The condensation of chondroprogenitor cells is one of the earliest steps in endochondral skeletal development. In the developing limb bud, the core of condensing mesenchymal cells subsequently differentiate into chondrocytes to give rise to the cartilage anlage. The cellular condensation step is crucial to chondrogenesis, since both in vivo and in vitro perturbations of condensation inhibit mesenchymal chondrogenesis. We have previously demonstrated that N-cadherin, a cell-cell adhesion protein, is functionally involved in limb mesenchymal condensation and chondrogenesis. Cadherins are membrane glycoproteins which mediate cell adhesion in a Ca2+-dependent, homotypic manner, and have been postulated to act as morphoregulatory molecules during development. Cadherins function as a complex with the cytosolic, alpha-, -beta- and gamma-catenins, and the complex is generally believed to function in adhesion-mediated signaling. In this proposal, we aim to further examine the mechanism and function of N-cadherin-mediated mesenchymal cell interactions in chondrogenesis, using two systems of mesenchymal chondrogenesis: 1) chick embryonic limb mesenchyme, which is primed to undergo chondrogenesis when cultured at high density; and 2) the murine multipotential cell line, C3H1OT1/2, which will undergo chondrogenesis when plated as high density micromass and treated with bone morphogenetic protein-2 (BMP-2). The high density requirement of both systems underscores the importance of intimate cell-cell interactions.
The specific aims are: 1) To determine the subcellular site of N-cadherin-mediated activity by examining the effects of expressing structural variants of N-cadherin; 2) To examine the functional role of beta-catenin in cellular condensation and chondrogenesis by analyzing its subcellular distribution, phosphorylation status, and association with N-cadherin, and the effect of expressing its structural variants; 3) To demonstrate that N- cadherin-mediated cell adhesion is pivotally involved in the effects of chondroinductive influences, including the activity of TGF-beta superfamily members, on mesenchymal cells; and 4) To test the hypothesis that N-cadherin expression/activity is a functional marker of chondroprogenitor cells residing in non skeletal tissues. These proposed studies will provide valuable information on the early events of mesenchymal chondrogenesis, and the cellular and molecular mechanisms of cell-cell interactions in general. Investigating the basic mechanism of cartilage development is highly relevant to understanding the etiology of congenital skeletal deformities and the biology of skeletal repair.
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