Hox Gene Regulation of Skeletal Repair

Hox 基因对骨骼修复的调控

• 批准号: 10550118
• 负责人: Katharine A. Hubert
• 金额: $ 3.49万
• 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 2024-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10550118
• 关键词: Acute; Adipocytes; Adult; Alleles; Animals; Anterior; Behavior; Biological; Biological Assay; Bone Marrow; Bone Matrix; Bone callus; Bone remodeling; Cartilage; Cell Culture Techniques; Cell Differentiation process; Cell Lineage; Cells; Chondrocytes; Chondrogenesis; Collagen; Collection; Control Animal; Data; Defect; Development; Developmental Process; Embryonic Development; Event; Exhibits; Fracture; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Recombination; Goals; Growth; Histocytochemistry; Histology; Homeobox Genes; Homeostasis; Injury; Knowledge; Label; Laboratories; Life; Maintenance; Measures; Mediating; Mesenchymal; Mesenchymal Stem Cells; Modeling; Molecular; Mus; Natural regeneration; Organ; Osteoblasts; Osteocytes; Osteogenesis; Pathway interactions; Pattern; Play; Population; Process; Radial; Reporter; Reporting; Roentgen Rays; Role; Skeleton; Small Interfering RNA; Stromal Cells; Testing; Time; Tomatoes; Work; base; bone; bone cell; bone fracture repair; cartilage cell; conditional mutant; differential expression; fibula; gene function; healing; injury and repair; insight; loss of function; microCT; mouse model; mutant; osteogenic; paralogous gene; postnatal; progenitor; repaired; response; response to injury; self-renewal; single cell analysis; single cell sequencing; skeletal; skeletal stem cell; stem; stem cell population; stem cells; tibia; tool; transcription factor; transcriptome sequencing; transcriptomics; ulna

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.

项目摘要/摘要 HOX基因是一组进化保守的转录因子，对几种发育很重要 过程，包括骨骼前后轴的图案。 HOX11寄生虫基因 在适当的适当 Zeugopod的图案。在过去的几年中，Wellik实验室的工作表明这些 发育中重要的HOX转录因子在一生中仍在骨骼中表达 特别是富含祖细胞的间充质干细胞（MSC）。严格的遗传谱系标记 实验室证明，这些细胞产生所有三个间充质谱系，成骨细胞，软骨细胞和 脂肪细胞和终身自我更新，提供了有力的证据，表明这种细胞是骨骼 干细胞。基于此信息的关键问题是HOX基因功能在这些词干中是否重要 一生的细胞。我们最近报告说，在成人阶段，HOX11的时间删除导致缺陷 成骨细胞生成，其中启动分化，但成骨细胞和骨细胞无法成熟。成人 HOX11功能的有条件丧失导致胶原蛋白不逐渐弱的骨基质 正确组装重塑骨。在这项研究中，我将使用临时控制的条件丧失 功能模型以响应裂缝修复来评估缺陷（AIM 1）。初步数据表明 暂时删除，robacreert2/+; Hoxa11EGFP/ - ; Hoxd11loxp/loxp小鼠在骨折后无法修复。 此外，初步数据表明成骨细胞和软骨细胞的种群似乎在 突变体异常。使用Hoxa11Creert2既有删除和谱系标记，我可以标记细胞 已经进行了重组以进行分离和转录组分析（Hoxa11EGFP/Creert2; Hoxd11loxp/loxp; rosatd-tomato/+，目标2）。断裂损伤引起急性反应，其中茎/祖细胞扩张和 与两个骨骼谱系的区分都在同时发生，提供了一个极好的模型来隔离 单细胞并确定在这些过程中调节HOX基因的途径和靶向。初步数据 表明大部分GFP+细胞可从骨折愈伤组织中收集，使单身 细胞测序不仅可能，而且是研究HOX-转录组变化的高效工具 表达和HOX-LINEGE细胞。该项目的总体目标是定义骨折中的HOX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。