ECM remodeling and crosstalk with cell fate in zebrafish ligament regeneration

项目总结/摘要 健康的关节对日常活动很重要，而导致残疾的主要原因是骨关节炎。 韧带损伤通过纤维性瘢痕组织解决，由于下部瘢痕组织使关节不稳定。有 由于我们的韧带细胞再生能力有限，因此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，并且在以下方面不足： 解决这个再生的挑战。我们的实验室开发了一种新的斑马鱼模型来研究基本的 眼间下颌骨（IOM）颅面韧带再生的生物学基础。我们的初步数据 研究表明，在IOM切断后，斑马鱼在一个月内再生了无疤痕的韧带。我们证明了 韧带细胞去分化并有助于再生韧带。这种严格调控的再生 反应的特征在于动态变化的细胞外基质（ECM）之间的恒定串扰， 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，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 重塑，我们表明，LGMN突变体愈合与一个错误的模式，疤痕韧带横断后。在 与WT再生相反，lgmn突变体纤维化愈合的特征在于韧带细胞的缺陷， 脱分化，新旧组织不能整合，以及整个过程中胶原蛋白紊乱。 韧带愈合为了研究Lgmn介导的再生的作用，本建议将鉴定Lgmn介导的亚细胞再生。 Lgmn在颅面韧带再生中的定位和分子机制。此外，本发明还 该建议还将表征斑马鱼IOM韧带在稳态期间的蛋白质组学谱， 再生和纤维化。这将产生新的知识差异表达的蛋白质在这些过程中， 不同的状态，可用于识别促再生微环境的有效分子调节剂。 我们初步的scRNAseq分析显示，在损伤部位有一个巨噬细胞亚群表达ECM， 包括LGMN的重塑因子。在IOM横断后，lgmn突变体具有较少的巨噬细胞存在于 受伤的地方。总之，这表明巨噬细胞衍生的LGMN在介导促GBM中的重要作用。 再生微环境为了测试这一点，我计划使用scRNAseq来表征巨噬细胞中的变化， WT和Igmn突变体之间的子集。此外，我将功能测试lgmn突变巨噬细胞和使用 过继性巨噬细胞转移以测试是否挽救了LGMN突变体瘢痕愈合。通过这些目标，我将 揭示Lgmn在韧带再生过程中介导损伤微环境的作用。下 乔安娜·斯米顿博士和劳拉·约翰斯顿博士沿着一起指导他们在斑马鱼方面的综合专业知识 生物学和遗传学的联合，我将为我下一个博士后研究员的角色做好准备。培训计划 概述将帮助我发展必要的技能，成功地作为一个独立的颅面研究科学家。

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.