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

项目摘要/摘要 哺乳动物胚胎发生的复杂性使得确定遗传扰动对 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。 可能影响成人的表型或导致疾病。为了实现这个长期目标，这个总的目标是 应用是开发高通量回溯性血统重建平台，以定量绘制发展图。 自闭症小鼠模型的视功能改变。这个平台将基于开发条形码的地方随机 在胚胎发育过程中，突变会累积在合成的基因座上。每个突变都是由细胞的后代继承的 每个后代都可以在它继承的基因基础上增加新的突变。此过程使用集合标记每个单元格 一种可以用来解析其血统的条形码。中心假设是高分辨率谱系 在单个细胞中进行空间排序的条形码可以用来追溯地绘制增殖和分化的图谱 神经前体细胞的动力学，以确定野生型和突变小鼠模型之间的差异。其基本原理是 这项研究表明，许多与出生缺陷有关的遗传风险因素在机制上仍然无法解释。 基于对终末分化组织的细胞和分子分析；这一平台将使回溯 在发育后绘制这些遗传效应的图谱，以确定它们影响哪些祖细胞，当它们影响那些 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。中心假设将是 通过追求三个具体目标进行了测试：1)建立组合和累积条带的高分辨率谱系记录- 嵌入在每个细胞基因组中的代码。这一目标将结合双链断裂的突变，这种突变占主导地位。 以正交诱导点突变建立超高分辨率谱系记录 在老鼠怀孕的整个过程中。2)直接从小鼠组织中建立原位单细胞条形码和身份读出。这 AIM将设计条形码基因座，以促进其在组织切片和分子切片中的扩增和测序 细胞状态的标记。结合细胞状态和谱系条形码将揭示细胞的增殖和分化动力学 他们的祖先。3)研究CHD8单倍体不足对小鼠脑发育的影响。 反思的血统重建。CHD8单倍体不足会导致自闭症，但它是如何改变神经发生的尚不清楚。 这一目标将量化CHD8单倍体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基因。这里提出的研究是创新的，因为它为 血统的高分辨率基因组条形码和这些条形码在组织中的高通量空间测序- 特兹。它进一步使用新的理论概念来转换终端细胞的谱系条形码和分子同一性信息。 关于他们祖先的量化洞察的信息。此外，它还在宫内分析CHD8单倍体是如何- 充分性改变了创造大脑的祖细胞场。这项研究意义重大，因为它将使我们能够确定 遗传扰动会改变哺乳动物的胚胎发育，导致自闭症等发育异常。

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.