Dissecting Stem Cell Heterogeneity in the Zebrafish Skeletal System

项目摘要/摘要： 骨骼组织提供了允许运动的结构，并保护身体的基本器官免受 损坏。尽管骨骼显示出一定的修复能力，但不可愈合的骨骼损伤仍然是主要的经济来源 和医疗负担。了解骨骼干细胞(SSCs)促进骨修复的潜力 因此蕴含着巨大的希望。颅面骨修复领域面临的一个挑战是，这些骨骼具有 与更多研究的四肢骨骼的发育轨迹不同，并经历直接骨化，而不是 软骨介导的损伤修复反应。因此，目前仍不清楚修复的程度 颅面膜内骨依赖于与四肢相同的SSCs组。在这 提议，我调查了一个假设，即骨骼中存在两个根本不同的SSCs来源：一个 胚胎软骨膜和骨膜中幼稚间充质细胞来源的SSC类型(PO- SSCs)和第二种来源于生长板肥大的软骨细胞，它们去分化 并进入骨髓腔(GP-SSCs)。使用跨部门遗传学、谱系追踪、条件细胞 消融，以及开放染色质的检测，我将测试GP-SSCs对软骨骨痂特别重要 由于从生长板来源(即表观遗传)保持可接近的软骨增强剂而形成的 内存)。在颅面膜内骨中，缺乏生长板，因此GP-SSCs将 在修复过程中会导致直接骨化。为了测试这些模型，我开发了创新的模型 成年斑马鱼头的膜内(前颌骨)和软骨内(角质)骨再生。 使用并行CRE-Lox和DRE-Lox系统，我将能够同时跟踪PO-SSC和GP-SSC 并评估它们对膜内与软骨内修复的贡献和需求 骨修复。总之，我的研究应该揭示SSCs在膜内再生的机制。 骨骼不同于软骨内骨骼，这将为专门修复 面部和头盖骨的膜内骨。我的导师盖奇·克兰普博士有出色的训练记录 负责我所属的发育、干细胞和再生医学项目。克伦普实验室 位于快速发展的远大干细胞研究所内，该研究所是一个高度协作和充满活力的 为我的科学发展创造环境。这些互动将帮助我适应新兴的技术 例如scRNAseq和ATACseq到我的新斑马鱼骨骼再生模型。一项培训计划 结合在斑马鱼遗传学和成像方面的技能，在基因组学方面的专门课程， 演讲和写作技能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一名成功的独立科学家。

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.