Analysis of the regulation of trabecular bone formation during mouse embryogenesis

小鼠胚胎发生过程中骨小梁形成的调控分析

• 批准号: 212381117
• 负责人: Professorin Dr. Christine Hartmann
• 金额: --
• 依托单位: Institut für Muskuloskelettale Medizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2015-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/212381117?language=en
• 关键词: Analysis; regulation; trabecular; bone; formation

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.

肥大软骨细胞（HTC）的分化和终末成熟是软骨内骨化过程中的重要步骤。 HTC 形成的延迟与成骨细胞分化和软骨模板重塑为骨的延迟有关。软骨骨前部的小梁骨形成取决于 HTC 的及时去除以及肥大软骨细胞产生的许多因素，这些因素控制着成骨细胞和可能的破骨细胞前体的局部分化。这两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 HTC 中的作用，另一方面我们将研究自噬对于及时清除 HTC 是否很重要。至于第一部分，我们对研究 β-catenin 在 HTC 中的作用感兴趣，因为 β-catenin 蛋白水平似乎相当低，表明不稳定。因此，我们决定使用 β-连环蛋白外显子 3 floxed 等位基因与 ColX-Cre 系相结合，对小鼠 HTC 中的 β-连环蛋白进行基因稳定。同时，我们将与 von der Mark 教授合作分析 HTC 中 β-连环蛋白功能的丧失。 HTC 中 β-连环蛋白活性丧失和增加时的表型变化将通过组织学、特定标记的原位杂交进行分析，并使用各自基因型的分离 HTC 通过 qRT-PCR 验证变化。我们的功能获得突变体分析的初步数据非常有趣，并暗示了骨保护素的潜在参与，骨保护素是一种负调节破骨细胞发育和成熟的分子。这将使用遗传方法进一步研究。其次，我们将再次使用 ColX-Cre 系，通过有条件地从 HTC 中删除 Atg5（一种自噬必需的基因），从基因角度测试自噬是否与 HTC 的存活/去除有关。将使用组织学、免疫组织化学和特定标记基因的原位杂交来表征表型变化。