Modular Control of Cranial Skeletal Connectivity through Joint-Specific Enhancers

项目摘要/摘要 颞下颌关节紊乱病(TMJ)和颅骨联合软骨性早熟只是两种 颅骨关节破裂造成的缺陷的例子。要求有合适的关节形式和功能 脊椎动物骨骼的连通性和灵活性。虽然头盖骨有大量的关节和亚型， 大多数关节生物学研究都集中在四肢上，在我们对颅骨关节如何 在它们的脑神经脊细胞贡献方面是独一无二的，发育。我们的实验室最近产生了单一的- 脑神经脊源性细胞在7个时间点的细胞转录组和染色质可及性数据 (胚胎到成年)在斑马鱼。我已经能够提取关于转录的初步全球信息 因子和顺式调节元件，或增强剂，将颅骨关节与其他类型的骨骼区分开来 纸巾。虽然以前的模型已经提出关节软骨仅仅是未成熟的软骨，但我的初步数据显示 几种增强剂仅在颅骨关节中驱动表达。此外，其他增强剂仅在 置换软骨。这些数据表明，存在两个完全独立的种群，具有关节 与置换软骨有明显区别。在这项提议中，我使用了斑马鱼强大的基因 研究增强子和转录因子区分颅骨关节置换的模型 软骨。我的初步生物信息学分析表明，AP-1转录因子(Jun/Fos)与 掌握软骨转录因子Sox9，一般指定关节软骨。有趣的是，几个基因突变 转录因子可以独立地导致特定颅骨关节的缺陷，提示是局部性的 转录因子可以专门化关节。这项提案中概述的目标是研究神经脊派生的 发育中的颅骨关节中的细胞(目标1)，它们是如何从替代软骨中分离出来的 (目标2)，以及区域特异性转录因子如何在不同的 头部和面部的部分(目标3)。我计划使用SnATAC-seq和Cut&Tag等技术来确定 如果在关节中唯一打开或激活了增强剂。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联合活动和身份的必要性和充分性，我将显著 提高对颅骨关节发育的认识。本奖学金期间的项目和活动计划如下 旨在为我的长期目标奠定基础，获得一份终身教授的职位 一流的学术研究机构。此外，在这个项目中生成的数据将为我生成 竞争激烈的K99应用程序。我将接受斑马鱼领域的顶尖科学家盖奇·克伦普博士的指导 头面部发育。这项提案中的实验将在健康科学校园 南加州大学，拥有最有经验的颅面和面部社区之一 这个国家的骨骼生物学家。

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.