Analysis of the regulation of trabecular bone formation during mouse embryogenesis

肥大软骨细胞（HTCs）的分化和终末成熟是软骨内成骨的重要步骤。HTCs的延迟形成与成骨细胞分化和软骨模板骨重塑的延迟有关。软骨-骨前缘的骨小梁形成依赖于HTCs的及时去除，以及由肥大软骨细胞产生的许多因素，其中包括控制成骨细胞和可能的破骨细胞前体的局部分化。这两个方面都没有得到很好的理解。在这个项目中，我们将一方面研究β -连环蛋白在HTCs中的作用，另一方面我们将研究自噬是否对它们的及时清除很重要。对于第一部分，我们对研究β -catenin在HTCs中的作用产生了兴趣，因为β -catenin蛋白水平似乎相当低，表明不稳定。因此，我们决定利用β -catenin外显子3柔化等位基因与ColX-Cre系结合，在小鼠HTCs中稳定β -catenin。与此同时，将与von der Mark教授合作分析β -连环蛋白在HTCs中的功能丧失。HTCs中β -连环蛋白活性丧失和获得时的表型变化将通过组织学分析，对特定标记物进行原位杂交，并使用分离的各自基因型的HTCs通过qRT-PCR验证变化。我们获得的功能突变分析的初步数据非常有趣，并暗示了骨保护素的潜在参与，骨保护素是一种负性调节破骨细胞发育和成熟的分子。这将使用遗传方法进行进一步研究。其次，我们将再次使用ColX-Cre系，通过有条件地从HTCs中删除Atg5（自噬所必需的基因），从基因上测试自噬是否参与HTCs的存活/移除。表型变化将使用组织学，免疫组织化学和特异性标记基因的原位杂交来表征。

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.