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

在胚胎发育期间，骨骼元素的软骨原骨需要重塑为骨，这一过程被称为软骨内成骨。为此，需要去除肥大的软骨细胞，以便形成骨髓腔。在这一过程中，完全成熟的肥大软骨细胞的矿化基质被遗留下来，为软骨膜来源的成骨细胞前体提供平台，这些前体与血管一起迁移到成熟和构建松质骨。去除肥大的软骨细胞可能需要不同细胞的协同作用，如内皮细胞、破骨细胞、软骨破骨细胞和间隔破骨细胞，因为缺乏任何一种细胞类型并不会导致骨髓腔形成的丧失。然而，某些细胞类型的丧失，如破骨细胞，会导致细胞环境的变化。因为这些缺乏破骨细胞分化所需因子的突变体，如c-fos或Rank受体，是破骨细胞缺陷的，但增加了骨髓中存在的巨噬细胞的数量。在过去的几年里，巨噬细胞受到了极大的关注。它们是非常异质、长寿命和生物合成活性的细胞，经典地知道它们在惰性和适应性免疫反应中的功能。它们还在发育和肿瘤发生的形态形成过程中发挥作用。对巨噬细胞在骨髓中的作用知之甚少；它们被认为与刺激成骨细胞活动和维持造血生态位有关。骨髓中的巨噬细胞与其他双重起源的器官中的巨噬细胞一样，常驻的巨噬细胞来自卵黄囊，其他巨噬细胞来自造血祖细胞。在拟议的项目中，我们想要对Rank突变小鼠的长骨发育进行比较分析，Rank突变小鼠的巨噬细胞数量大约是野生小鼠的3倍，而PU.1缺陷小鼠总共缺乏巨噬细胞。初步数据显示，在E18.5的PU.1突变体中没有观察到的等级新生儿突变体的生长板变化。我们还想说明在Rank突变体中增加的巨噬细胞种群的起源，并分析它们相对于M1或M2种群的组成是否发生了变化。此外，作为这个拟议项目的一部分，我们想要分析等级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，我们想要研究使用可诱导的、有条件的CRE/FLOX方法有条件地消除卵黄囊来源的巨噬细胞群对胚胎骨骼发育和出生后维持的功能后果。总之，我们期待从这些实验中对巨噬细胞在正常和基因改变条件下的作用有新的见解。

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.