The role of macrophages during embryonic endochondralen ossification in wild-type and osteoclast deficient mice.

在胚胎发育过程中，骨骼元素的软骨原基需要重塑成骨，这一过程称为软骨内骨化。为此，需要去除肥大的软骨细胞，以便形成骨髓腔。在此过程中，完全成熟的肥大软骨细胞的矿化基质被留下，并作为软骨膜衍生的成骨细胞前体的平台，这些前体与血管一起迁移成熟并构建骨小梁。肥大软骨细胞的去除可能需要不同细胞如内皮细胞、破骨细胞、破软骨细胞和破隔细胞的协同作用，因为单独缺乏任何一种细胞类型不会导致骨髓腔形成的损失。然而，一些细胞类型如破骨细胞的损失导致细胞环境的变化。因此，缺乏破骨细胞分化所需的因子（例如c-fos或Rank受体）的突变体是破骨细胞缺陷型的，但骨髓中存在的巨噬细胞数量增加。在过去的几年里，巨噬细胞得到了很多关注。它们是非常异质的、长寿的和生物合成活性的细胞，其在惰性和适应性免疫应答中的功能是经典的。它们还在发育和肿瘤发生的形态发生过程中发挥作用。关于巨噬细胞在骨髓中的作用知之甚少;它们与刺激成骨细胞活性和维持造血生态位有关。骨髓中的巨噬细胞与其他双重来源的器官中的巨噬细胞一样，驻留的巨噬细胞是卵黄囊来源的，而其他巨噬细胞则来源于造血髓样细胞谱系。在拟议的项目中，我们希望对Rank突变小鼠的长骨发育进行比较分析，这些小鼠的巨噬细胞比野生型小鼠多3倍，而Pu.1缺陷小鼠缺乏巨噬细胞。初步数据显示，Rank新生突变体的生长板改变在E18.5时的Pu.1突变体中未观察到。我们还想探讨在Rank突变体中增加的巨噬细胞群体的起源，并分析它们相对于M1或M2群体的组成是否改变。此外，作为该项目的一部分，我们希望分析Rank突变体中生长板改变的潜在机制。最后但并非最不重要的是，我们要检查胚胎骨发育和产后维护的功能后果后，有条件地消除卵黄囊衍生的巨噬细胞群体使用诱导，条件Cre/flox方法。总之，我们期望从这些实验中对正常和遗传改变条件下巨噬细胞的作用有新的认识。

During embryonic development the cartilage anlage of the skeletal element needs to be remodeled into bone, a process referred to as endochondral ossification. For this the hypertrophic chondrocytes need to be removed so that a bone marrow cavity can form. The mineralized matrix of fully maturated hypertrophic chondrocytes is left behind in this process and serves as a platform for perichondrium-derived osteoblast precursors that migrate in together with blood vessels to mature and to build the trabecular bone. The removal of hypertrophic chondrocytes probably requires the concerted action of different cells, such as endothelial cells, osteoclasts, chondroclasts and septoclasts as the lack of any one of the cell types alone does not result in a loss of bone marrow cavity formation. Nevertheless, the loss of some cell types such as the osteoclasts leads to changes in the cellular environment. As such mutants lacking factors required for osteoclast differentiation, e.g. c-fos or Rank receptor, are osteoclast-deficient, but have increased numbers of macrophages present in the bone marrow. In the past years macrophages got a lot of attention. They are very heterogenous, long-lived, and biosynthetically active cells that are classically known for their functions in inert and adaptive immune responses. They also play a role in morphogentic processes in development and tumorigenesis. Little is known about the role of macrophages in the bone marrow; they have been implicated in stimulating osteoblast activity and the maintenance of the hematopoietic niche. The macrophages in the bone marrow are like in other organs of dual origin, the resident macrophages are yolk sac derived and others are derived from the hematopoietic myloid lineage. In the proposed project, we want to perform a comparative analysis of long bone development in Rank mutant mice, which have about 3 times more macrophages than wild types, with Pu.1 deficent mice that lack macrophages all together. Preliminary data revealed growth plate alterations in Rank newborn mutants that are not observed in Pu.1 mutants at E18.5. We also want to adress the origin of the macrophage population that is increased in the Rank mutant and analyse whether their composition with regard to M1 or M2 populations is altered. In addition, as part of this proposed project we want to analyse the underlying mechanisms of growth plate alteration in the Rank mutant. Last but not least we want to examine the functional consequences on embryonic bone development and postnatal maintenance upon conditionally eliminating the yolk sac derived macrophage population using an inducible, conditional Cre/flox approach. All together we expect from these experiments new insights into the role of macrophages under normal and genetically altered conditions.