of column formation during chondrocyte maturation and to determine the relationship between these processes and the morphology of cartilage elements. To achieve these objectives, the Specific Aims of this proposal are designed to: (1) determine if Fzd signaling regulates the rearrangement of proliferative chondrocytes into columns, (2) define a role for the Fzd-dependent noncanonical signaling pathways in regulating column formation, and (3) determine whether chondrocyte columns define vectors of growth in developing cartilage elements. The experiments described in this proposal combine the strength of retrovirus based gene expression and lineage analysis in the chick with a powerful method for determining the orientation of cell division in fixed tissue sections. More specifically, lineage analysis in the chick will be used to describe the organization of cells in clones in long bones and irregular bones and, in combination with ectopic gene expression, to determine the specific effects of interfering with Fzd signaling on chondrocyte morphogenesis. These studies will determine whether the formation of chondrocyte columns is a regulated process that correlates with cartilage morphology. This idea will be tested further by combining these approaches with morphometric and histological analysis to determine if cartilage morphology depends on the specific arrangement of chondrocytes. Preliminary data demonstrate that Fzd signaling regulates the arrangement of chondrocytes in concert with the plane of cell division in chick long bones via the noncanonical Fzd signaling pathways. This possibility will be formally tested by determining the arrangement of cells in clones of proliferative chondrocytes expressing mutant forms of Dishevelled that interfere with noncanonical Fzd signaling. Collectively, the proposed experiments will rigorously test the hypothesis that the morphology of cartilage elements depends on the regulation of chondrocyte organization by noncanonical Fzd signaling pathways and will potentially establish a new paradigm in which regulated cell polarity defines vectors of growth in the developing skeleton.
Characterization of novel signaling pathways that regulate cartilage morphogenesis will provide new insights into the regulation of cartilage growth, and will identify molecular targets for the prevention of birth defects and the treatment of bone injury and disease. In the future robust therapies for the treatment of bone disorders are likely to involve approaches that include tissue engineering. Importantly, studies of cartilage growth will play a prominent role in the development of novel methods of tissue engineering to treat orthopedic disorders.
