Cell biological mechanisms that regulate development of cartilage architecture

AbstractChondrodysplasias comprise a class of heritable defects that occur in 1/2500 births and disrupt skeletalgrowth, resulting in dwarfism or asymmetric limb length. Therapeutic options for chondrodysplasias are limitedin part because the underlying mechanisms that regulate cartilage architecture are poorly understood.Anisotropic processes that increase cell volume and deposit cartilage matrix are largely responsible for thegrowth of long bones. The inherent anisotropies in growth plate cartilage are mirrored by the arrangement ofchondrocytes in the developing bone. Specifically, the architecture of cartilage arises from disordered restingprogenitor cells that enter a transit amplifying phase in which clonal expansion generates columns of discoidcells that resemble stacks of coins that are aligned with the longitudinal axis of the bone. Column formationoccurs in a process involving planar cell division followed by rearrangement of daughter cells. Interestingly,genetic studies in model organisms have shown many chondrodysplasia phenotypes are associated withdefects in chondrocyte column formation. These studies also revealed that defects in cell signaling and cellpolarity pathways, cell adhesion, and extracellular matrix structure each disrupt column formation and producechondrodysplasia. However, a major gap in knowledge exists regarding how these distinct molecular functionsare integrated to promote cartilage architecture. This proposal introduces a novel live-cell imaging method thatallows quantitative analysis of column formation to test the innovative hypothesis that antagonism betweenPthrp and Wnt5a signaling regulates myosin II motor protein activity at the cell-cell and cell-matrixinterfaces to produce the anisotropic forces required to rearrange daughter chondrocytes into acolumn. This hypothesis is based in part on the observations of phospho-myosin light chain at the cell-matrixinterface during column formation and the relocalization of this activity to the cell-cell interface in Wnt5amutants and following activation of Pthrp signaling in chondroctyes. The proposed experiments utilize the live-cell imaging system in conjunction with immunofluorescence, electron microscopy, and biochemical analysesto: (1) defining the roles of cell-cell and cell-matrix adhesion in column formation, (2) determining the pathwaythrough which Wnt5a regulates myosin activity in chondrocytes, and (3) testing if Pthrp signaling alters thebalance between Rho and Rac GTPase activity at the cell-matrix and cell-cell interfaces, respectively. Resultsof these studies will advance the development of novel therapeutics, including growth plate cartilageengineering, to treat chondrodysplasias by providing crucial information that provide mechanistic links betweenknown regulators of chondrocyte maturation and cell mechanics that are crucial for generating cartilage tissuearchitecture and growth.