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: 10840553
• 负责人: Deneen M Wellik
• 金额: $ 10.82万
• 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/10840553
• 关键词: ATAC-seq; Adipocytes; Adult; Affect; Alleles; Binding; Binding Sites; Biological Assay; Bone remodeling; Chondrocytes; Chromatin; Development; Development Plans; Elements; Embryo; Embryonic Development; Epitopes; Family; Forelimb; Genes; Genetic; Genetic Transcription; Growth; Hindlimb; Homeobox Genes; Impairment; Laboratories; Life; Limb Development; Limb structure; Maintenance; Mediating; Mentors; Methods; Modeling; Mus; Natural regeneration; Osteoblasts; Pattern; Population; Radial; Reporter; Research; Site; Skeletal Development; Skeleton; Work; bone fracture repair; bone repair; career development; epigenomics; experimental study; fibula; genomic locus; histone modification; mutant; novel; nuclease; osteochondral tissue; paralogous gene; progenitor; repaired; self-renewal; skeletal; skeletal stem cell; stem; stem cell differentiation; stem cell population; stem cells; tibia; transcription factor; transcriptome sequencing; ulna

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.

项目概要/摘要 Hox 基因是一个进化上保守的转录因子家族，对于正确的转录因子至关重要。 胚胎发生过程中中轴和附肢骨骼的发育。后 Hox 基因 (Hox9-13) 是 对于沿着近远轴构建肢体的骨骼元素至关重要。具体来说，Hox11 旁系同源基因塑造了 zeugopod，它由桡骨/尺骨和胫骨/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/祖细胞群能够自我更新并产生成骨细胞、软骨细胞和 zeugopod 一生中的脂肪细胞。骨骼干祖细胞中 Hox11 功能的丧失会损害 它们分化成成骨细胞的能力；随后影响骨的重塑和修复。更远， Hoxa11eGFP 报告基因表达表明 Hox11 突变干/祖细胞关闭 Hox 表达，离开茎/祖细胞库并开始分化，但未能分化为成熟 成骨细胞。这些发现与需要 Hox11 功能才能建立 谱系定型期间的分化潜力。尽管人们已充分了解遗传对肢体的贡献 发育、维持和修复，Hox11 调节骨骼干细胞的机制 分化仍然未知。我目前在 Wellik 实验室的工作重点是识别 Hox11 结合 基因组位点与 RNA-Seq 分析一起识别潜在的调节下游靶基因 干/祖细胞分化。利用两个新生成的表位标记的小鼠等位基因 （Hoxa11-3XFLAG 和 Hoxd11-3XFLAG）并使用目标下的切割和使用核酸酶的释放 (CUT&RUN) 分析中，我已经在干细胞/祖细胞中鉴定出数百个 Hox11 结合位点。然而，如何 Hox11 转录功能调节染色质状态，这些位点的可及性仍不清楚。 该提案旨在了解 Hox11 在骨软骨发育过程中调控的表观基因组景观 差异化。在拟议的实验（目标 1）中，我将利用一种新颖的染色质分析方法，即多重染色质分析方法 胚胎控制和 Hox11 突变干祖细胞中的标记 (MulTI-Tag) 目标识别 细胞通过探测几种组蛋白修饰来定义其染色质状态。此外，我将研究如何 Hox11 功能在骨软骨分化过程中调节染色质可及性，使用 ATAC-Seq 胚胎对照和 Hox11 突变干祖细胞（目标 2）。综上所述，所提出的实验 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。