Hox-Regulated MSCs in Skeletal Development, Growth and Fracture Healing

Hox 调节的 MSC 在骨骼发育、生长和骨折愈合中的作用

• 批准号: 10662574
• 负责人: Deneen M Wellik
• 金额: $ 45.76万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-08 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10662574
• 关键词: Address; Adipocytes; Adult; Alleles; Animal Model; Animals; Apoptosis; Binding; Binding Sites; Biology; Biometry; Bypass; Candidate Disease Gene; Cartilage; Cell Culture Techniques; Cell Differentiation process; Cells; Chondrocytes; Chondrogenesis; Chromatin; Collection; Control Animal; Data; Defect; Differentiation Antigens; Embryo; Embryonic Development; Epitopes; Event; Forelimb; Fracture; Gene Expression Profiling; Genes; Genetic; Genomics; Goals; Growth; Growth and Development function; Homeobox Genes; Homeostasis; Human; In Vitro; Injury; Knock-in; Knowledge; Label; Laboratories; Life; Maps; Mediating; Mission; Modeling; Molecular; Morphology; Mus; Osteoblasts; Osteocalcin; Osteocytes; Osteogenesis; Pathway interactions; Pattern; Phenotype; Play; Population; Positioning Attribute; Process; Publishing; Radial; Regulation; Reporter; Reporting; Research; Research Personnel; Research Proposals; Resolution; Role; Site; Skeletal Development; Skeleton; Testing; Time; Tissue-Specific Gene Expression; United States National Institutes of Health; Vertebrates; Visualization; Work; bone; bone fracture repair; comparative; conditional mutant; experimental study; gene function; in vivo; innovation; loss of function; morphogens; mutant; osteogenic; osteopontin; progenitor; repaired; response to injury; self-renewal; skeletal; skeletal stem cell; stem; stem cells; tool; transcription factor; transcriptomics; ulna; virtual

PROJECT SUMMARY/ABSTRACT Although the critical roles Hox genes play in establishing skeletal morphology has been known for decades, virtually nothing is understood regarding the molecular mechanisms by which Hox genes function in the skeleton. Utilizing a unique collection of genetic tools that permit live visualization of Hox expression (Hoxa11eGFP), Cre-mediated lineage labeling and/or conditional deletion (HoxCreERT2, Hoxd11LoxP/LoxP) and assessment of Hox11 chromatin binding sites (unpublished, validated Hoxa113XFLAG and Hoxd113XFLAG alleles), the overall objective of this application is to dissect the pathways and targets regulated by Hox transcription factors in skeletal stem/progenitor cells to regulate osteogenic and chondrogenesis differentiation. Previous work has demonstrated that Hox-expressing stem/progenitors are maintained in the skeleton in the absence of Hox function, and osteo- and chondrogenic lineages continue to emerge (Sox9-, Osx-/Runx2-expressing), but differentiation is incomplete. Osteoblasts do not progress to mature stages, and chondrocytes fail to undergo normal apoptosis and replacement by bony matrix in Hox mutants. This differentiation defect can be recapitulated in vitro. Based on previously published work and preliminary data, the central hypothesis is that Hox transcription factors regulate critical downstream events at the top of the hierarchy during osteochondrogenic differentiation from skeletal stem/progenitor cells in parallel with canonical differentiation factors. This project will utilize the Hoxa11eGFP reporter and Hoxa11CreERT2-mediated lineage labeling in the presence and absence of adult conditional deletion of Hoxd11 to probe the single cell trajectories of Hox11- expressing progenitors as they expand and differentiate into cartilage and bone in response to injury (Aim 1). The recapitulation of osteo- and chondrogenic differentiation defects in Hox11 mutants in vitro permits a comparative assessment of differential gene expression during temporally controlled differentiation (Aim 2). Newly generated and validated Hoxa113XFLAG; Hoxd113XFLAG epitope-tagged alleles will be utilized to interrogate the sites of chromatin binding in Hox-expressing progenitors and early differentiating cells (Aim 3). The research proposal is innovative in its use of sophisticated genetic tools generated by the research team, the combined in vivo and in vitro approaches, and critical inclusion of a co-investigator and her team with biostatistics expertise. The proposed research is significant as it addresses the longstanding and highly significant question of the molecular mechanism of Hox function in the skeleton. As Hox expression is only observed in skeletal stem/progenitors and early differentiation markers initiate as cells exit the Hox lineage, dissecting the downstream targets and pathways regulated by Hox that are critical to complete successful osteogenic and chondrogenic differentiation will provide impactful new knowledge of skeletal biology.

项目概要/摘要 尽管 Hox 基因在建立骨骼形态方面发挥的关键作用已众所周知 几十年来，对于 Hox 基因在体内发挥作用的分子机制几乎一无所知。 骨架。利用一系列独特的遗传工具来实时可视化 Hox 表达 (Hoxa11eGFP)、Cre 介导的谱系标记和/或条件删除 (HoxCreERT2、Hoxd11LoxP/LoxP) 和 评估 Hox11 染色质结合位点（未发表、经过验证的 Hoxa113XFLAG 和 Hoxd113XFLAG 等位基因）， 该应用程序的总体目标是剖析 Hox 转录调控的途径和靶标 骨骼干/祖细胞中调节成骨和软骨形成分化的因子。以前的 研究表明，在缺乏 Hox 功能以及骨和软骨形成谱系不断出现（Sox9-、Osx-/Runx2-表达），但是 分化不完全。成骨细胞不会进展到成熟阶段，软骨细胞也无法经历 Hox 突变体中的正常细胞凋亡和骨基质替代。这种分化缺陷可以是 体外重现。根据之前发表的工作和初步数据，中心假设是 Hox 转录因子在层次结构顶部调节关键的下游事件 骨骼干细胞/祖细胞的骨软骨分化与典型分化并行 因素。该项目将利用 Hoxa11eGFP 报告基因和 Hoxa11CreERT2 介导的谱系标记 成人条件性删除 Hoxd11 的存在和不存在，以探测 Hox11-的单细胞轨迹 当祖细胞响应损伤而扩展并分化为软骨和骨时表达祖细胞（目标 1）。 Hox11 突变体体外骨和软骨分化缺陷的重现允许 时间控制分化过程中差异基因表达的比较评估（目标 2）。 新生成并经过验证的 Hoxa113XFLAG； Hoxd113XFLAG 表位标记的等位基因将用于询问 表达 Hox 的祖细胞和早期分化细胞中染色质结合的位点（目标 3）。这 研究提案的创新在于使用了研究团队开发的复杂遗传工具， 结合体内和体外方法，以及联合研究者和她的团队的关键参与 生物统计学专业知识。拟议的研究意义重大，因为它解决了长期存在且高度重视的问题 骨架中 Hox 功能的分子机制的重要问题。由于 Hox 表达式仅 在骨骼干/祖细胞中观察到，早期分化标记随着细胞退出 Hox 谱系而启动， 剖析 Hox 调控的下游目标和途径，这对于成功至关重要 成骨和软骨分化将提供有影响力的骨骼生物学新知识。