Transcriptional Casualty in Embryonic Morphogenesis: from the Specifications GRN

胚胎形态发生中的转录伤亡：来自 GRN 规范

• 批准号: 8092700
• 负责人: DAVID MCCLAY
• 金额: $ 25.45万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8092700
• 关键词: Adhesions; Adult; Area; Biological Assay; Boxing; Cell Nucleus; Cell physiology; Cells; Cellular biology; Collaborations; Communication; Complex; Coupled; DNA Sequence Rearrangement; Data; Databases; Development; Devices; Ectoderm; Ectoderm Cell; Embryo; Endoderm; Endomesoderm; Event; Exhibits; Figs - dietary; Gastrocoele; Gene Expression; Genes; Genome; Germ Layers; Goals; Hearing; Hour; Individual; Laboratories; Link; Maps; Mesoderm; Mesoderm Cell; Modeling; Morphogenesis; Movement; Nuclear; Operating System; Oral; Organism; Paper; Pathway Analysis; Pattern; Physiology; Property; Proteomics; Publishing; Reaction; Regulator Genes; Relative (related person); Research; Role; Running; Sea Urchins; Series; Signal Transduction; Signaling Molecule; Solutions; Source; Specific qualifier value; Students; System; Systems Analysis; Technology; Testing; Thick; Time; Work; base; cell behavior; cell motility; daughter cell; embryo cell; gastrulation; improved; insight; member; network models; next generation; novel; programs; prototype; reproductive; research study; tool; transcription factor

hi 2002 the first provisional version of the endomesoderm GRN was published (Davidson et al, 2002a; Davidson et al., 2002b), and in the past year the first drafts of the ectoderm GRN were published, work that included contributions from the McClay lab (Bradham and McClay, 2006); see http://sugp.caltech.edu/endomes/#EctoNetworkDiagram (Figure la, lb). The Network models were constracted by Eric Davidson based on data from many laboratories with major contributions from the Davidson and the McClay labs. I spent my sabbatical year in the Davidson lab in 2002, a stay that has resulted in a long-term collaboration with Eric Davidson and members of his laboratorv. To date 9 papers have come from that collaboration with a number of additional papers linked to this proposal, and to ongoing research (Amore et al., 2003; Davidson et al, 2003; Davidson et al., 2002a; Davidson et al., 2002b; Oliveri et al., 2003; Oliveri et al., 2006; Otim et al, 2004; Sodergren et al., 2006). The original goal was to constract a GRN that reflected the sequence of endomesoderm specification during the first 30 hours (up until the beginning of gastmlation). The network assembly was restricted to transcription factors and to signal transductions. This was because we knew that to include all of cell biology as well as physiology of development was beyond reach at that time. We also wanted to constract the GRNs in such a way that each edge and each node of the network could be authenticated experimentally. Technologies were developed so that each component of the current GRNs generally have at least three independent sources of experimental support. In some cases predicted franscriptional inputs were verified at the cis-regulatory level, a connection that "hardwires" the previous predictions of the Network (those cis-regulatory coimections that have been verified in this way are indicated by the thick edges in Fig. la). As seen in the Davidson component of this Program Project, efforts continue to add more cis-regulatory confirmation, and to develop new strategies that allow more rapid analyses to this tedious, but cracial component of network solutions. The McClay component of the project has been to coimect signal transduction devices to the transcriptional network components, and to provide a number of experimental embryological approaches to validate network predictions, and connect the GRN to morphogenesis. That effort required a number of new assays and has led to the current view of the Network as outlined in this Proposal. Why, it might be asked, would one want such a detailed look at how an embryo is specified? After all, when the GRN is displayed to students, an audible gasp at the complexity is heard. Despite that reaction, the reality is that the mechanisms of specification in all cells are highly complex interactions of many transcription factors and many signaling devices. Inside each nucleus of every cell there is an operational network of transcription factors governing the progression of development and the physiology of that functioning cell. Each time two daughter cells assume different identities, a single network state must diverge into two different network states. The arrangement and distribution of cell network states in an embryo