Dissecting Stem Cell Heterogeneity in the Zebrafish Skeletal System

剖析斑马鱼骨骼系统中的干细胞异质性

• 批准号: 10561710
• 负责人: Claire Elizabeth Arata
• 金额: $ 4.85万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10561710
• 关键词: ATAC-seq; Ablation; Address; Adult; Animals; Automobile Driving; Behavior; Biological Assay; Biology; Bone Injury; Bone Marrow; Bone Regeneration; Bone callus; Cartilage; Cell Lineage; Cells; Chondrocytes; Chromatin; Clinic; Cre-LoxP; Data; Development; Embryo; Endosteum; Enhancers; Environment; Epiphysial cartilage; Face; Fracture; Gene Expression; Genes; Genetic; Genomics; Goals; Head; Heterogeneity; Human; Image; Imaging Device; Injury; Label; Limb structure; Maintenance; Marrow; Mediating; Medical; Memory; Mentors; Mesenchymal; Modeling; Movement; Musculoskeletal; Organ; Periosteum; Physiologic Ossification; Population; Quality of life; Regenerative Medicine; Research; Role; Running; Scientist; Skeletal system; Skeleton; Structure; System; Techniques; Testing; Training; Work; Writing; Zebrafish; bone; bone repair; career development; cell type; craniofacial; craniofacial bone; craniofacial repair; cranium; epigenetic memory; experimental study; genetic approach; improved; innovation; intramembranous bone; limb bone; novel; premaxilla; programs; recombinase; regeneration model; regenerative; repaired; response to injury; single-cell RNA sequencing; skeletal; skeletal stem cell; skeletal tissue; skill acquisition; stem cell growth; stem cell self renewal; stem cells; transcriptome; wound

PROJECT SUMMARY/ABSTRACT: Skeletal tissues provide structure that allows movement and protects essential organs in the body from damage. Whereas bone displays some capacity for repair, non-healing bone injuries remain a major financial and medical burden. Understanding the potential for skeletal stem cells (SSCs) to improve bone repair therefore holds great promise. A challenge for the field of craniofacial bone repair is that these bones have a different developmental trajectory from the more studied limb bones and undergo direct ossification rather than cartilage-mediated repair in response to injury. Thus, it remains unclear the extent to which the repair of craniofacial intramembranous bones depends on the same suites of SSCs as those of the limbs. In this proposal, I investigate a hypothesis that there are two fundamentally distinct origins of SSCs in bones: one type of SSC derived from naïve mesenchymal cells in the embryonic perichondrium and periosteum (PO- SSCs) and a second type derived from hypertrophic chondrocytes of the growth plate, which dedifferentiate and move into the marrow cavity (GP-SSCs). Using intersectional genetics, lineage tracing, conditional cell ablation, and assays of open chromatin, I will test that GP-SSCs are especially important for cartilage callus formation due to maintenance of accessible cartilage enhancers from their growth plate origin (i.e. epigenetic memory). In the craniofacial intramembranous bones, the lack of growth plates and hence GP-SSCs would result in direct ossification during repair. To test these models, I have developed innovative models of intramembranous (premaxilla) and endochondral (ceratohyal) bone regeneration in the adult zebrafish head. Using parallel Cre-Lox and Dre-Lox systems, I will be able to simultaneously trace PO-SSCs and GP-SSCs and assess their contributions to and requirements for the repair of intramembranous versus endochondral bone repair. Together, my studies should reveal mechanisms by which SSCs regenerate intramembranous bones differently than endochondral bones, which will inform approaches to specifically repair the intramembranous bones of the face and skull. My mentor, Dr. Gage Crump, has an exceptional training record and runs the Development, Stem Cells, and Regenerative Medicine program to which I belong. The Crump lab is located within the rapidly growing Broad Stem Cell Institute, which is a highly collaborative and dynamic environment for my scientific development. These interactions will help me in adapting emerging techniques such as scRNAseq and ATACseq to my novel zebrafish bone regeneration models. A training plan that incorporates acquisition of skillsets in zebrafish genetics and imaging, specialized coursework in genomics, the honing of presentation and writing skills, and career development will help me in achieving my goal of becoming a successful independent scientist in the field of craniofacial regenerative medicine.

项目概要/摘要： 骨骼组织提供允许运动的结构并保护身体的重要器官免受 损害。尽管骨骼显示出一定的修复能力，但不愈合的骨损伤仍然是一个主要的经济问题 和医疗负担。了解骨骼干细胞 (SSC) 改善骨修复的潜力 因此前景广阔。颅面骨修复领域面临的挑战是这些骨头具有 与更多研究的四肢骨不同的发育轨迹，并且经历直接骨化而不是 软骨介导的损伤修复。因此，目前还不清楚修复的程度。 颅面膜内骨依赖于与四肢相同的 SSC 套件。在这个 提案中，我研究了一个假设，即骨骼中的 SSC 有两种根本不同的起源：一是 源自胚胎软骨膜和骨膜中幼稚间充质细胞的 SSC 类型（PO- SSC）和源自生长板肥大软骨细胞的第二种类型，其去分化 并进入骨髓腔（GP-SSC）。使用交叉遗传学、谱系追踪、条件细胞 消融和开放染色质测定，我将测试 GP-SSC 对软骨愈伤组织特别重要 由于从其生长板起源维持可接近的软骨增强剂（即表观遗传）而形成 记忆）。在颅面膜内骨中，缺乏生长板，因此缺乏 GP-SSCs 导致修复过程中直接骨化。为了测试这些模型，我开发了创新模型 成年斑马鱼头部膜内（前上颌骨）和软骨内（角软骨）骨再生。 使用并行 Cre-Lox 和 Dre-Lox 系统，我将能够同时追踪 PO-SSC 和 GP-SSC 并评估它们对膜内修复与软骨内修复的贡献和要求 骨骼修复。总之，我的研究应该揭示 SSC 膜内再生的机制 骨骼与软骨内骨骼不同，这将为专门修复软骨的方法提供信息 面部和颅骨的膜内骨。我的导师盖奇·克伦普 (Gage Crump) 博士拥有出色的培训记录 并负责我所属的开发、干细胞和再生医学项目。 The Crump lab 位于快速发展的布罗德干细胞研究所内，这是一个高度协作和充满活力的研究所 科学发展的环境。这些互动将帮助我适应新兴技术 例如 scRNAseq 和 ATACseq 到我的新型斑马鱼骨再生模型。一个培训计划 包括斑马鱼遗传学和成像技能的获取、基因组学专业课程、 演讲和写作技巧的磨练以及职业发展将帮助我实现我的目标 成为颅面再生医学领域成功的独立科学家。