Analysis of the regulation of trabecular bone formation during mouse embryogenesis

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Differentiation and terminal maturation of hypertrophic chondrocytes (HTCs) is an essential step dur-ing endochondral ossification. Delayed formation of HTCs is associated with a delay in osteoblast differentiation and remodeling of the cartilage template into bone. Trabecular bone formation at the chondro-osseous front is dependent on timely removal of HTCs and on a number of factors produced by hypertrophic chondrocytes controlling, amongst others, local differentiation of osteoblastic and probably osteoclastic precursors. Both aspects are not well understood. In this project we will study on one hand the role of beta-catenin in HTCs and on the other had we will examine whether autophagy is important for their timely removal. As for the first part, we got interested in studying the role of beta-catenin in HTCs as beta-catenin protein levels appear to be rather low, suggesting a destabilization. Hence, we decided to genetically stabilize betacatenin in HTCs in the mouse using the beta-catenin exon3 floxed allele in combination with a ColX-Cre line. In parallel loss-of function of beta-catenin in HTCs will be analyzed in collaboration with Prof. von der Mark. Phenotypic changes upon loss- and gain-of beta-catenin activity in HTCs will be analyzed by histology, in situ hybridization for specific markers, and changes will be verified by qRT-PCR using isolated HTCs of the respective genotypes. Preliminary data from our gain-of function mutant analysis are highly interesting and hint at a potential involvement of osteoprotegerin, a molecule that negatively regulates development and maturation of osteoclasts. This will be further investigated using a genetic appraoch. Secondly we will genetically test whether autophagy is involved in the survival / removal of HTCs, by conditionally deleting Atg5, a gene essential for autophagy, from HTCs using again the ColX-Cre line. Phenotypic changes will be characterized using histology, immunohistochemistry and in situ hybridization for specific marker genes.