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

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育的影响具有挑战性。 发展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， 可能影响成人表型或引起疾病。为了实现这一长期目标， 应用程序是开发一个高通量回顾性谱系重建的平台，以定量地映射发育， 自闭症小鼠模型的视觉改变。该平台将基于开发条形码，其中随机 突变在胚胎发生期间在合成基因座中积累。每一个突变都由细胞的后代遗传， 每一个后代都可以在继承的基础上添加新的突变。此过程使用集合标记每个单元格 一种条形码，可以用来解析它的血统。核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映射增殖和分化 神经祖细胞的动力学，以确定野生型和突变小鼠模型之间的差异。的理由 这项研究表明，许多与出生缺陷相关的遗传风险因素仍然无法解释， 基于终末分化组织的细胞和分子分析;该平台将使回顾性 在发育后绘制这些遗传效应，以确定它们影响哪些祖细胞，何时影响这些祖细胞， 祖细胞，以及它们如何影响这些祖细胞在发育过程中的行为。核心假设是 通过追求三个具体目标进行测试：1）在组合和累积酒吧中建立高分辨率谱系记录， 每个细胞基因组中的编码这一目标将联合收割机从双链断裂诱变，这占主导地位， Nantly导致indel，正交诱导点突变，以建立高分辨率谱系记录 整个小鼠妊娠期。2)直接从小鼠组织建立原位单细胞条形码和身份读数。这 Aim将设计条形码基因座，以促进其在组织切片中的扩增和测序， 细胞状态的标志物。组合细胞状态和谱系条形码将揭示细胞增殖和分化动力学。 他们的祖先。3)用逆转录-聚合酶链反应技术测定Chd 8单倍不足对小鼠脑发育的影响。 血统重建chd8单倍不足导致自闭症，但它如何改变神经发生仍不清楚。 这一目标将量化Chd8单倍不足对脑前体细胞增殖和分化参数的影响。 小鼠神经发生过程中的生殖细胞。这里提出的研究是创新的，因为它建立了新的战略， 谱系的高分辨率基因组条形码化和组织中这些条形码的高通量空间测序， 选择。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， 信息到关于它们的祖先的定量见解。此外，它还在子宫内分析了Chd8单倍蛋白- 充足改变了创造大脑的祖先场。这项研究意义重大，因为它将有助于确定如何 遗传干扰改变哺乳动物胚胎发生，导致发育异常，如自闭症。

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.