Single-cell transcriptomic analysis of sibling progenitors with differential Notch activity

项目总结 已经在不同物种中观察到胚胎放射状胶质神经前体细胞经历不对称细胞。 分裂(ACD)以产生具有不同Notch活性的子代细胞。野池的女儿经历了自我 更新，而Notchlo的女儿则开始分化。能够从分子上定义这种不同的 各国将极大地促进我们对自我更新和差异化如何监管的理解。 尽管胚胎子代之间的相对Notch活性水平与其自身的 更新与分化潜能相比，Notch的绝对活性在祖细胞之间是不同的 人口。这使得不可能简单地基于 Notch效应器的表达水平(例如，他/她的转录水平)。因此，追踪血统很重要 在scRNA-seq实验中，祖细胞之间精确地在兄弟细胞水平上的关系。 这一探索性的R21应用，受一个重要的生物学问题的激励，旨在建立高 解析同胞血统追踪并与scRNA-seq结合。这代表着一项技术突破， 将能够比较兄弟细胞之间的基因表达谱。 预期结果和影响：如果成功，该项目将建立一个新的和广泛的 可以在体内和在转录水平上比较同胞细胞状态的适用方法 在试管中。通过将这种方法应用于正在经历不对称细胞的胚胎放射状胶质祖细胞 在活跃的神经发生过程中，我们希望发现进化上保守的核心基因和途径 区分在兄弟姐妹谱系中共享的Notchhi和Notchlo兄弟姐妹状态。自从Notchhi和Notchlo姐妹 在胚胎祖细胞中，状态分别与自我更新和分化有关，我们预计 了解是否以及如何将自我更新和分化描述为不同的细胞状态 在转录水平上，这些基因与特定的血统结果无关。我们将把这部小说 具有精确克隆跟踪的数据集广泛适用于广泛的研究社区。 该项目将为未来的R01奠定基础，该R01旨在剖析签名基因的功能 以及定义Notchhi和Notchlo细胞状态的通路。从长远来看，新的关于 潜在的遗传程序将有助于开发新的治疗想法。高风险、高回报的本质 拟议工作的一部分，使这一应用程序非常适合R21机制。

PROJECT SUMMARY It has been observed across species that embryonic radial glia neural progenitors undergo asymmetric cell division (ACD) to generate daughter cells with different Notch activity. The Notchhi daughter undergoes self- renewal, whereas the Notchlo daughter embarks on differentiation. Being able to molecularly define such different states will significantly advance our understanding of how self-renewal and differentiation are regulated. Although the relative Notch activity levels between embryonic daughters are correlated with their self- renewing vs. differentiation potential, the absolute Notch activity is heterogeneous across the progenitor population. This makes it impossible to simply sort single-cell RNA-seq (scRNA-seq) data based on the expression levels of Notch effectors (e.g. hes/her transcript levels). It is therefore important to track the lineage relationships among progenitors precisely at the level of sibling cells in scRNA-seq experiments. This exploratory R21 application, motivated by an important biological problem, aims to establish high resolution sib lineage-tracing and combine it with scRNA-seq. This represents a technological breakthrough that will enable comparison of gene expression profiles between sibling cells. Expected outcomes and impact: If successful, this project will establish a new and broadly applicable method in which sib cell states can be compared at the transcriptomic level both in vivo and in vitro. By applying this method to embryonic radial glia progenitors that are undergoing asymmetric cell division during active neurogenesis, we expect to uncover evolutionarily conserved core genes and pathways distinguishing Notchhi and Notchlo sib states that are shared across sib-lineages. Since Notchhi and Notchlo sib states are associated with self-renewal and differentiation respectively in embryonic progenitors, we expect to gain a glimpse into whether and how self-renewal and differentiation as distinct cellular states can be depicted at the transcriptomic level that are uncoupled from specific lineage outcomes. We will make this novel dataset with precise clonal tracking widely available to the broad research community. This project will lay foundation for a future R01, which aims to dissect the function of signature genes and pathways that define Notchhi vs. Notchlo cell states. In the long run, new basic knowledge about the underlying genetic programs will aid in developing new therapeutic ideas. The high risk and high reward nature of the proposed work makes this application well suited for the R21 mechanism.