GLOBAL GENE REGULATORY NETWORKS FOR SPECIFIC CELL TYPES OF THE SEA URCHIN EMBRYO

描述（由申请人提供）：动物细胞的所有发育、形态发生和分化功能都是通过大型的、专门部署的电池和下游蛋白质编码基因盒来执行的。近年来，生物科学的一个重大成功是系统级阐明了控制模式形成和决定身体计划发展的上游基因调控网络（grn）。grn由编码转录因子和信号分子的基因以及这些基因之间的转录联系组成，它们决定了每个细胞在发育时间的每个点的调控状态。因此，它们包括所有下游基因的控制输入，这些基因负责细胞的工作。但是，现在在理解上存在一个巨大的鸿沟，将我们开始了解的上游grn与细胞功能的下游效应物分开：决定这些下游基因部署的实际控制电路是什么？grn究竟是如何与细胞分化和形态发生的功能产生因果关系的？原则上，已知上游GRN的知识和各种新的可用技术，可以缩小这一差距，并在系统级别解决问题，这是本提案的特定对象。我们将选择发育中的海胆胚胎的三种细胞类型，每一种都有普遍的兴趣。这个胚胎的上游grn的知识比任何其他系统都要先进。靶细胞类型为胚胎免疫细胞，其下游效应基因编码多种先天免疫蛋白；原肠胚内胚层细胞，其中下游基因介导胃内陷；而在幼虫期产生成体计划的胚胎体腔囊的全能性预留细胞，其中下游的效应细胞包括那些保持全能性的细胞。先天免疫、胃内陷和全能细胞系都是泛双侧的特征。简而言之，该方法将是使用特定调控基因表达和FACS分离每种细胞类型；深度转录组测序；生物信息学预测;并通过高通量顺式调控分析验证了与特异性表达基因的因果调控联系。该项目将为任何正在开发的系统提供第一个全球上下游GRN。它将告知下游基因磁带组织的基本原则。它还将作为哺乳动物系统类似方法的技术示范项目。

DESCRIPTION (provided by applicant): All developmental, morphogenetic, and differentiation functions of animal cells are executed by large, specifically deployed batteries and cassettes of downstream protein coding genes. A major success of bioscience in recent years has been system level elucidation of the upstream gene regulatory networks (GRNs) that control pattern formation and determine development of the body plan. GRNs consist of genes encoding transcription factors and signaling molecules, plus the transcriptional linkages among these genes, and they determine the regulatory state of every cell at every point in developmental time. Therefore they include the control inputs into all the downstream genes that do the work of the cell. But an enormously important gap in understanding now separates the upstream GRNs that we are beginning to learn about from the downstream effectors of cell function: What is the actual control circuitry that determines the deployment of these downstream genes? How, exactly, are GRNs causally connected to cellular functions of differentiation and morphogenesis? In principle, given knowledge of the upstream GRN and various newly available technologies, this gap can be closed, and the problem solved at a system level, and this is the particular object of the present proposal. We will choose three cell types of the developing sea urchin embryo, each of general interest. Knowledge of the upstream GRNs of this embryo is more advanced than for any other system. The target cell types are the immune cells of the embryo, where the downstream effector genes encode a great variety of innate immunity proteins; gastrulating endoderm cells, where the downstream genes mediate gastrular invagination; and the totipotent set-aside cells of the embryonic coelomic pouches that in larval stage produce the adult body plan, where the downstream effector cells include those that maintain totipotency. Innate immunity, gastrular invagination, and totipotent cell lineages are all pan-bilaterian features. The approach, briefly, will be isolation of each cell type using specific regulatory gene expression and FACS; deep transcriptome sequencing; bioinformatic prediction; and validation of causal regulatory connections to specifically expressed genes by high throughput cis- regulatory analysis. This project will provide the first global upstream-downstream GRN for any developing system. It will inform as to basic principles by which downstream gene cassettes are organized. It will also serve as a technological demonstration project for similar approaches to mammalian systems. PUBLIC HEALTH RELEVANCE: This work is about finding the causal lines of control that determine how fundamental life processes are executed according to the instructions encoded in the genomic regulatory system. The most powerful approach to general solutions to complex disease states requires solid understanding of their control circuitry. Our practice must get beyond struggling to ameliorate effects rather than altering causes. This research shows the way to discovery of structure and function in causal genomic control systems.