Spatiotemporal mapping of enhancer activity in developing frog embryos

项目概要： 生物体中的所有细胞类型都共享相同的基本遗传信息，但它们却表现得非常出色。 不同的基因表达程序和行为。这种多样性大部分来自细胞类型特异性 和条件特异性使用基因远端调控区称为顺式调控模块（CRM） (also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，这些基因组距离共同作用， 极其复杂的基因表达模式目前，我们对生物学作用的理解， 这些CRM中的大多数仍然有限。例如，如何选择特定的CRM来调节给定的 吉恩的表情？不同的CRM如何协调和激活不同的靶基因在空间上 和时间上的规范我们缺乏了解在很大程度上是由于目前的限制， 提供大量CRM活动的同时实验测量， 动物发展。这项建议的主要目标是成功地利用非洲爪蟾原肠胚阶段 胚胎作为模型系统，用于量化CRM活动， 靶基因的表达模式。虽然CRM活动将在全基因组水平上进行测定， 它们相关的基因调控机制将用单细胞分辨率来确定。应用 先进的计算生物学算法的数据将提供CRM活动的映射足够 推断转录因子和相关的信号传导过程参与调节不同的细胞状态， 原肠胚在单细胞分辨率。我们提出两个具体目标。首先，我们将使用修改后的 STARR-seq方法鉴定调控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结合位点，以帮助鉴定它们的生物学特性。 功能协调发展的其次，我们将联合收割机scRNA-seq与STARR-seq结合，作为一种揭示CRM中心的手段。 基因调控结构对原肠期胚胎中每个细胞的细胞状态至关重要。在 总之，我们将使用热带爪蟾原肠胚期胚胎作为模型系统，用于产生 由于关键的系统发育位置， 两栖动物在脊椎动物进化谱系中占有一席之地。这里提出的方法将是非常重要的 在哺乳动物中进行更困难，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用性是限制率 一些基因组研究

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.