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 健康的关节对日常活动很重要，而导致残疾的主要原因是骨性关节炎。 韧带损伤以纤维疤痕组织解决，由于下部瘢痕组织而使关节不稳定。确实有 由于我们韧带细胞再生的能力有限，没有适当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性愈合，并且在 应对这一再生挑战。我们实验室开发了一种新的斑马鱼模型来研究基本的 盖间下颌(IOM)颅面韧带再生的生物学基础我们的初步数据 结果显示，IOM切断后，斑马鱼在一个月内再生出无疤痕的韧带。我们证明了 韧带细胞去分化，并促进再生的韧带。这种严格监管的再生 反应的特征是动态变化的细胞外基质(ECM)之间的持续串扰 损伤微环境中存在不同的细胞群。为了进行比较分析，我们有 开发了一种豆类(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控。LGMn是一种参与细胞外基质的半胱氨酸蛋白酶 重塑和我们的研究表明，IGMN突变体在横断后愈合了一个图案错误的、伤痕累累的韧带。在……里面 与WT再生相比，IgMN突变型纤维化愈合的特征是韧带细胞的缺陷。 在整个过程中，去分化，未能整合新旧组织，以及紊乱的胶原 韧带愈合。为了研究LGMN介导的再生的作用，本方案将鉴定亚细胞 LGMN在头面部韧带再生中的定位和分子机制。另外， 这一建议还将描述斑马鱼IOM韧带在动态平衡期间的蛋白质组学特征， 再生和纤维化。这将产生关于在这些过程中差异表达蛋白质的新知识 不同的状态，可用于识别促进再生微环境的有效分子调节器。 我们的初步scRNAseq分析显示损伤部位表达ECM的巨噬细胞亚群 重塑因子包括IGMN。IOM横切后，lgMn突变体的巨噬细胞较少。 受伤的地方。综上所述，这表明巨噬细胞来源的IgMn在介导一种促性腺激素释放的过程中起重要作用。 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突变体之间的亚群。此外，我将对IgMN突变巨噬细胞进行功能性测试，并使用 过继巨噬细胞转移以检测是否挽救了lgMn突变型瘢痕愈合。通过这些目标，我将 揭示LGMN在韧带再生过程中对损伤微环境的调节作用。在……下面 乔安娜·斯米顿博士和劳拉·约翰斯顿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，我将为我的下一个角色做好准备，成为博士后研究员。培训计划 大纲将帮助我培养必要的技能，以成功地成为一名独立的头面部研究科学家。