must constantly be coordinated and many signaling inputs have been discovered to accommodate those requirements. In short, to understand development it is essential to explore gene regulatory network assemblies, mechanisms of divergence, interconnection through signaling between cells, and subcircuits that control the progression of an embryo toward adulthood and a new reproductive cycle. This is a daimting challenge, but if one seeks to really know how the system operates, a detailed analysis of gene regulatory networks is essential. Accepting this, the next question is " how best to analyze network circuitry?" Currently many laboratories ask this question. Some utilize microarray analyses, proteomic assays, ChlP-Chip assays and other high throughput approaches to identify candidate molecules for networks. While these approaches provide candidates for networks, and while they produce diagrams that look quite complex, often they are not authenticated cormections. The challenge and the goal must be to authenticate each connection as it actually works in the organism. Only when a network can be authenticated in the organism, with signaling inputs rationalized, can one begin to understand how the system actually works. That is the goal of this project. We seek to understand how the complexity and dynamics of gene regulatory networks program the early cells of the embryo and then drive those cells through the morphogenetic movements of gastmlation. By the time gastralation begins, the ectoderm, mesoderm, and endoderm in each deuterostome is at least partially specified. The long-term goal of this project has been to build and understand how Gene Regulatory Networks (GRNs) work in governing the specification of germ layers using the sea urchin embryo as a model. In this application, we extend that goal to understand how the ectoderm and endomesoderm GRNs cormect to, and control the events of archenteron invagination and ectoderm patteming. The sea urchin is used as a model for this effort because it is well suited for interrogation of the specification mechanisms, and the relative simplicity of gastmlation in this embryo is the prototype for deuterostome early development. In the first seven years as this project unfolded in the Davidson and McClay labs, with additional contributions from the sea urchin commxmity, more than 80 transcription factors (with perhaps on the order of 80 more yet to add) and a number of signal transduction inputs were identified and incorporated into a nuclear view of triploblastic specification in this organism. The GRN as currently modeled (Figure la,b below) provides the template for the next generation of studies that are proposed in this Program Project. Here, in this sub-project, three goals will advance the Network studies into novel areas to establish "next generation" approaches. First, changes in the progression of the GRN currently are based on data collected at intervals. An important goal of this proposal is to establish tools for gathering GRN states in individual cells. This goal will better enable us to leam how the endoderm and mesoderm cells prepare for and then execute morphogenesis. As detailed in the Davidson project Genomicists view the entire GRN for their purposes (VfA), while developmental biologists prefer to view the subcircuits of that network that ran in each cell as development progresses (VfN). This is because the information relevant to the developmental biologist are the GRN states in each nucleus that progress toward, and control morphogenesis. A large number of pubhcations have provide anecdotal information on how archenteron invagination works (always with a black-box approach). Here the exciting challenge is to discover how those properties are controlled at the Network level. This effort will merely be a begiiming of what will be a major effort of many people to understand how a complex and dynamic rearrangement of cells is controlled at a franscriptional and signaling level. Further, we will expand the GRN exploration into a cormection with patteming. The ectoderm subdivides into oral and aboral halves with a ciliary band separating them. During the 2006 Genome Annotation project we led the effort to annotate all known signaling molecules. The third aim will take advantage of that effort and the ability to identify signaling inputs functionally. Our effort, combined with the Davidson lab's advances in understanding the ectoderm GRN, will provide insight into how patteming information is produced and distributed between the ca. 500 cells of the ectoderm.

