Transcriptional regulation of progenitor cell fate in craniofacial ligament regeneration

项目摘要/摘要 韧带损伤因再次损伤和最终骨性关节炎的风险急剧增加而变得复杂。这些 风险源于分化失败；撕裂的哺乳动物韧带用纤维疤痕组织而不是 与真韧带细胞结合，改变修复后韧带的生物力学性能，破坏韧带的稳定性 附近的小酒馆。而目前研究韧带修复的动物模型(小鼠和兔)主要是对韧带的概括。 由于伤痕累累，它们不太可能对再生疗法的目标产生新的见解。要解决这个问题 缺点是，我们实验室已经开发出高度再生的斑马鱼作为头面部韧带的模型。 再生。斑马鱼有非凡的能力再生它们的韧带，而不会在 月份。我们的初步数据表明，斑马鱼的下颌关节支撑互操作(IOP)韧带 横断后28天即显示与未损伤韧带相同的形态。在此期间， 损伤的韧带被致密的间质取代，间质沉积复杂的细胞外基质以重建 功能性韧带。此外，初步的血统追踪实验表明，未受伤的韧带 再生性间充质来源于颅神经脊区。这个项目的目的是确定 改造韧带的间质细胞，以及促进韧带的遗传和表观遗传变化 韧带细胞的命运。在目标1中，我将使用在韧带受伤后增加可访问性的增强剂峰，以首先 将再生间充质的谱系追溯到再生韧带，然后追溯谱系 通过再生来修复骨膜。在目标2中，我将分析我的单核多倍体(基因表达 和每个细胞的染色质可访问性)数据 候选转录因子的简短列表，使用基序可获得性、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 采用韧带细胞命运，并产生敲除斑马鱼品系，以评估是否每个转录因子 对于韧带细胞的命运来说是必要的。通过这些目标，我们将揭示一种新的细胞群体，能够 再生韧带，以及韧带细胞分化所需的转录因子。 通过本计划概述的培训计划将发展技术、指导和沟通 作为一名研究颅面再生的独立科学家，建立我未来职业生涯所需的技能。 通过Joanna Smeeton博士(赞助商)、Stavros Thomopoulos博士(共同赞助商)和我的 论文咨询委员会，我将为我过渡到一个领先的博士后研究员职位做好准备。 生物医学机构。斯米顿博士对斑马鱼作为颅面模型有广泛的了解 Thomopoulos博士是翻译小鼠肌腱开发方面的专家，具有出色的 导师关系记录。在他们的共同赞助下，我将学习尖端的计算和体内 遗传学，在再生颅面生物学领域发展富有成效的职业。

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.