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: 10594903
• 负责人: Kelsey Elliott
• 金额: $ 6.99万
• 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/10594903
• 关键词: Adult; Affect; Alleles; Animal Model; Binding; Binding Sites; Bioinformatics; Biological Assay; Biology; California; Cartilage; Cells; Cephalic; Chromatin; Classification; Communities; Country; Craniofacial Abnormalities; Craniosynostosis; DNA Binding; Data; Data Set; Defect; Development; Disease; Dominant-Negative Mutation; Ear; Embryo; Emerging Technologies; Enhancers; Exclusion; 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; Transcription Factor AP-1; Transgenic Organisms; Universities; Vertebral column; Work; Zebrafish; arthropathies; bone; cartilaginous; cell type; cost; craniofacial; craniofacial development; craniofacial disorder; cranium; design; experience; experimental study; flexibility; genetic testing; 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.

项目总结/摘要 颞下颌关节紊乱病（TMJ）和颅骨软骨结合的早熟骨化只是其中的两种 由颅关节破裂引起的缺陷的例子。需要关节的适当形式和功能， 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关节亚型， 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的理解存在空白， 其在颅神经嵴细胞贡献方面是独特的。我们的实验室最近制造了一个- 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 （胚胎到成年）。我已经能够提取出关于转录的初步全球信息 因子和顺式调节元件或增强子，其将颅关节与其他类型的骨骼关节分开。 组织中虽然先前的模型提出关节软骨只是不成熟的软骨，我的初步数据显示， 几种增强子仅在颅关节中驱动表达。此外，其他增强子仅在 替换软骨这些数据表明存在两个完全独立的种群， 与替代软骨不同。在这个提议中，我使用了斑马鱼强大的遗传学 模型来研究什么增强子和转录因子区分颅关节和置换 软骨我的初步生物信息学分析表明，Ap-1转录因子（Jun/Fos）与转录因子的作用有关。 主软骨转录因子Sox 9通常指定关节软骨。有趣的是，几个基因的突变 转录因子可以独立地导致缺陷，只有特定的颅关节，这表明局部 转录因子可以使关节特化。本提案中概述的目标是研究神经嵴源性 颅关节发育中的细胞（目标1），它们如何与替代软骨分开形成独特的图案 (Aim 2），以及区域特异性转录因子如何负责在不同的颅关节特化 头部和面部（目标3）。我计划利用snATAC-seq和CUT & Tag等技术来确定 如果增强子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是否是联合活动和身份所必需和充分的，我将显著地 加深我们对颅关节发育的理解本项目和研究金期间的活动计划是 旨在为我的长期目标奠定基础，即获得一个终身教授的职位， 一流的学术研究机构。此外，在这个项目中生成的数据将准备我生成 有竞争力的K99应用程序。我将接受盖奇·克伦普博士的指导，他是研究斑马鱼的顶尖科学家 颅面发育该提案中的实验将在健康科学校园进行， 南加州大学，拥有最有经验的颅面和 最好的骨骼生物学家