Mapping the dynamics of mouse neurogenesis in autism models using high-resolution genomic barcoding technologies

使用高分辨率基因组条形码技术绘制自闭症模型中小鼠神经发生的动态图

• 批准号: 10705779
• 负责人: Reza Kalhor
• 金额: $ 52.04万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-16 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10705779
• 关键词: Address; Adult; Affect; Bar Codes; Behavior; Biological Models; Birth; Brain; Caenorhabditis elegans; Cell Differentiation process; Cell Lineage; Cell Proliferation; Cells; Chromosome Mapping; Complex; Congenital Abnormality; Data; Development; Developmental Process; Disease; Embryonic Development; Engineering; Environmental Risk Factor; Event; Gene Mutation; Genes; Genetic; Genetic Variation; Genome; Genomics; Goals; Health; Heritability; Human; In Situ; Inherited; Label; Lead; Macrocephaly; Mammals; Maps; Measurement; Mission; Modeling; Molecular; Molecular Analysis; Mus; Mutagenesis; Mutation; Nematoda; Neurodevelopmental Disorder; Organism; Outcome; Phenotype; Point Mutation; Positioning Attribute; Pregnancy; Process; Proliferating; Public Health; Research; Resolution; System; Technology; Testing; Tissue Differentiation; Tissues; United States; United States National Institutes of Health; Work; autism spectrum disorder; base editor; combinatorial; computerized tools; disorder risk; genetic risk factor; genome wide association study; in situ sequencing; in utero; in vivo; innovation; insertion/deletion mutation; insight; molecular marker; mouse development; mouse model; mutant mouse model; nerve stem cell; neurodevelopment; neurogenesis; novel; progenitor; reconstruction; transcriptome; ultra high resolution

PROJECT SUMMARY/ABSTRACT The complexity of mammalian embryogenesis makes it challenging to determine the effect of genetic perturbations on development. The long-term goal is to better understand how genetic and environmental factors alter mammalian devel- opment to affect adult phenotypes or cause diseases. Toward achieving this long-term goal, the overall objective of this application is to develop a platform for high-throughput retrospective lineage reconstruction to quantitatively map devel- opmental alterations in a mouse model of autism. This platform will be based on developmental barcoding where random mutations accumulate in synthetic loci during embryogenesis. Each mutation is inherited by the descendants of the cell in which it occurs; each descendant can add new mutations to the ones it inherited. This process marks each cell with a set of mutations—a barcode—that can be used to resolve its lineage. The central hypothesis is that high-resolution lineage barcodes that are sequenced spatially in single cells can be used to retrospectively map proliferation and differentiation dynamics of neural progenitors to identify the differences between wildtype and mutant mouse models. The rationale for this research is that many genetic risk factors that are associated with birth defects remain mechanistically inexplicable based on cellular and molecular analyses of terminally differentiated tissues; this platform would enable retrospective mapping of these genetic effects after development to determine which progenitors they affect, when they affect those progenitors, and how they affect the behavior of those progenitors during development. The central hypothesis will be tested by pursuing three specific aims: 1) Establish high-resolution lineage recording in combinatorial and cumulative bar- codes embedded in each cell’s genome. This Aim will combine mutagenesis from double-strand breaks, which predomi- nantly lead to indels, with orthogonally induced point mutations to establish ultrahigh resolution lineage recording throughout mouse gestation. 2) Establish in situ single-cell barcode and identity readout directly from mouse tissues. This Aim will engineer barcoding loci to facilitate their amplification and sequencing in tissue sections together with molecular markers of cell state. Combining cell state and lineage barcodes will reveal proliferation and differentiation dynamics of their progenitors. 3) Determine the effects of Chd8 haploinsufficiency on the development of mouse brain using retro- spective lineage reconstruction. Chd8 haploinsufficiency causes autism but how it alters neurogenesis remains unclear. This aim will quantify the effects of Chd8 haploinsufficiency on proliferation and differentiation parameters of brain pro- genitors during mouse neurogenesis. The research proposed here is innovative because it establishes new strategies for high-resolution genomic barcoding of lineages and high-throughput spatial sequencing of these barcodes in tissue sec- tions. It further uses new theoretical concepts to convert terminal cells’ lineage barcodes and molecular identity infor- mation to quantitative insights about their progenitors. Additionally, it carries out in utero analysis of how Chd8 haploin- sufficiency alters progenitor fields that create the brain. This research is significant because it will enable determining how genetic perturbations alter mammalian embryogenesis to cause developmental anomalies such as autism.

项目概要/摘要 哺乳动物胚胎发生的复杂性使得确定遗传扰动对胚胎发生的影响具有挑战性。 长期目标是更好地了解遗传和环境因素如何改变哺乳动物的发育。 影响成人表型或引起疾病的机会 为了实现这一长期目标，这是本次会议的总体目标。 应用程序是开发一个高通量回顾性谱系重建平台，以定量图谱开发 该平台将基于随机的发育条形码。 突变在胚胎发生过程中积累在合成基因座中，每个突变都会由细胞的后代遗传。 它发生；每个后代都可以在其继承的突变中添加新的突变。这个过程用一组标记每个细胞。 突变的条形码——可用于解析其谱系。中心假设是高分辨率谱系。 在单细胞中进行空间测序的条形码可用于回顾性地绘制增殖和分化图谱 神经祖细胞的动力学以确定野生型和突变型小鼠模型之间的差异。 这项研究表明，许多与出生缺陷相关的遗传风险因素在机械上仍然无法解释 基于终末分化组织的细胞和分子分析；该平台将实现回顾性 在发育后绘制这些遗传效应，以确定它们影响哪些祖细胞，何时影响那些祖细胞 祖细胞，以及它们在发育过程中如何影响这些祖细胞的行为。 测试所追求的三个具体目标：1）在组合和累积条中建立高分辨率谱系记录 嵌入每个细胞基因组中的代码将结合双链断裂的诱变，这主要是 很容易导致插入缺失，并通过正交诱导点突变建立超高分辨率谱系记录 2) 直接从小鼠组织中建立原位单细胞条形码和身份读出。 Aim 将设计条形码位点，以促进其在组织切片中的扩增和测序以及分子 结合细胞状态和谱系条形码将揭示细胞的增殖和分化动态。 3) 使用逆转录法确定 Chd8 单倍体不足对小鼠大脑发育的影响。 Chd8 单倍体不足会导致自闭症，但它如何改变神经发生仍不清楚。 这一目标将量化 Chd8 单倍体不足对大脑前体细胞增殖和分化参数的影响。 这里提出的研究具有创新性，因为它建立了新的策略。 谱系的高分辨率基因组条形码以及组织秒中这些条形码的高通量空间测序 它进一步使用新的理论概念来转换终末细胞的谱系条形码和分子身份信息。 此外，它还对 Chd8 单倍体如何进行子宫内分析。 这项研究意义重大，因为它将能够确定如何改变创造脑区的祖细胞。 遗传扰动改变哺乳动物胚胎发生，导致自闭症等发育异常。