Endochondral bone formation is a complex process in which mesenchyrnal cells differentiate into chondrocytes; the chondrocytes undergo maturation, becoming hypertrophic and initiating production of type X collagen; the cartilage matrix then becomes calcified and the mineralized matrix is replaced by bone. The rate at which chondrocytes progress through these steps must be precisely regulated, since this process determines the ultimate size and shape of the bone. The signaling molecules Indian hedgehog (Ihh) and parathyroid hormone related peptide (PTHrP) prevent progression of chondrocytes to the hypertropbic state, while retinoic acid (RA), bone morphogenetic proteins (BMPs) and the transcription factor Cbfal accelerate the transition. Little is known about the integration of these signaling pathways or the molecular mechanisms by which they regulate chondrocyte maturation. Our preliminary results suggest a model in which RA regulates chondrocyte maturation by blocking the inhibitory Ihh/PTHrP signaling pathway and activating the stimulatory BMP/Cbfal signaling pathway. Specifically, RA may inhibit production of Ihh, reducing PTHrP and thus relieving the repression of hypertrophy, and induce BMP signaling and Cbfal, thus accelerating maturation. We will test this hypothesis using transcription of the type X collagen gene as a paradigm for control of chondrocyte maturation. We propose to identify the DNA sequences and transcription factors required for stimulation of type X collagen gene transcription by RA and BMPs and for repression by PTHrP We will also examine the effects of RA on components of the BMP and PTHrP signaling pathways and determine whether inhibitors of BMP and PTHrP signaling interfere with RA modulation of type X collagen promoter function. These experiments are important for understanding endochondral bone formation during embryonic development, and may also provide insight into the mechanisms that regulate fracture repair, which occurs in part by a recapitulation of endochondral ossification. In addition, these studies may increase insight into the mechanisms that lead to a hypertrophic-like state in osteoarthritis.