Decoding the dynamics and regulation of parental allelic expression at single-cell resolution

以单细胞分辨率解码亲本等位基因表达的动态和调节

• 批准号: 10376247
• 负责人: Chen Cao
• 金额: $ 11.56万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376247
• 关键词: 3-Dimensional; ATAC-seq; Alleles; Allelic Imbalance; Animals; Bar Codes; Biological Assay; Cell Count; Cell Lineage; Cells; Characteristics; Chromatin; Ciona intestinalis; Complex; DNA Sequence; Data; Development; Development Plans; Developmental Process; Dimensions; Disease; Embryo; Embryonic Development; Etiology; Four-dimensional; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Genome; Genomics; Haplotypes; Human; Hybrids; Individual; Interdisciplinary Study; Kinetics; Link; Maps; Measures; Mentorship; Methods; Morphogenesis; Neurons; Organ; Organism; Parents; Pattern; Phase; Population; Positioning Attribute; Process; Rana; Regulation; Resolution; Role; Single Nucleotide Polymorphism; Source; Swimming; System; Tadpoles; Techniques; Technology; Time; Tissues; To specify; Training; Variant; Vertebrates; Work; ascidian; base; career development; cell type; disorder risk; experimental study; genetic architecture; genome wide association study; innovation; microscopic imaging; novel strategies; programs; single cell sequencing; single-cell RNA sequencing; transcriptome; transcriptomics; vertebrate embryos

Project Summary The inheritance of both the maternal and paternal genes is required for normal development to proceed. Yet, we are not equal products of both parents’ genes. With exclusive expression of specific genes from only one parent, allelic effects have been suggested to influence genetic architecture and disease risk, but little is known about their contributions to development. Fundamental obstacles of comprehensively understanding the parental genetic inheritance and its regulation during development are limitations in incomplete coverage of each cell within developing embryos, and the accuracy of differentiation between allele sequences. As the closest living relatives of vertebrates, the ascidians C. intestinalis and C. savignyi provide the right degree of simplicity among multicellular organisms and extreme divergence of the maternal and paternal genomes for overcoming these limitations. Here I propose to use hybrid ascidians to elucidate the regulatory grammar of allele-specific gene expression during development at single cell resolution. During the K99 phase, I will work on Aim 1 and initiate Aim 2. Based on my preliminary results, I have reconstructed the reference lineage map of temporal expression profiles for hybrid embryos using single cell RNA-Seq technology. Due to the small cell numbers of ascidians, I expect to obtain comprehensive coverage of every cell type during development, including rare neuronal subtypes. In Aim 1, based on the extreme divergence between parental genomic sequences in hybrid embryos, I will characterize allele-specific expression of each defined cell lineage, and develop novel approaches that are able to simultaneously profile gene expression and chromatin accessibility in single cells, which will link parental expression patterns with putative cis-regulatory DNA sequences. Upon the completion of Aim 1, I expect to establish potential associations of allelic-specific expression and the specification and morphogenesis of individual cells and cell lineages. During the R00 phase, based on my training gained during the K99 phase, I will work on the highly innovative Aim 2, in which I plan to develop the spatial transcriptomics technique in order to add another dimension to dissect the allele-specific expression pattern and its regulation in the context of cell-cell contacts. Upon successful completion of Aim 1 and Aim 2, this data-rich approach will greatly enhance our understanding of parental genetic inheritance and its regulation, and eventually let us move closer to a holistic and quantitative understanding of embryonic development. It also outlines an extensive career development plan for me to complete my training under the mentorship of Prof. Levine and the transition to an independent academic position by establishing a multi-disciplinary research program.

项目摘要 母亲和父亲基因的遗传是正常发育所必需的。然而， 我们不是父母基因的平等产物。只从一个基因中 父母，等位基因效应已被认为影响遗传结构和疾病风险，但很少有人被认为是遗传结构和疾病风险的影响。 了解他们对发展的贡献。全面认识的根本障碍 亲本遗传及其在发育过程中的调节是不完全覆盖的限制 每个细胞在发育中的胚胎，和等位基因序列之间的差异的准确性。为 海鞘是脊椎动物现存的近亲，C.和C. savignyi提供正确的程度 多细胞生物之间的简单性和母亲和父亲基因组的极端分歧， 克服这些限制。在这里，我建议使用杂交海鞘阐明的监管语法， 等位基因特异性基因表达在发展过程中在单细胞分辨率。 在K99阶段，我将致力于目标1并启动目标2。根据我的初步结果， 利用单细胞克隆技术构建了杂种胚胎时间表达谱的参考谱系图 RNA-Seq技术由于海鞘细胞数量少，我希望能获得全面的覆盖 包括罕见的神经元亚型。在目标1中，基于极端 杂交胚胎中亲本基因组序列之间的差异，我将描述等位基因特异性 表达的每一个定义的细胞谱系，并开发新的方法，能够同时分析 单细胞中的基因表达和染色质可及性，这将把亲本表达模式与 推定的顺式调节DNA序列。在目标1完成后，我希望建立潜力 等位基因特异性表达与个体细胞和细胞的特化和形态发生的关系 血统 在R 00阶段，基于我在K99阶段获得的培训，我将致力于高度创新的 目标2，我计划开发空间转录组学技术，以便为 剖析等位基因特异性表达模式及其在细胞-细胞接触中的调节。后 成功完成目标1和目标2后，这种数据丰富的方法将大大提高我们对 父母的遗传及其调控，并最终让我们更接近一个整体和定量 了解胚胎发育。它还为我概述了一个广泛的职业发展计划， 在莱文教授的指导下完成我的培训，并过渡到一个独立的学术 通过建立多学科的研究计划。