The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a carefully regulated program of chondrocyte maturation. Identification of the genes and signaling molecules that control hypertrophic chondrocyte maturation and the regulatory interactions among them is crucial to understanding bone growth and development. The goal of this research is to investigate the hypothesis that the homeodomain transcription factor Dlx5 is a key positive regulator of chondrocyte maturation during endochondral ossification, and to unravel the relationships between Dlx5 and other factors involved in controlling the process. Dlx5 is expressed during the conversion of immature proliferating chondrocytes to post-mitotic prehypertrophic chondrocytes, a critical step in maturation. Retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in formation of severely shortened skeletal elements that contain excess numbers of hypertrophying chondrocytes, expanded and upregulated domains of expression of some molecular markers of hypertrophic differentiation including osteopontin and type X collagen, and expansion of mineralized cartilage matrix. Dlx5 misexpression also markedly reduces cell proliferation concomitant with promoting hypertrophic maturation. These results suggest Dlx5 positively regulates maturation at least in part by promoting conversion of immature proliferating chondrocytes to hypertrophying chondrocytes. The role of Dlx5 in chondrocyte maturation will be further investigated by examining effects of Dlx5 misexpression in the chick limb on expression of additional markers of hypertrophic maturation, and determining if chondrocyte maturation is promoted when Dlx5 misexpression is specifically targeted to the cartilage models of the limbs of transgenic mouse embryos. It will also be determined if maturation is promoted by Dlx6, a putative functionally redundant member of the Dlx family. The mechanism of Dlx5 regulation of maturation will be investigated using gain- and loss-of-function approaches in limb mesenchymal cell and chondrocyte model culture systems. The possible regulatory relationships between Dlx5 and other positive regulators of chondrocyte maturation including RMPs, Cbfal/Runx2, and (-catenin-mediated Wnt signaling will be investigated. A variety of molecular agonists and antagonists will be used to begin to unravel how Dlx5 and the other factors interact and cooperate in regulating hypertrophic maturation. It will also be determined if Dlx5 participates in transcriptional regulation of the osteopontin, bone sialoprotein, and type X collagen genes during chondrocyte maturation. The possibility that Dlx5 interacts and cooperates with the BMP signaling pathway and/or Cbfal/Runx2 in the transcriptional regulation of the promoters of one or more of these candidate target genes will be investigated.