Dissecting Stem Cell Heterogeneity in the Zebrafish Skeletal System

项目总结/摘要： 骨骼组织提供了允许运动的结构，并保护身体中的重要器官免受 损害虽然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。 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的一个挑战是这些骨具有 不同的发展轨迹，从更多的研究肢骨，并进行直接骨化，而不是 软骨介导的损伤修复。因此，目前尚不清楚修复的程度， 颅面膜内骨依赖于与四肢相同的SSC套件。在这 根据我的建议，我调查了一个假设，即骨骼中的SSCs有两个根本不同的起源：一个是 型SSC来源于胚胎软骨膜和骨膜中的幼稚间充质细胞（PO- 第二种类型来源于生长板的肥大软骨细胞， 并进入骨髓腔（GP-SSC）。利用交叉遗传学，谱系追踪，条件细胞 消融和开放染色质的测定，我将测试GP-SSCs对软骨愈伤组织特别重要 由于维持来自其生长板来源的可接近的软骨增强子（即表观遗传的 内存）。在颅面膜内骨中，缺乏生长板，因此GP-SSC将 导致修复过程中直接骨化。为了测试这些模型，我开发了 膜内（前颌骨）和软骨内（ceratohyal）骨再生在成年斑马鱼头部。 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要求， 骨修复总之，我的研究应该揭示了精原干细胞膜内再生的机制。 骨与软骨内骨不同，这将为专门修复骨的方法提供信息。 面部和头骨的膜内骨。我的导师，盖奇·克博士，有着出色的训练记录 并运行开发，干细胞，和再生医学程序，我属于。Crump实验室 位于快速发展的布罗德干细胞研究所，这是一个高度协作和动态的 为我的科学发展创造环境。这些互动将帮助我适应新兴技术 如scRNAseq和ATACseq应用于我的新斑马鱼骨再生模型。培训计划， 结合了斑马鱼遗传学和成像技能的获取，基因组学的专业课程， 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。

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.