ECM remodeling and crosstalk with cell fate in zebrafish ligament regeneration

斑马鱼韧带再生中 ECM 重塑和细胞命运的串扰

• 批准号: 10748627
• 负责人: Julia Mo
• 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: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10748627
• 关键词: Acute; Address; Arthralgia; Arthritis; Automobile Driving; Biochemical; Bioinformatics; Biology; Biophysics; Caspase; Cell Cycle; Cell Nucleus; Cell surface; Cells; Chronic; Cicatrix; Collagen; Coupled; Cues; Data; Defect; Degenerative polyarthritis; Down-Regulation; Event; Extracellular Matrix; Failure; Fibrosis; Future; Genetic; Genetic Transcription; Health; Homeostasis; Immune response; Inferior; Injury; Invaded; Joints; Knowledge; Ligaments; Macrophage; Mandible; Mechanics; Mediating; Mentorship; Mesenchymal; Modeling; Molecular; Movement; Natural regeneration; Neural Crest; Patient-Focused Outcomes; Pattern; Phagocytosis; Population; Postdoctoral Fellow; Process; Proliferating; Proteins; Proteome; Proteomics; Regenerative response; Regulation; Research; Risk; Role; Scientist; Signal Transduction; Site; Synovial joint; System; Testing; Tissue Grafts; Tissues; Training; Validation; Work; Zebrafish; asparaginylendopeptidase; cell dedifferentiation; cell growth regulation; cell type; comparative; craniofacial; cytokine; differential expression; disability; experience; extracellular; functional improvement; healing; improved; injured; insight; jaw movement; joint mobilization; ligament injury; migration; mutant; novel; preservation; prevent; programs; reconstruction; regenerative; repaired; response; response to injury; scleraxis; single-cell RNA sequencing; skills

PROJECT SUMMARY/ABSTRACT Healthy joints are important for everyday activities and the leading cause of disability is osteoarthritis. Ligament injury resolves with fibrous scar tissue that destabilizes the joint due to inferior scar tissue. There are no adequate treatments to address this problem due to the limited capacity of our ligamentocytes to regenerate native tissue. Current models used to study ligament repair experience fibrotic healing and are insufficient in addressing this challenge to regenerate. Our lab has developed a novel zebrafish model to study the basic biology underlying interopercular mandibular (IOM) craniofacial ligament regeneration. Our preliminary data shows that after IOM transection, zebrafish regenerate a scar-free ligament within a month. We show that ligamentocytes dedifferentiate and contribute to the regenerated ligament. This tightly regulated regenerative response is characterized by constant crosstalk between the dynamically changing extracellular matrix (ECM) and the different cell populations present in the injury microenvironment. For comparative analysis, we have developed a legumain (lgmn) mutant zebrafish model that experiences fibrotic healing to gain insight into the molecular and cellular regulation of regeneration vs. fibrosis. Lgmn is a cysteine protease involved in ECM remodeling and we show that lgmn mutants heal with a mis-patterned, scarred ligament after transection. In contrast to WT regeneration, lgmn mutant fibrotic healing is characterized by a defect in ligamentocyte dedifferentiation, failure to integrate new and old tissue, and disorganized collagen throughout the course of ligament healing. To investigate the role of Lgmn-mediated regeneration, this proposal will identify the subcellular localization and molecular mechanism of Lgmn in the context of craniofacial ligament regeneration. Additionally, this proposal will also characterize the proteomic profile of the zebrafish IOM ligament during homeostasis, regeneration, and fibrosis. This will yield new knowledge on differentially expressed proteins during these different states that can be used to identify potent molecular regulators of a pro-regenerative microenvironment. Our preliminary scRNAseq analysis shows a subset of macrophages at the injury site that express ECM remodeling factors including lgmn. Following IOM transection, lgmn mutants have less macrophages present at the injury site. Together, this indicates an important role of macrophage-derived lgmn in mediating a pro- regenerative microenvironment. To test this, I plan to use scRNAseq to characterize changes in macrophage subsets between WT and lgmn mutants. Further, I will functionally test lgmn mutant macrophages and use adoptive macrophage transfer to test if lgmn mutant scarred healing is rescued. Through these aims, I will uncover the role of Lgmn in mediating the injury microenvironment during ligament regeneration. Under the mentorship of Dr. Joanna Smeeton and Dr. Laura Johnston along with their combined expertise in zebrafish joint biology and genetics, I will be well prepared for my next role as a post-doctoral fellow. The training plan outlined will help me develop the necessary skills to succeed as an independent craniofacial research scientist.

项目概要/摘要 健康的关节对于日常活动很重要，而导致残疾的主要原因是骨关节炎。 韧带损伤会随着纤维疤痕组织的消失而消失，纤维疤痕组织会因劣质疤痕组织而使关节不稳定。有 由于我们的韧带细胞再生能力有限，没有足够的治疗方法来解决这个问题 天然组织。目前用于研究韧带修复的模型经历了纤维化愈合，并且在 应对这一挑战以实现复兴。我们的实验室开发了一种新型斑马鱼模型来研究斑马鱼的基本原理 鳃间下颌（IOM）颅面韧带再生的生物学基础。我们的初步数据 结果显示，在 IOM 横断后，斑马鱼在一个月内再生出无疤痕韧带。我们表明 韧带细胞去分化并有助于韧带再生。这种严格调控的再生 响应的特点是动态变化的细胞外基质 (ECM) 之间的持续串扰 以及损伤微环境中存在的不同细胞群。为了进行比较分析，我们有 开发了一个legumain（lgmn）突变斑马鱼模型，该模型经历纤维化愈合，以深入了解 再生与纤维化的分子和细胞调节。 Lgmn 是一种参与 ECM 的半胱氨酸蛋白酶 我们发现，lgmn 突变体在横断后会以错误模式、疤痕累累的韧带愈合。在 与 WT 再生相比，lgmn 突变体纤维化愈合的特点是韧带细胞缺陷 在整个过程中去分化、新旧组织整合失败以及胶原蛋白紊乱 韧带愈合。为了研究 Lgmn 介导的再生的作用，该提案将鉴定亚细胞 Lgmn 在颅面韧带再生中的定位和分子机制。此外， 该提案还将描述斑马鱼 IOM 韧带在稳态过程中的蛋白质组学特征， 再生、纤维化。这将产生关于这些过程中差异表达蛋白质的新知识 不同的状态可用于识别促再生微环境的有效分子调节剂。 我们的初步 scRNAseq 分析显示损伤部位表达 ECM 的巨噬细胞子集 重塑因子包括lgmn。 IOM 横切后，lgmn 突变体的巨噬细胞减少 受伤部位。总之，这表明巨噬细胞衍生的 lgmn 在介导亲- 再生微环境。为了测试这一点，我计划使用 scRNAseq 来表征巨噬细胞的变化 WT 和 lgmn 突变体之间的子集。此外，我将对lgmn突变型巨噬细胞进行功能测试并使用 过继性巨噬细胞转移以测试lgmn突变体疤痕愈合是否得到挽救。通过这些目标，我将 揭示 Lgmn 在介导韧带再生过程中损伤微环境中的作用。在下面 Joanna Smeeton 博士和 Laura Johnston 博士的指导以及他们在斑马鱼方面的综合专业知识 联合生物学和遗传学，我将为我的下一个博士后职位做好充分准备。培训计划 概述将帮助我培养作为一名独立颅面研究科学家取得成功所需的技能。