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

项目摘要/摘要 尽管HOX基因在建立骨骼形态中扮演的关键角色已经为人所知 几十年来，关于HOX基因在体内发挥作用的分子机制几乎一无所知 那副骷髅。利用独特的基因工具集合，实现HOX表达的实时可视化 (Hoxa11eGFP)、Cre介导的谱系标记和/或条件缺失(HoxCreERT2、Hoxd11LoxP/loxP)和 Hox11染色质结合位点的评估(未发表的、有效的Hoxa113XFLAG和Hoxd113XFLAG等位基因)， 这个应用程序的总体目标是分析HOX转录调控的途径和靶点 骨骼干/祖细胞中调节成骨和软骨分化的因子。上一首 研究表明，在没有HOX的情况下，表达HOX的干/祖细胞保持在骨骼中 HOX功能，以及成骨和成软骨的谱系继续出现(表达Sox9，OSX-/Runx2)，但是 差异化不完整。成骨细胞不会发展到成熟阶段，软骨细胞也不会经历 HOX突变体的正常细胞凋亡和骨基质替代。这种差异化缺陷可能是 在体外重述。根据之前发表的工作和初步数据，中心假设是 HOX转录因子在结构的顶端调节关键的下游事件 骨干/祖细胞向成骨软骨分化与规范分化并行 各种因素。该项目将利用Hoxa11eGFP报告和Hoxa11CreERT2介导的谱系标记 存在和不存在成人条件缺失Hoxd11以探索Hox11的单细胞轨迹- 祖细胞在损伤后扩张和分化为软骨和骨骼时表达(目标1)。 在体外对Hox11突变体骨和软骨细胞分化缺陷的概括允许 时间控制分化过程中差异基因表达的比较评估(目标2)。 新产生和验证的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置(目标3)。这个 研究提案的创新之处在于它使用了由研究团队产生的复杂的基因工具， 体内和体外相结合的方法，以及关键的纳入合作研究人员和她的团队 生物统计学专业知识。这项拟议的研究具有重要意义，因为它解决了长期和高度 骨骼中HOX功能的分子机制的重要问题。AS HOX表达式仅为 在骨骼干/祖细胞中观察到，早期分化标志物在细胞退出HOX谱系时启动， 剖析HOX调控的下游目标和途径，这些是成功完成的关键 成骨和成软骨分化将为骨骼生物学提供有影响力的新知识。

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labeling and/or conditional deletion (HoxCreERT2, Hoxd11LoxP/LoxP) and assessment of Hox11 chromatin binding sites (unpublished, validated Hoxa113XFLAG and Hoxd113XFLAG alleles), the overall objective of this application is to dissect the pathways and targets regulated by Hox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.