Hox Gene Regulation of Skeletal Repair

Hox 基因对骨骼修复的调控

• 批准号: 10312868
• 负责人: Katharine A. Hubert
• 金额: $ 3.42万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10312868
• 关键词: Hox; Gene; Regulation; Skeletal; Repair

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.

项目总结/摘要 Hox基因是一组进化上保守的转录因子，对几种发育过程起重要作用。 过程，包括骨骼前后轴的图案。Hox 11同源基因 组，这是在zeugopod地区（桡骨/尺骨和腓骨/胫骨）表达，是必要的适当 这是一种共足类动物的模式。在过去的几年里，Wellik实验室的工作表明， 发育重要的Hox转录因子在整个生命过程中在骨骼中保持表达， 特别是在祖细胞富集的间充质干细胞（MSC）中。严格的遗传谱系标记， 实验室证明，这些细胞产生所有三种间充质谱系，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 干细胞基于这些信息的一个关键问题是Hox基因功能在这些茎中是否重要 细胞一生我们最近报道了Hox 11在成体阶段的暂时缺失导致了在胚胎发育中的缺陷。 成骨细胞发生，其中分化开始，但成骨细胞和骨细胞不能成熟。成人 Hox 11功能的条件性丧失导致骨基质逐渐变弱，而胶原蛋白不 在重塑骨中正确组装。在这项研究中，我将使用一个时间控制的，有条件的损失- 功能模型，以评估响应骨折修复的缺陷（目标1）。初步数据显示， 暂时缺失的ROSACreERT 2/+; Hoxa 11 eGFP/-; Hoxd 11 LoxP/LoxP小鼠在骨折后不能修复。 此外，初步数据表明，成骨细胞和软骨细胞的群体似乎是在 变种人的异常使用Hoxa 11 CreERT 2来制定缺失和谱系标记，我可以标记细胞， 进行了重组分离和转录组学分析（Hoxa 11 eGFP/CreERT 2; Hoxd 11 LoxP/LoxP; ROSAtd-Tomato/+，Aim 2）。骨折损伤诱导急性反应，其中干/祖细胞扩张和 同时向两种骨骼谱系分化，提供了一个很好的模型来分离 单细胞，并确定Hox基因在这些过程中调节的途径和靶点。初步数据 显示大比例的GFP+细胞可用于从骨折愈伤组织收集， 细胞测序不仅是可能的，而且是研究Hox转录组变化的高效工具， 表达和Hox谱系细胞。该项目的总体目标是确定Hox在骨折中的遗传功能 修复，并确定Hox基因在此过程中调节骨骼行为的分子机制。