A Single-Cell Resolution Enhancer Atlas of Craniofacial Development

颅面发育的单细胞分辨率增强图谱

• 批准号: 10615069
• 负责人: Axel Visel
• 金额: $ 79.8万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-05-01 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10615069
• 关键词: 3-Dimensional; 8q24; ATAC-seq; Alleles; Anatomy; Atlases; Biological Assay; Breathing; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Communities; Complement; Congenital Abnormality; Craniofacial Abnormalities; DNA Sequence; Data; Data Set; Development; Developmental Gene; Developmental Process; Dissection; Distant; Embryo; Engineering; Enhancers; Etiology; Face; FaceBase; Gene Activation; Gene Expression; Gene Expression Regulation; Gene Transfer Techniques; Generations; Genes; Genetic; Genetic study; Histologic; Human; Human Genetics; Individual; Knock-in; Knock-in Mouse; Label; LacZ Genes; Letters; Link; Location; Maps; Methods; Molecular; Morphology; Mus; Mutation; Operative Surgical Procedures; Optical reporter; Optics; Pathway interactions; Pattern; Phenotype; Population; Process; Regulator Genes; Reporter; Research Personnel; Resolution; Resources; Risk; Role; Site; Specificity; Speech; Stains; Techniques; Testing; Time; Tissues; Transcript; Transgenic Organisms; Untranslated RNA; Variant; cell type; cleft lip and palate; comparative; cost; craniofacial; craniofacial development; data portal; epigenome; feeding; functional genomics; genome-wide; genome-wide analysis; genomic tools; in vivo; insight; member; migration; mouse model; new technology; novel; orofacial cleft; prenatal; psychosocial; risk variant; single cell analysis; single-cell RNA sequencing; temporal measurement; tomography; tool; trait; transcription factor; transcriptome; transcriptome sequencing; transcriptomics

PROJECT SUMMARY Craniofacial development requires the precisely orchestrated differentiation and migration of many different cell populations in time and space. Understanding the gene regulatory control of this dynamic process is key for deciphering the genetic basis of craniofacial birth defects such as clefts of the lip and palate. We previously demonstrated the critical role of distant-acting enhancers in controlling craniofacial development and, as members of FaceBase, generated genome-wide maps of enhancers active during this process. However, our studies also highlighted the limited resolution of genome-wide transcriptome and epigenome mapping from RNA- seq and ChIP-seq of primary bulk tissues. New technologies now make it possible to map gene expression and enhancer activities at single-cell resolution and enable the testing of hypotheses regarding the role of cell type- specific enhancers in craniofacial development. In preliminary studies, we profiled the transcriptomes of 28,000 single craniofacial cells, assigned enhancers to distinct cell populations, integrated single-cell transcriptome and Optical Projection Tomography (OPT) data to map enhancer-labeled single-cell populations onto three- dimensional anatomy, and used single-nucleus ATAC-seq to map open chromatin at single-cell resolution. Here we propose to expand on these studies to generate a three-dimensional, single-cell resolution enhancer and transcriptome atlas of craniofacial development. We will use the latest generation of single-cell profiling tools, a suite of unique mouse engineering techniques, and a vast molecular toolbox of >300 craniofacial enhancers we characterized previously in vivo. The resulting data sets, which will also be made available through the FaceBase data portal, will create a vast community resource for studies of craniofacial genes, enhancers, and pathways and provide a much-needed framework for the interpretation of non-coding sequence changes responsible for craniofacial birth defects. The specific aims are to: 1) Create a single-cell resolution transcriptome and open chromatin compendium of craniofacial development. This reference will include transcriptomes from >1 million cells, as well as in vivo activity data for 150 craniofacial enhancers mapped onto this high-resolution data through single-cell analysis of purpose-engineered reporter mice. 2) Perform integrative analysis of single-cell transcriptomic, accessible chromatin, and three-dimensional transgenic reporter OPT data to generate a cohesive, spatially and temporally resolved atlas linking enhancers and cell populations to specific subregions of the developing face. 3) Leverage these data sets to identify enhancer variants associated with human craniofacial malformations and assess a causal role of these variants through single-cell characterization and phenotyping of knock-in mice with human risk and control alleles. These studies will provide insight into the cellular and gene regulatory basis of craniofacial development at unprecedented resolution and establish the use of single-cell methods for elucidating how human non-coding variants mechanistically contribute to the risk for craniofacial birth defects.

项目摘要 颅面的发育需要许多不同细胞的精确协调的分化和迁移 在时间和空间上的人口。了解这个动态过程的基因调控是 破译唇腭裂等颅面先天缺陷的遗传基础。我们之前 证明了远距离作用增强剂在控制颅面发育中的关键作用， FaceBase的成员，生成了在此过程中活跃的增强子的全基因组图谱。但我们的 研究还强调了全基因组转录组和RNA表观基因组作图的有限分辨率， 原代散装组织的seq和ChIP-seq。新技术使绘制基因表达图谱成为可能， 增强子活性在单细胞分辨率，并使有关细胞类型的作用假设的测试- 在颅面发育中的特殊增强剂。在初步研究中，我们分析了28000个 单个颅面细胞，为不同的细胞群体分配增强子，整合单细胞转录组， 光学投影断层扫描（OPT）数据将增强子标记的单细胞群体映射到三个- 三维解剖学，并使用单核ATAC-seq以单细胞分辨率绘制开放染色质。这里 我们建议在这些研究的基础上进行扩展，以产生一种三维的单细胞分辨率增强器 和颅面发育的转录组图谱。我们将使用最新一代的单细胞分析 工具，一套独特的小鼠工程技术，和一个巨大的分子工具箱， 增强子，我们以前在体内表征。由此产生的数据集也将通过 FaceBase数据门户将为颅面基因，增强子， 并为解释非编码序列的变化提供了一个急需的框架 导致颅面先天缺陷具体目标是：1）创建单细胞分辨率 转录组和开放染色质纲要颅面发育。这一参考将包括 来自超过100万个细胞的转录组，以及映射到150个颅面增强子的体内活性数据 这种高分辨率的数据是通过目的工程报告小鼠的单细胞分析获得的。2)执行 单细胞转录组、可接近染色质和三维转基因整合分析 报告基因OPT数据，以产生连接增强子和细胞的内聚的、空间和时间分辨的图谱 人口向发展中国家的特定次区域转移。3)利用这些数据集确定增强剂 与人类颅面畸形相关的变异，并评估这些变异的因果作用 通过对具有人类风险和对照等位基因的基因敲入小鼠进行单细胞表征和表型分析。这些 研究将提供深入了解颅面发育的细胞和基因调控基础， 前所未有的解决方案，并建立使用单细胞方法来阐明人类非编码 变异在机械上会增加颅面出生缺陷的风险。