Spatiotemporal mapping of enhancer activity in developing frog embryos

项目总结： 生物体中的所有细胞类型共享相同的基本遗传信息，但它们的执行速度非常快 不同的基因表达程序和行为。这种多样性很大程度上来自于特定类型的细胞 以及被称为顺式调节模块(CRM)的基因末端调节区的特定条件使用 (也称为增强剂)。典型的哺乳动物基因组包含数十万个CRM 分布在大的基因组距离上，共同和协作地产生差异 极其复杂的基因表达模式。目前，我们对血管内皮细胞的生物学作用 这些条件反应机制中的大多数仍然有限。例如，如何选择特定的CRM来规范给定的 吉恩的表情？不同的CRM如何在空间上协调和激活不同的靶基因 和受时间限制的方式？我们缺乏了解在很大程度上是由于目前 提供对大量CRM活动的同时实验测量 动物发育。这项建议的主要目标是成功利用非洲爪哇原肠幼虫阶段 胚胎作为量化客户关系管理活动的模型系统，涉及精确的时空传递 靶基因的表达模式。虽然将在全基因组水平上分析CRM活性， 它们相关的基因调控机制将通过单细胞分辨率来确定。施药 对该数据的高级计算生物学算法将提供足够的CRM活动映射 为了推断转录因子和相关的信号过程参与调节不同的细胞状态 单细胞分辨率的原肠胚。我们建议解决两个具体目标。首先，我们将应用修改后的 用STARR-SEQ方法鉴定调节原肠胚时空基因表达模式的CRM 胚胎。我们计划突变CRM中潜在的Tf结合部位，以帮助鉴定它们的生物学特性 功能。其次，我们将把scRNA-seq和starr-seq结合起来，作为一种揭示以crm为中心的方法。 基因调控结构对原肠胚期胚胎中每个细胞的细胞状态起关键作用。在……里面 总之，我们将使用非洲爪哇原肠胚期胚胎作为模型系统来产生 由于关键的系统发育地位，对单细胞中CRM活动的系统水平的理解 两栖动物在脊椎动物的进化谱系中占有一席之地。这里提出的方法将是 在哺乳动物身上更难实现，因为它们的胚胎不容易获得 必要的实验操作，因为哺乳动物胚胎的可获得性对 一些基因组工作。

Project Summary: All cell types in an organism share the same basic genetic information, yet they execute remarkably diverse gene expression programs and behaviors. Much of this diversity is derived from cell type-specific and condition-specific usage of gene-distal regulatory regions known as cis-regulatory modules (CRMs) (also known as enhancers). Typical mammalian genomes contain hundreds of thousands of CRMs distributed across large genomic distances that act collectively and collaboratively to produce differential gene expression patterns of extreme complexity. At present, our understanding of the biological roles of most of these CRMs remains limited. For instance, how are specific CRMs selected to regulate a given gene's expression? How do different CRMs coordinate and activate various target genes in a spatially and temporally regulated manner? Our lack of understanding is in large part due to current limitations in delivering simultaneous experimental measurements of the activities of large numbers of CRMs during animal development. The major goal of this proposal is the successful use of Xenopus gastrula-stage embryos as a model system for quantifying CRM activities involved in delivering precise spatiotemporal expression patterns of target genes. While CRM activities are to be assayed at a genome-wide level, their associated gene regulatory mechanisms would be determined with single-cell resolution. Applying advanced computational biology algorithms to that data will deliver mapping of CRM activities sufficient to infer the TFs and associated signaling processes involved in regulation of differential cell states in gastrulae at single-cell resolution. We propose to address two specific aims. First, we will apply a modified STARR-seq approach to identify CRMs that regulate spatiotemporal gene expression patterns in gastrula embryos. We plan to mutate potential TF binding sites within CRMs to aid in identifying their biological functions. Second, we will combine scRNA-seq with STARR-seq as a means of uncovering CRM-centric gene regulatory structures critical for specifying cell states for every cell in gastrula-stage embryos. In sum, we will use Xenopus tropicalis gastrula-stage embryos as the model system for generating a system-level understanding of CRM activities in single cells because of the key phylogenetic position amphibians occupy in the vertebrate evolutionary lineage. The approach proposed here would be much more difficult to perform in mammals, because their embryos are not easily accessed for certain necessary experimental manipulations, and because mammalian embryo availability is rate limiting for some genomic work.