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.

项目概要/摘要 韧带损伤会因再次受伤和最终骨关节炎的风险急剧增加而变得更加复杂。这些 差异化失败导致风险；哺乳动物撕裂的韧带会通过纤维疤痕组织而不是 与真正的韧带细胞一起，改变修复韧带的生物力学特性并破坏韧带的稳定性 附近的关节。目前用于研究韧带修复的模型（小鼠和兔子）重演韧带 疤痕，它们不太可能对再生治疗的目标产生新的见解。为了解决这个问题 针对这一缺点，我们实验室开发了高度再生的斑马鱼作为颅面韧带模型 再生。斑马鱼具有非凡的韧带再生能力，并且不会在一定条件下留下疤痕。 月。我们的初步数据表明，斑马鱼的颌关节支撑间鳃（IOP）韧带 横断后 28 天，韧带就显示出与未受伤韧带相同的形态。在此期间， 受伤的韧带被致密的间质取代，沉积复杂的细胞外基质以重塑 功能性韧带。此外，初步的谱系追踪实验表明，未受伤的韧带 再生间充质来源于颅神经嵴。该项目旨在确定 改造韧带的间充质细胞，以及促进韧带修复的遗传和表观遗传变化 韧带细胞的命运。在目标 1 中，我将首先使用韧带损伤后具有更高可达性的增强峰 追踪再生间质到再生韧带的谱系，其次追踪谱系 通过再生骨膜。在目标 2 中，我将分析我的单核多组学（基因表达 以及每个细胞的染色质可及性）来自颌关节的数据通过 IOP 韧带再生生成 使用基序可及性、基因表达和特征的选择漏斗列出候选转录因子的简短列表 转录因子结合。然后我将在体内评估这些转录因子之前是否表达过 采用韧带细胞命运，并生成敲除斑马鱼系以评估每个转录因子是否 韧带细胞命运所必需的。通过这些目标，我们将推出一个新的细胞群，能够 再生韧带，以及韧带细胞分化所需的转录因子。 本提案概述的培训计划将开发技术、指导和沟通 建立我未来作为一名研究颅面再生的独立科学家的职业所需的技能。 在 Joanna Smeeton 博士（发起人）、Stavros Thomopoulos 博士（联合发起人）和我的指导下 论文咨询委员会，我将为过渡到领先的博士后奖学金做好充分准备 生物医学机构。 Smeeton 博士对斑马鱼作为颅面模型有丰富的了解 Thomopoulos 博士是小鼠肌腱转化领域的专家，具有杰出的再生能力 辅导记录。在他们的联合赞助下，我将学习最前沿的计算和体内 遗传学在再生颅面生物学领域发展富有成效的职业生涯。