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.

项目摘要/摘要 颞下颌关节障碍（TMJ）和颅同点骨的早熟骨化仅为两个 由颅关节中断引起的缺陷的例子。需要适当的关节形式和功能 脊椎动物骨骼的连通性和灵活性。颅骨有大量和亚型的关节，但 大多数联合生物学研究都集中在四肢上，在我们对颅关节的理解中留下了差距 在其颅神经rest细胞的贡献中是独一无二的。我们的实验室最近生成了单个 - 在7个时点，颅神经衍生的细胞的细胞转录组和染色质可及性数据 （成人胚胎）在斑马鱼中。我已经能够提取有关转录的初步全球信息 因素和顺式调节元素或增强子，将颅关节与其他类型的骨骼分开 组织。虽然先前的模型已提出的关节软骨只是未成熟的软骨，但我的初步数据显示 几个增强剂仅在颅关节中驱动表达。此外，其他增强剂仅在 替换软骨。这些数据表明存在两个完全独立的人群，关节 明确指定的是替换软骨。在此提案中，我使用了斑马鱼的强大遗传学 模型以研究哪些增强剂和转录因子将颅关节与替代区分开 软骨。我的初步生物信息学分析表明，AP-1转录因子（JUN/FOS）与 主要软骨转录因子SOX9通常指定关节软骨。有趣的是，几个突变 转录因子只能独立导致缺陷，仅引起特定的颅关节，这表明本地化 转录因子可能专门为关节。该提案中概述的目的调查了神经rest衍生的 开发颅关节的细胞（AIM 1），它们如何与替换软骨分开唯一形成图案 （AIM 2），以及区域特异性转录因子如何负责专业化不同的颅关节 头部和脸部的一部分（AIM 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唯一打开或激活。通过将这些大数据集与转基因测定 为了确认转录因子基序是否需要和足够的关节活动和身份，我将显着 增进我们对颅联发育的理解。该研究金期的项目和活动计划是 旨在为我的长期目标奠定基础 顶级学术研究机构。此外，该项目中生成的数据将使我为生成 竞争性的K99申请。我将获得斑马鱼的主要科学家Gage Crump博士的指导 颅面发展。该提案中的实验将在健康科学校园进行 南加州大学，它是经验最丰富的颅面社区之一 该国的骨骼生物学家。