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 正常发育继续进行母体和父亲基因的遗传。然而， 我们不是父母双方基因的平等产物。仅来自一个特定基因的独家表达 父母，已经提出等位基因影响会影响遗传结构和疾病风险，但几乎没有 知道他们对发展的贡献。全面理解的根本障碍 父母的遗传遗传及其在发展过程中的调节是覆盖不完全的局限性 在发育胚胎中的每个细胞以及等位基因序列之间分化的准确性。作为 脊椎动物的最亲近的亲属，海沿河肠菌和C. savignyi提供了正确的程度 多细胞生物之间的简单性以及母体和父亲基因组的极端差异 克服这些限制。在这里，我建议使用杂交海腹人阐明法规语法 在单细胞分辨率下发育过程中的等位基因特异性基因表达。 在K99阶段，我将研究AIM 1并启动AIM 2。根据我的初步结果，我有 使用单个细胞重建了混合胚胎的临时表达曲线的参考谱系图 RNA-seq技术。由于海沿岸的细胞数量很少，我希望获得全面的覆盖范围 发育过程中每种细胞类型的菜单，包括罕见的神经元亚型。在AIM 1中，基于极端 父母基因组序列在混合胚胎中，我将表征特定于等位基因特异性 每个定义的细胞谱系的表达，并开发能够轻松概况的新方法 单细胞中的基因表达和染色质可及性，该细胞将将父母表达模式与 推定的顺式调节DNA序列。 AIM 1完成后，我希望建立潜力 等位基因表达的关联以及单个细胞和细胞的规范和形态发生 血统。 在R00阶段，根据我在K99阶段获得的培训，我将研究高度创新的 AIM 2，我计划开发空间转录组技术，以便将另一个维度添加到 在细胞细胞接触的背景下剖析等位基因特异性表达模式及其调节。之上 成功完成AIM 1和AIM 2，这种数据丰富的方法将大大增强我们对 父母的遗传遗传及其调节，有时让我们更接近整体和定量 了解胚胎发展。它还概述了我的广泛的职业发展计划 在莱文教授的指导下完成我的培训以及向独立学者的过渡 通过建立多学科研究计划来定位。