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.

项目摘要/摘要 健康关节对于日常活动很重要，残疾的主要原因是骨关节炎。 韧带损伤用纤维疤痕组织消退，由于疤痕组织下部，破坏了关节的稳定。有 由于我们的韧带再生能力有限，没有足够的治疗方法来解决此问题 天然组织。用于研究韧带修复经验纤维化愈合的当前模型，不足 应对重生的挑战。我们的实验室开发了一种新颖的斑马鱼模型来研究基本 生物学基础界面下颌（IOM）颅韧带再生。我们的初步数据 表明，在IOM横向后，斑马鱼在一个月内再生无疤痕的韧带。我们表明 韧带细胞去分化并有助于再生韧带。这种受严格调节的再生 反应的特征是动态变化的细胞外基质（ECM）之间的恒定串扰。 损伤微环境中存在的不同细胞群体。对于比较分析，我们有 开发了一种豆科蛋白酶（LGMN）突变斑马鱼模型，该模型经历了纤维化愈合，以深入了解 再生与纤维化的分子和细胞调节。 LGMN是参与ECM的半胱氨酸蛋白酶 重塑，我们表明LGMN突变体在过渡后用错误的，疤痕的韧带愈合。在 与WT再生形成对比，LGMN突变体纤维化愈合的特征是韧带细胞缺陷 在整个过程中，去分化，未能整合新的和旧的组织以及混乱的胶原蛋白 韧带愈合。为了研究LGMN介导的再生的作用，该建议将确定亚细胞 LGMN的定位和分子机制在颅面韧带再生的背景下。此外， 该建议还将表征斑马鱼IOM韧带的蛋白质组学特征， 再生和纤维化。在这些过程中，这将产生有关差异表达蛋白的新知识 可用于鉴定促再生微环境的有效分子调节剂的不同状态。 我们的初步SCRNASEQ分析显示了表达ECM的损伤部位的一部分巨噬细胞 重塑因素，包括LGMN。在IOM横断下，LGMN突变体的巨噬细胞较少 伤害部位。总之，这表明了巨噬细胞衍生的LGMN在介导研究中的重要作用 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，我将在功能上测试LGMN突变体巨噬细胞并使用 收养巨噬细胞的转移以测试LGMN突变体是否挽救了疤痕。通过这些目标，我会 发现LGMN在韧带再生过程中介导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的联合专业知识 联合生物学和遗传学，我将为我作为博士后研究员的下一个角色做好充分的准备。培训计划 概述将帮助我发展成为独立颅面研究科学家成功的必要技能。