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 颅面开发需要许多不同细胞的精确分化和迁移 时间和空间中的种群。了解这种动态过程的基因调节控制是关键 破译颅面先天缺陷的遗传基础，例如嘴唇和pa裂。我们以前 展示了远处施加增强剂在控制颅面发育中的关键作用，并 Facebase的成员在此过程中生成了活跃的增强子的全基因组图。但是，我们的 研究还强调了从RNA-的全基因组转录组和表观基因组映射的有限分辨率 原代散装组织的Seq和Chip-Seq。现在，新技术使映射基因表达和 在单细胞分辨率下的增强剂活性，并能够测试有关细胞类型的作用的假设 颅面发育中的特定增强子。在初步研究中，我们介绍了28,000的转录组 单个颅面细胞，分配给不同细胞种群的增强子，整合的单细胞转录组和 光学投影层析成像（OPT）数据以将增强子标签的单细胞种群映射到三个 尺寸解剖结构，并使用单核ATAC-SEQ以单细胞分辨率绘制开放染色质。这里 我们建议对这些研究进行扩展，以生成三维的单细胞分辨率增强子 和颅面发育的转录组图集。我们将使用最新一代的单细胞分析 工具，一套独特的鼠标工程技术以及> 300颅面的庞大分子工具箱 我们先前在体内表征的增强剂。结果数据集，也将通过 Facebase数据门户将为颅面基因，增强剂，，创造庞大的社区资源 和途径，并为解释非编码序列的解释提供了急需的框架 负责颅面出生缺陷。具体目的是：1）创建单细胞分辨率 颅面发育的转录组和开放染色质汇编。此参考将包括 来自> 100万个细胞的转录组以及150个颅面增强剂的体内活性数据 该高分辨率数据通过针对目的工程的报道小鼠的单细胞分析。 2）执行 单细胞转录组，可访问染色质和三维转基因的综合分析 记者选择数据以生成一个凝聚力，空间和时间分析的地图集，将增强剂和单元格连接 人群到发展面的特定子区域。 3）利用这些数据集识别增强器 与人类颅面畸形相关的变体并评估这些变体的因果作用 通过具有人类风险和控制等位基因的单细胞表征和表型。这些 研究将提供有关颅面发育的细胞和基因调节基础的见解 前所未有的分辨率，并建立使用单细胞方法来阐明人类非编码的方式 机械上的变体有助于颅面先天缺陷的风险。