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

项目总结/摘要 尽管Hox基因在建立骨骼形态中的关键作用已经被认为是 几十年来，几乎没有什么是了解的分子机制，Hox基因的功能，在 骨架。利用独特的遗传工具集合，允许Hox表达的实时可视化 （Hoxa 11 eGFP）、Cre介导的谱系标记和/或条件性缺失（HoxCreERT 2、Hoxd 11 LoxP/LoxP）和 评估Hox 11染色质结合位点（未发表、经验证的Hoxa 113 XFLAG和Hoxd 113 XFLAG等位基因）， 本申请的总体目标是剖析Hox转录调控的途径和靶点 因子在骨骼干/祖细胞调节成骨和软骨分化。先前 工作已经证明，表达Hox的干/祖细胞在缺乏Hox的情况下维持在骨骼中。 Hox功能，骨和软骨形成谱系继续出现（Sox 9-，Osx-/Runx 2-表达），但 分化是不完全的。成骨细胞不能进展到成熟阶段，软骨细胞不能经历 Hox突变体中正常的细胞凋亡和骨基质替代。这种分化缺陷可能是 在体外重现。基于先前发表的工作和初步数据，中心假设是， Hox转录因子在转录过程中调节位于层级顶部的关键下游事件。 骨干/祖细胞向骨软骨细胞的分化与典型分化平行 因素该项目将利用Hoxa 11 eGFP报告基因和Hoxa 11 CreERT 2介导的谱系标记， 存在和不存在Hoxd 11的成人条件性缺失，以探测Hox 11- 表达祖细胞，因为它们响应于损伤而扩增并分化成软骨和骨（Aim 1）。 Hox 11突变体体外成骨和软骨分化缺陷的重演允许 在时间控制分化期间差异基因表达的比较评估（目的2）。 新生成并验证的Hoxa 113 XFLAG; Hoxd 113 XFLAG表位标记的等位基因将用于询问 在表达Hox的祖细胞和早期分化细胞中染色质结合的位点（Aim 3）。的 这项研究计划是创新的，它使用了研究小组开发的复杂的遗传工具， 结合体内和体外方法，以及关键的共同研究者和她的团队， 生物统计学专业知识。拟议的研究是重要的，因为它解决了长期和高度 Hox在骨骼中功能的分子机制的重要问题。由于Hox表达式仅 在骨骼干细胞/祖细胞中观察到，早期分化标志物在细胞退出Hox谱系时开始， 剖析下游目标和Hox调控的途径，这是完成成功的关键， 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labeling and/or conditional deletion (HoxCreERT2, Hoxd11LoxP/LoxP) and assessment of Hox11 chromatin binding sites (unpublished, validated Hoxa113XFLAG and Hoxd113XFLAG alleles), the overall objective of this application is to dissect the pathways and targets regulated by Hox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.