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.

项目概要/摘要 Hox 基因是一组进化上保守的转录因子，对多种发育非常重要 过程，包括骨骼前后轴的图案。 Hox11 旁系同源基因 组，在 zeugopod 区域（桡骨/尺骨和腓骨/胫骨）中表达，对于正确的 zeugopod 的图案。在过去几年中，Wellik 实验室的工作表明，这些 对发育至关重要的 Hox 转录因子在整个生命周期中都在骨骼中保持表达， 特别是在富含祖细胞的间充质干细胞（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，并表现出终生的自我更新，提供了强有力的证据表明该细胞群是骨骼细胞 干细胞。基于此信息的一个关键问题是 Hox 基因功能在这些茎中是否重要 细胞贯穿一生。我们最近报道，成年阶段 Hox11 的暂时缺失会导致 成骨细胞发生，其中分化开始，但成骨细胞和骨细胞未能成熟。成人 Hox11 功能的有条件丧失会导致骨基质逐渐减弱，而胶原蛋白则不会 正确组装在重塑骨中。在这项研究中，我将使用时间控制的、有条件的损失- 评估骨折修复缺陷的函数模型（目标 1）。初步数据表明 暂时缺失的 ROSACreERT2/+;Hoxa11eGFP/-;Hoxd11LoxP/LoxP 小鼠骨折后无法修复。 此外，初步数据表明，成骨细胞和软骨细胞的数量似乎处于 突变体中异常。使用 Hoxa11CreERT2 进行删除和谱系标记，我可以标记那些 已进行重组以进行分离和转录组分析（Hoxa11eGFP/CreERT2；Hoxd11LoxP/LoxP； ROSAtd-番茄/+，目标 2)。骨折损伤会引起急性反应，其中干细胞/祖细胞扩张并 两种骨骼谱系的分化同时发生，提供了一个很好的模型来分离 单细胞并识别 Hox 基因在这些过程中调节的途径和目标。初步数据 显示大部分 GFP+ 细胞可从骨折愈伤组织中收集，从而使单个 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 表达细胞和Hox谱系细胞。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。