Modular Control of Cranial Skeletal Connectivity through Joint-Specific Enhancers

通过关节特异性增强器对颅骨连接进行模块化控制

• 批准号: 10462414
• 负责人: Kelsey Elliott
• 金额: $ 6.76万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10462414
• 关键词: Adult; Affect; Alleles; Animal Model; Binding; Binding Sites; Bioinformatics; Biological Assay; Biology; California; Cartilage; Cells; Cephalic; Chromatin; Communities; Country; Craniofacial Abnormalities; Craniosynostosis; DNA Binding; Data; Data Set; Defect; Development; Disease; Dominant-Negative Mutation; Ear; Embryo; Emerging Technologies; Enhancers; Face; Fellowship; Fishes; Gene Expression; Gene Expression Profiling; Gene Transfer Techniques; Genes; Genetic; Genomics; Goals; Head; Health Sciences; Human; In Situ Hybridization; Institution; Jaw; Joints; Label; Limb structure; Location; Maintenance; Mediating; Mentorship; Modeling; Mutation; Natural regeneration; Neural Crest; Neural Crest Cell; Pattern; Physiologic Ossification; Play; Population; Positioning Attribute; Regulator Genes; Regulatory Element; Replacement Arthroplasty; Research; Resolution; Role; Scientist; Site; Skeletal Development; Skeleton; Specific qualifier value; Surgical sutures; Synovial joint; Techniques; Temporomandibular Joint; Temporomandibular Joint Disorders; Testing; To specify; Transcription Factor AP-1; Transgenic Organisms; Universities; Vertebral column; Work; Zebrafish; arthropathies; bone; cartilaginous; cell type; cost; craniofacial; craniofacial development; craniofacial disorder; cranium; design; developmental genetics; experience; experimental study; flexibility; improved; innovation; joint formation; joint function; large datasets; morphogens; mutant; novel; precursor cell; prevent; professor; promoter; single cell analysis; skeletal; skeletal tissue; skull base; tenure track; transcription factor; transcriptome; transgene expression

PROJECT SUMMARY/ABSTRACT Temporomandibular Joint Disorder (TMJ) and precocious ossification of cranial synchondroses are just two examples of defects caused by disruptions to cranial joints. Proper form and function of joints are required for connectivity and flexibility of the vertebrate skeleton. While the cranium has a vast number and subtype of joints, most joint biology studies have focused on the limbs, leaving a gap in our understanding of how the cranial joints, which are unique in their cranial neural crest cell contribution, develop. Our lab has recently generated single - cell transcriptome and chromatin accessibility data for cranial neural crest-derived cells across 7 timepoints (embryo to adult) in zebrafish. I have been able to extract preliminary global information about the transcription factors and cis-regulatory elements, or enhancers, which separate cranial joints from other types of skeletal tissues. While prior models have proposed joint cartilage is simply immature cartilage, my preliminary data shows several enhancers drive expression in only cranial joints. Additionally, other enhancers drive expression in only replacement cartilage. These data suggest the existence of two completely separate populations, with joints being specified distinctly from replacement cartilage. In this proposal, I use the powerful genetics of the zebrafish model to investigate what enhancers and transcription factors differentiate cranial joints from replacement cartilage. My preliminary bioinformatic analyses suggest that Ap-1 transcription factors (Jun/Fos) work with the master cartilage transcription factor Sox9 to generally specify joint cartilage. Interestingly, mutations in several transcription factors can independently cause defects to only specific cranial joints, suggesting localized transcription factors may specialize joints. The aims outlined in this proposal investigate the neural crest-derived cells in developing cranial joints (Aim 1), how they are uniquely patterned separately from replacement cartilage (Aim 2), and how region-specific transcription factors are responsible for specializing cranial joints in different parts of the head and face (Aim 3). I plan to utilize techniques such as snATAC-seq and CUT&Tag to determine if enhancers are uniquely opened or activated in joints. By combining these large datasets with transgenic assays to confirm if transcription factor motifs are necessary and sufficient for joint activity and identity, I will significantly enhance our understanding of cranial joint development. This project and activity plan for fellowship period are designed to lay the groundwork for my long-term goal of obtaining a position as a tenure-track Professor at a top-tier academic research institution. Furthermore, the data generated in this project will prepare me to generate a competitive K99 application. I will receive mentorship from Dr. Gage Crump, a leading scientist in zebrafish craniofacial development. The experiments in this proposal will take place on the Health Sciences Campus of the University of Southern California, which hosts one of the most experienced communities of craniofacial and skeletal biologists in the country.

项目概要/摘要 颞下颌关节紊乱病 (TMJ) 和颅骨软骨病早熟骨化只是其中的两个 由颅骨关节破坏引起的缺陷的例子。关节的正确形式和功能是必需的 脊椎动物骨骼的连接性和灵活性。虽然颅骨有大量的关节和亚型的关节， 大多数关节生物学研究都集中在四肢上，这使得我们对颅关节如何 其颅神经嵴细胞的贡献是独一无二的，并且发育。我们的实验室最近生成了单 - 7 个时间点颅神经嵴衍生细胞的细胞转录组和染色质可及性数据 （胚胎到成体）斑马鱼。我已经能够提取有关转录的初步全局信息 将颅关节与其他类型的骨骼区分开的因子和顺式调节元件或增强剂 组织。虽然之前的模型提出关节软骨只是未成熟的软骨，但我的初步数据显示 一些增强子仅在颅关节中驱动表达。此外，其他增强子仅驱动表达 置换软骨。这些数据表明存在两个完全独立的种群，具有联合 与置换软骨不同。在这个提案中，我利用了斑马鱼强大的遗传学 模型来研究哪些增强子和转录因子将颅关节与置换关节区分开来 软骨。我的初步生物信息分析表明 Ap-1 转录因子 (Jun/Fos) 与 掌握软骨转录因子Sox9来全面指定关节软骨。有趣的是，一些突变 转录因子可以独立地仅导致特定颅关节的缺陷，这表明局部 转录因子可能使关节特化。本提案概述的目标是研究神经嵴衍生的 发育中的颅关节中的细胞（目标 1），它们如何与替代软骨分开形成独特的图案 （目标 2），以及区域特异性转录因子如何负责不同部位的颅关节专门化 头部和脸部的部分（目标 3）。我计划利用 snATAC-seq 和 CUT&Tag 等技术来确定 如果增强剂在关节中被独特地打开或激活。通过将这些大型数据集与转基因检测相结合 为了确认转录因子基序对于关节活动和身份是否是必要和充分的，我将显着 增强我们对颅骨联合发育的理解。本项目及联谊期间活动计划为 旨在为我在某大学获得终身教授职位的长期目标奠定基础 一流的学术研究机构。此外，该项目中生成的数据将使我准备好生成 具有竞争力的 K99 应用程序。我将接受斑马鱼领域领先科学家 Gage Crump 博士的指导 颅面发育。本提案中的实验将在健康科学园区进行 南加州大学拥有最有经验的颅面和面部护理社区之一 该国的骨骼生物学家。