Hox Gene Regulation of Skeletal Repair

项目摘要/摘要 HOX基因是一组进化上保守的转录因子，对几个发育阶段都很重要 过程，包括骨骼前后轴的图案化。Hox11等位基因 在斑足动物区(尺骨/桡骨和腓骨/胫骨)表达的基因群，是正常 斑足类动物的图案。在过去的几年里，韦尔利克实验室的研究表明，这些 发育上重要的HOX转录因子在整个生命过程中仍在骨骼中表达， 尤其是在祖细胞富集间充质干细胞(MSCs)中。严格的遗传谱系标记来自于 实验室证明，这些细胞可分化成三种间充质细胞，即成骨细胞、软骨细胞和 脂肪细胞，并表现出终身自我更新，提供了强有力的证据，表明这一群体的细胞是骨骼 干细胞。基于这一信息的一个关键问题是，Hox基因的功能在这些干细胞中是否重要 整个生命中的细胞。我们最近报道，在成人期，Hox11基因的临时缺失会导致脑细胞缺陷 成骨细胞形成：分化开始，但成骨细胞和骨细胞不成熟。成虫 Hox11功能的条件性丧失会导致骨基质逐渐减弱，而胶原蛋白则不会 在重塑的骨骼中正确组装。在这项研究中，我将使用时间控制的、有条件的损失 评估骨折修复反应中缺陷的功能模型(目标1)。初步数据显示， 暂时缺失的ROSACreERT2/+；Hoxa11eGFP/-；Hoxd11LoxP/loxP小鼠骨折后无法修复。 此外，初步数据显示，成骨细胞和软骨细胞的数量似乎在 在突变体中不正常。使用Hoxa11CreERT2同时执行删除和谱系标记，我可以标记 进行重组以进行分离和转录分析(Hoxa11eGFP/CreERT2；Hoxd11LoxP/loxP； ROSATD-番茄/+，目标2)。骨折损伤引起急性反应，在这种反应中，干细胞/祖细胞扩张和 向两个骨骼谱系的分化同时发生，为分离提供了一个极好的模型 并确定HOX基因在这些过程中调节的途径和靶点。初步数据 显示有很大比例的GFP+细胞可从骨折骨痂中收集，使单个 细胞测序不仅是可能的，而且是研究HOX转录变化的高效工具- 表达和HOX谱系细胞。该项目的总体目标是确定HOX基因在骨折中的作用 并确定HOX基因在这一过程中调节骨骼行为的分子机制。

PROJECT SUMMARY/ABSTRACT Hox genes are a group of evolutionarily conserved transcription factors important for several developmental processes, including patterning of the anterior-posterior axis of the skeleton. The Hox11 paralogous gene group, which is expressed in the zeugopod region (radius/ulna and fibula/tibia), are necessary for proper patterning of the zeugopod. In the past few years, work from the Wellik laboratory has shown that these developmentally important Hox transcription factors remain expressed in the skeleton throughout life, specifically in progenitor-enriched mesenchymal stem cells (MSCs). Rigorous genetic lineage labeling from the lab demonstrated that these cells give rise to all three mesenchymal lineages, osteoblasts, chondrocytes and adipocytes, and exhibit life-long self-renewal, providing strong evidence that this population of cells are skeletal stem cells. A key question based on this information is whether Hox gene function is important in these stem cells throughout life. We recently reported that temporal deletion of Hox11 at adult stages results in defects in osteoblastogenesis, wherein differentiation is initiated, but osteoblasts and osteocytes fail to mature. Adult conditional loss of Hox11 function results in a progressively weakened bone matrix where collagen does not properly assemble in remodeling bone. In this study, I will use a temporally-controlled, conditional loss-of- function model to assess defects in response to fracture repair (Aim 1). Preliminary data shows that temporally-deleted, ROSACreERT2/+;Hoxa11eGFP/-;Hoxd11LoxP/LoxP mice are unable to repair after fracture. Additionally, preliminary data suggest that the populations of osteoblasts and chondrocytes appear to be in abnormal in mutants. Using Hoxa11CreERT2 to enact both deletion and lineage labeling, I can mark the cells that have undergone recombination for isolation and transcriptomic analyses (Hoxa11eGFP/CreERT2;Hoxd11LoxP/LoxP; ROSAtd-Tomato/+, Aim 2). Fracture injury induces an acute response in which stem/progenitor expansion and differentiation to both skeletal lineages is occurring simultaneously, providing an excellent model to isolate single cells and identify the pathways and targets Hox genes regulate in these processes. Preliminary data shows that a large proportion of GFP+ cells are available for collection from the fracture callus, making single cell sequencing not only possible, but a highly effective tool to investigate transcriptomic change in Hox- expressing and Hox-lineage cells. The overall goal of this project is to define Hox genetic function in fracture repair and to identify the molecular mechanisms by which Hox genes regulate skeletal behavior in this process.