Functional analysis of the role of Cdc42 in mammalian skeletal development

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Development of the mammalian skeleton is a complex, temporally and spatially organized process during which mesenchymal tissues either directly differentiate into bone (intramembranous ossification) or first form a cartilaginous intermediate which is subsequently replaced by bone (endochondral ossification). Skeletal morphogenesis is strongly influenced by cell-matrix interactions and tightly regulated via extracellular proteins, growth factors, hormones, transcription factors and intracellular signalling molecules. Mutations in a broad range of genes result in various osteochondrodysplasias, genetic diseases charactherized by growth and skeletal abnormalities. ß1 integrins, the major adhesion receptors that mediate cell-matrix interactions during skeletogenesis, have been shown to be crucial for both types of ossification processes regulating extracellular matrix assembly, cell survival, proliferation, differentiation and cytoskeletal organization. However, relativelly less is known about the intracellular signalling pathways directing these integrin functions in vivo. The Rho GTPases constitute a family of intracellular molecules involved in signalling from adhesive as well as nonadhesive receptors. Cdc42, a prototype member of the Rho family, is implicated in skeletal development but its function has not been studied in animal models. In order to elucidate the role of Cdc42 during skeletogenesis we have conditionally inactivated the mouse Cdc42 gene in mesenchymal precursor cells of osteoblasts and chondrocytes using the Cre-loxP system. Our preliminary data revealed severe skeletal defects of the mutant mice including both cartilaginous and bony tissues. Present application aims 1) to charactherize these defects in detail; 2) to identify Cdc42-dependent signaling pathways that affect skeletogenesis; 3) and to provide a complex mechanistic view how Cdc42 integrates extra- and/or intracellular signals during bone formation. The project involves histological, ultrastructural and immunohistochemical analyses as well as biochemical, cellular and gene expression approaches.