The role of osteoclasts and blood vessels as mediators downstream of beta-catenin in the differentiation of osteoblasts derived from two origins – chondrocytes and perichondrium

骨稳态依赖于成骨细胞和破骨细胞的相互作用。成骨细胞来自胚胎中两种不同的来源，肥大的软骨细胞和软骨膜，而破骨细胞则来自造血细胞。在影响关节的疾病中，会观察到成骨细胞和破骨细胞之间的平衡偏离，如骨关节炎和类风湿性关节炎，并导致骨质疏松或骨化症等骨骼疾病。Wnt/β-catenin通路参与了骨骼发生的不同方面，包括骨内环境的稳定。我们最近的工作表明，肥大软骨细胞中的β-连环蛋白(由CTNNB1基因编码)的共转录功能一方面对软骨细胞来源的成骨细胞的分化起重要作用，另一方面负向控制软骨骨前的破骨细胞生成。在肥大的软骨细胞中CTNNB1缺乏的小鼠是骨量减少的，反映在骨小梁的严重减少。然而，令人惊讶的是，在这些小鼠中，软骨膜来源的成骨细胞的分化也受到影响，这表明涉及非细胞自主机制。在小鼠肥大的软骨细胞中表达稳定形式的β-连环蛋白，导致Wnt/β-连环蛋白途径的激活，我们观察到软骨细胞来源的成骨细胞数量增加，但我们未能在这些细胞中检测到高水平的β-连环蛋白，支持从两个来源调控成骨细胞形成的非细胞自主机制的想法。根据初步数据，我们推测部分表型改变涉及破骨细胞和/或可能由于H型和L型血管成分的改变。更准确地说，在缺乏破骨细胞的小鼠中，我们观察到软骨细胞和软骨膜来源的成骨细胞的分化增加，这表明破骨细胞要么分泌/存在一种因子，要么通过它们的活性从细胞外基质中释放一种因子，对这两种来源的成骨细胞的分化产生负面影响。破骨细胞产生的这样一种可能的因子是Semaphorin 4D。为了检验是否需要破骨细胞活性，我们还将使用破骨细胞存在但不活跃的小鼠模型来研究来自这两种来源的成骨细胞的分化。此外，还将使用单细胞RNA序列方法在一些小鼠模型中检查血管和骨髓成分。综上所述，在这个项目中，我们的目标是从两个来源识别作用于β-连环蛋白下游的调节成骨细胞分化的分泌因子，并了解破骨细胞和血管作为可能的介体所起的作用。

Bone homeostasis depends on the interplay of osteoblasts and osteoclasts. Osteoblasts are derived from two different origins in the embryo, from hypertrophic chondrocytes and the perichondrium, while osteoclasts are of hematopoietic origin. Deviations from the equilibrium between osteoblasts and osteoclasts are observed in diseases affecting the joint, like osteoarthritis and rheumatoid arthritis, and lead to bone diseases like osteoporosis or osteopetrosis. The Wnt/beta-catenin pathway has been implicated in different aspects of skeletogenesis including bone homeostasis. Our recent work has shown that the co-transcriptional function of beta-catenin (encoded by the Ctnnb1 gene) in hypertrophic chondrocytes is, on the one hand, important for the differentiation of chondro¬cyte-derived osteoblasts and, on the other hand, negatively controls osteoclasto¬genesis at the chondro-osseous front. Mice deficient for Ctnnb1 in hypertrophic chondrocytes are osteopenic, reflected in the severe reduction of trabecular bone. Yet, surprisingly, in these mice, the differentiation of perichondrium-derived osteoblasts is also affected, suggesting the involve¬ment of non-cell-autonomous mechanisms. In mice expressing a stabilized form of beta-catenin in hypertrophic chondrocytes leading to an activation of the Wnt/beta-catenin pathway, we observed that the chondrocyte-derived osteoblasts increased in number but we failed to detect high levels of beta-catenin protein in these cells, supporting the idea of non-cell-autonomous mechanisms regulating osteoblastogenesis from both origins. Based on preliminary data, we propose that part of the phenotypic changes involve osteoclasts and/or maybe due to changes in the composition of the H- and L-type blood vessels. More precisely, in mouse mutants lacking osteoclasts we observed that the differentiation of chondrocyte- and perichondrium-derived osteoblasts were increased suggesting that osteoclasts either secret/present a factor or through their activity a factor is released from the extracellular matrix affecting osteoblast differentiation from both origins negatively. Such a possible factor produced by osteoclasts is Semaphorin 4D. To examine whether osteoclast activity is required, we will also examine the differentiation of osteoblasts derived from the two origins using a mouse model in which osteoclasts are present but inactive. In addition, the blood vessel and also bone marrow composition will be examined in some of the mouse models using a single-cell RNA seq approach. Taken together, in this project we aim to identify secreted factors acting downstream of beta-catenin regulating osteoblast differentiation from both origins and understand the role of osteoclasts and blood vessels as possible mediators.