Transcriptional regulation of progenitor cell fate in craniofacial ligament regeneration

颅面韧带再生中祖细胞命运的转录调控

• 批准号: 10709889
• 负责人: Troy Anderson
• 金额: $ 4.85万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10709889
• 关键词: ATAC-seq; Acute; Address; Adopted; Adult; Advisory Committees; Automobile Driving; Binding; Biological Assay; Biology; Biomechanics; Candidate Disease Gene; Cell Differentiation process; Cell Nucleus; Cells; Cephalic; Chromatin; Cicatrix; Clustered Regularly Interspaced Short Palindromic Repeats; Communication; Complex; Data; Degenerative polyarthritis; Deposition; Development; Enhancers; Epigenetic Process; Extracellular Matrix; Failure; Fellowship; Future; Gene Expression; Genetic; Genetic Transcription; Genomic Segment; Human; Injury; Institution; Joint Instability; Joint repair; Joints; Knock-out; Knockout Mice; Knowledge; Learning; Life; Ligaments; Mandible; Mentorship; Mesenchymal; Mesenchyme; Modeling; Molecular Target; Morphology; Mus; Mutation; Natural regeneration; Neural Crest; Operative Surgical Procedures; Oryctolagus cuniculus; Patient-Focused Outcomes; Periosteal Cell; Periosteum; Population; Postdoctoral Fellow; Productivity; Property; Recurrence; Risk; Scientist; Signal Transduction; Source; Specific qualifier value; Techniques; Temporomandibular Joint; Testing; Therapeutic; Time; Tissues; Training; Transcriptional Regulation; Translating; Transposase; Zebrafish; career; cell regeneration; cell type; craniofacial; differential expression; experimental study; gene regulatory network; healing; improved; in vivo; insight; ligament development; ligament injury; loss of function; multiple omics; mutant; novel; osteogenic; overexpression; progenitor; regenerative; regenerative therapy; repair model; repaired; scleraxis; single nucleus RNA-sequencing; skeletal; skills; stem cells; tendon development; transcription factor; translational study

PROJECT SUMMARY/ABSTRACT Ligament injuries are compounded by the drastically increased risks of reinjury and eventual osteoarthritis. These risks result from a failure of differentiation; torn mammalian ligaments reform with fibrous scar tissue rather than with true ligamentocytes, altering the biomechanical properties of the repaired ligament and destabilizing the nearby joint. While the current models (mice and rabbits) used to study ligament repair recapitulate ligament scarring, they are unlikely to yield novel insights into targets for regenerative therapeutics. To address this shortcoming, our lab has developed the highly regenerative zebrafish as a model for craniofacial ligament regeneration. Zebrafish have the remarkable capacity to regenerate their ligaments without scarring in under a month. Our preliminary data demonstrate that the jaw joint-supporting interopercle (IOP) ligament in zebrafish shows identical morphology to the uninjured ligament as soon as 28 days after transection. During this time, the injured ligament is replaced with a dense mesenchyme which deposits a complex extracellular matrix to remake a functional ligament. Additionally, preliminary lineage tracing experiments show that both the uninjured ligament and the regenerative mesenchyme are cranial neural crest-derived. This project aims to identify the source of the mesenchymal cells which reform the ligament, as well as the genetic and epigenetic changes which promote ligamentocyte fate. In Aim 1, I will use enhancer peaks with increased accessibility after ligament injury to firstly trace the lineage of the regenerative mesenchyme to the regenerated ligament, and secondly trace the lineage of the periosteum through regeneration. In Aim 2, I will analyze my single-nuclei multiomic (gene expression and chromatin accessibility for each cell) data from jaw joints through IOP ligament regeneration to generate a short list of candidate transcription factors using a selection funnel of motif accessibility, gene expression, and transcription factor binding. I will then assess in vivo if these transcription factors are expressed before ligamentocyte fate is adopted, and generate knockout zebrafish lines to assess if each transcription factor is necessary for ligamentocyte fate. Through these aims, we will unveil a novel population of cells capable of regenerating ligaments, as well as the transcription factors necessary for ligamentocyte differentiation. The training plan outlined through this proposal will develop the techniques, mentorship, and communication skills necessary to establish my future career as an independent scientist studying craniofacial regeneration. Through the mentorship of Dr. Joanna Smeeton (Sponsor), Dr. Stavros Thomopoulos (Co-sponsor), and my thesis advisory committee, I will be well prepared for my transition to a postdoctoral fellowship at a leading biomedical institution. Dr. Smeeton has extensive knowledge of the zebrafish as a model for craniofacial regeneration, and Dr. Thomopoulos is an expert in translational mouse tendon development with an outstanding mentorship record. Under their combined sponsorship, I will learn cutting-edge computational and in vivo genetics to develop a productive career in regenerative craniofacial biology.

项目摘要/摘要 韧带损伤大大增加了重伤和最终骨关节炎的风险。这些 风险是由于分化失败而导致的；用纤维疤痕组织撕裂的哺乳动物韧带改革而不是 使用真正的韧带细胞，改变修复韧带的生物力学特性并破坏稳定 附近的关节。而目前用于研究韧带修复的当前模型（小鼠和兔子）概括了韧带 疤痕，它们不太可能对再生治疗剂的目标产生新颖的见解。解决这个问题 缺点，我们的实验室开发了高度再生斑马鱼作为颅面韧带的模型 再生。斑马鱼具有重生韧带的非凡能力，而不会在 月。我们的初步数据表明，斑马鱼中的下颌关节支持互动（IOP）韧带 在横切后28天后，显示出与未受伤的韧带相同的形态。在此期间， 受伤的韧带被密集的间充质代替，该密质沉积了复杂的细胞外基质以重塑 功能性韧带。此外，初步的谱系跟踪实验表明，这两种韧带 再生间充质是颅神经rest衍生的。该项目旨在确定 改革韧带的间充质细胞以及促进的遗传和表观遗传变化 韧带细胞命运。在AIM 1中，我将使用韧带受伤后增加可及性的增强峰首先 将再生间充质的谱系追溯到再生韧带，其次追踪谱系 通过再生的骨膜。在AIM 2中，我将分析我的单核多核酸（基因表达） 每个单元格的染色质可及性）来自颌关节到IOP韧带再生的数据 候选转录因子的简短列表，使用基序可访问性，基因表达和 转录因子结合。然后，我将在体内评估这些转录因子是否在表达之前 采用韧带卵细胞命运，并生成敲除斑马鱼线以评估每个转录因子是否为 韧带细胞命运所必需的。通过这些目标，我们将推出一个能够 再生韧带，以及韧带分化所需的转录因子。 通过此提案概述的培训计划将开发技术，指导和沟通 确立我作为研究颅面再生的独立科学家未来职业所必需的技能。 通过Joanna Smeeton博士（赞助商），Stavros Thomopoulos博士（共同赞助者）的指导 论文咨询委员会，我将为过渡到领先的博士后奖学金做好准备 生物医学机构。 Smeeton博士对斑马鱼有广泛的了解作为颅面的典范 再生，Thomopoulos博士是翻译老鼠肌腱开发的专家 指导记录。在他们的共同赞助下，我将学习尖端计算和体内 遗传学在再生颅面生物学领域发展生产力。