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

项目摘要/摘要 HOX基因是一类进化上保守的转录因子家族，对正常的 胚胎发育过程中轴骨和附属骨的发育。后位HOX基因(Hox9-13)是 对于沿着近远端轴线构图肢体的骨骼元素至关重要。具体地说，Hox11 类似的基因形成了斑足类动物的模式，它由尺骨/桡骨和胫骨/腓骨组成。 分别是前肢和后肢。最近的研究表明，Hox11基因在 骨骼干细胞/祖细胞群体，可自我更新并产生成骨细胞、软骨细胞和 斑足类动物一生中的脂肪细胞。骨骼干祖细胞Hox11功能丧失损害 它们分化为成骨细胞的能力；随后影响骨骼重塑和修复。此外， Hoxa11eGFP报告表达证明Hox11突变的干/祖细胞关闭HOX 表达，离开干/祖细胞库并启动分化，但未能分化为成熟 成骨细胞。这些发现与需要Hox11功能来建立 血统承诺过程中的分化潜力。尽管对肢体的遗传贡献已为人所知 发育、维持和修复，Hox11调节骨骼干细胞的机制 分化尚不清楚。我目前在韦利克实验室的工作重点是识别Hox11结合 基因组座位和RNA-Seq分析，以确定潜在的下游调控靶基因 干细胞/祖细胞分化。利用两个新产生的表位标记的小鼠等位基因 (Hoxa11-3XFLAG和Hoxd11-3XFLAG)和使用靶下切割和使用核酸酶释放 (Cut&Run)分析，我已经在干细胞/祖细胞中确定了数百个Hox11结合位点。然而，如何 Hox11的转录功能调节染色质状态，这些位点的可及性尚不清楚。 这一建议试图了解Hox11在骨软骨过程中调节的表观基因组图谱 差异化。在拟议的实验(目标1)中，我将使用一种新的染色质分析方法，即多个 标记(多标签)在胚胎对照和Hox11突变干祖细胞中的靶标识别 通过探测几个组蛋白修饰来确定细胞的染色质状态。此外，我将调查如何 Hox11功能调节骨软骨分化过程中染色质的可及性，使用ATAC-Seq 胚胎对照和Hox11突变干祖细胞(目标2)。综上所述，拟议的实验 将阐明Hox11功能调节骨骼干细胞/祖细胞分化的机制 骨骼的发育和再生。

PROJECT SUMMARY/ABSTRACT Hox genes are a family of evolutionarily conserved transcription factors that are essential for the proper development of the axial and appendicular skeleton during embryogenesis. Posterior Hox genes (Hox9-13) are critical for patterning the skeletal elements of the limb along the proximodistal axis. Specifically, Hox11 paralogous genes pattern the zeugopod, which is comprised of the radius/ulna and the tibia/fibula in the forelimb and hindlimb, respectively. Recent studies have demonstrated that Hox11 genes are expressed in skeletal stem/progenitor cell population that self-renews and gives rise to osteoblasts, chondrocytes, and adipocytes of the zeugopod throughout life. Loss of Hox11 function in skeletal stem progenitor cells impairs their capacity to differentiate into osteoblasts; subsequently affecting bone remodeling and repair. Further, Hoxa11eGFP reporter expression demonstrates that Hox11 mutant stem/progenitor cells turn off Hox expression, leave the stem/progenitor pool and initiate differentiation but fail to differentiate into mature osteoblasts. These findings are consistent with a model in which Hox11 function is required to establish the differentiation potential during lineage commitment. Despite well understood genetic contributions to limb development, maintenance and repair, the mechanisms by which Hox11 regulates skeletal stem cell differentiation remain unknown. My current work in the Wellik laboratory focuses on identifying Hox11-bound genomic loci along with RNA-Seq analyses to identify potential downstream target genes that regulate stem/progenitor cell differentiation. Taking advantage of two newly generated epitope-tagged mouse alleles (Hoxa11-3XFLAG and Hoxd11-3XFLAG) and using Cleavage Under Targets and Release Using Nuclease (CUT&RUN) assays, I have identified hundreds of Hox11 binding sites in stem/progenitor cells. However, how Hox11 transcriptional functions regulate the chromatin state and accessibility of these sites remains unclear. This proposal seeks to understand the epigenomic landscape regulated by Hox11 during osteochondral differentiation. In the proposed experiments (Aim 1), I will utilize a novel chromatin profiling method, Multiple Target Identification by Tagmentation (MulTI-Tag) in embryonic control and Hox11 mutant stem progenitor cells to define their chromatin status by probing for several histone modifications. Further, I will investigate how Hox11 function regulates chromatin accessibility during osteochondral differentiation, using ATAC-Seq in embryonic control and Hox11 mutant stem progenitor cells (Aim 2). Taken together, the proposed experiments will elucidate the mechanism by which Hox11 function regulates skeletal stem/progenitor cell differentiation in the development and regeneration of the skeleton.