2002 年，内中胚层 GRN 的第一个临时版本是 发表（Davidson 等人，2002a；Davidson 等人，2002b），并在去年发表了外胚层 GRN 的初稿，其中包括 McClay 实验室的贡献（Bradham 和 McClay，2006）；参见http://sugp.caltech.edu/endomes/#EctoNetworkDiagram（图la、lb）。该网络模型是由 Eric Davidson 根据许多实验室的数据构建的，其中 Davidson 和 McClay 实验室的主要贡献。 2002 年，我在戴维森实验室度过了休假一年，这次停留使我与埃里克戴维森及其实验室成员进行了长期合作。目前已收到9篇论文 与该提案以及正在进行的研究相关的许多其他论文的合作（Amore et al., 2003; Davidson et al., 2003; Davidson et al., 2002a; Davidson et al., 2002b; Oliveri et al., 2003; Oliveri et al., 2006; Otim et al., 2004; Sodergren et al., 2006）。最初的目标是构建一个 GRN，反映前 30 小时（直到开始时）内中胚层规范的序列。 胃化）。网络组装仅限于转录因子和信号转导。这是因为我们知道，要涵盖所有细胞生物学以及发育生理学在当时是遥不可及的。我们还希望以这样的方式构建 GRN，即网络的每个边和每个节点 可以通过实验来验证。技术的开发使得当前 GRN 的每个组件通常都具有至少三个独立的实验支持来源。在某些情况下，预测的转录输入在顺式监管水平上得到验证，这种连接“硬连线”了网络先前的预测（那些已经以这种方式验证的顺式监管连接由图1a中的粗边表示）。正如本计划项目的戴维森部分所示，我们继续努力增加 更多的顺式监管确认，并制定新的策略，以便对网络解决方案中这一繁琐但重要的组成部分进行更快速的分析。该项目的 McClay 部分是将信号转导装置与转录网络组件相连接，并提供许多实验胚胎学方法来验证网络预测，并将 GRN 连接到形态发生。这项工作需要进行一些新的分析，并形成了本提案中概述的当前网络观点。 可能有人会问，为什么人们想要如此详细地了解胚胎是如何被指定的呢？毕竟，当向学生展示 GRN 时，他们会听到对其复杂性的惊叹。尽管有这种反应，但事实是所有细胞的规范机制都是许多转录因子和许多信号装置之间高度复杂的相互作用。每个细胞的每个细胞核内都有一个转录因子的操作网络，控制着该功能细胞的发育进程和生理学。 每次两个子单元采用不同的身份时，单个网络状态必须分化为两个不同的网络状态。胚胎中细胞网络状态的排列和分布必须不断协调，并且已发现许多信号输入来满足这些要求。简而言之，要了解发育，必须探索基因调控网络组件、分化机制、细胞之间通过信号传导的互连以及控制发育进程的子电路。 胚胎走向成年和新的生殖周期。这是一项艰巨的挑战，但如果想真正了解该系统如何运作，对基因调控网络的详细分析是必不可少的。接受这一点，下一个问题是“如何最好地分析网络电路？”目前许多实验室都会问这个问题。有些利用微阵列分析、蛋白质组分析、ChlP 芯片分析和其他高通量方法来识别网络的候选分子。虽然这些方法为网络提供了候选者，同时 他们生成的图表看起来相当复杂，通常它们不是经过验证的修正。挑战和目标必须是验证每个连接在生物体中的实际作用。只有当网络能够在生物体中得到验证并且信号输入合理化时，人们才能开始理解系统的实际工作原理。这就是这个项目的目标。我们试图了解基因调控网络的复杂性和动态性如何对胚胎的早期细胞进行编程，然后驱动这些细胞通过 胃化的形态发生运动。 当原肠胚形成开始时，每个后口动物的外胚层、中胚层和内胚层至少部分被指定。该项目的长期目标是建立并了解基因调控网络（GRN）如何以海胆胚胎为模型来管理胚层的规范。 在此应用中，我们将这一目标扩展到了解外胚层和内中胚层 GRN 如何连接并控制原肠内陷和外胚层模式事件。海胆被用作这项工作的模型，因为它非常适合询问规范机制，并且该胚胎中胃化的相对简单是后口动物早期发育的原型。在戴维森和麦克莱实验室开展该项目的最初七年里，海胆界也做出了额外的贡献，其中包括 80 多个转录因子（可能还有 80 个尚未添加）和 确定了许多信号转导输入并将其纳入该生物体三叶细胞规格的核视图中。当前建模的 GRN（下图 la、b）为本计划项目中提出的下一代研究提供了模板。在这个子项目中，三个目标将把网络研究推进到新的领域，以建立“下一代”方法。首先，目前 GRN 进展的变化是基于定期收集的数据。此次活动的一个重要目标 提案是建立用于收集各个单元中的 GRN 状态的工具。这一目标将使我们能够更好地了解内胚层和中胚层细胞如何准备并执行形态发生。正如戴维森项目中详细描述的，基因组学家根据他们的目的查看整个 GRN（VfA），而发育生物学家更喜欢查看随着发育进展在每个细胞中运行的网络子电路（VfN）。这是因为与发育生物学家相关的信息是每个细胞核中进展并控制形态发生的 GRN 状态。 大量出版物提供了有关 archenteron 内陷如何发挥作用的轶事信息（始终采用黑盒方法）。这里令人兴奋的挑战是发现如何在网络级别控制这些属性。这项努力只是许多人的主要努力的开始，以了解如何在转录和信号水平上控制细胞的复杂和动态重排。此外，我们将把 GRN 探索扩展到与模式的结合。外胚层 分为口部和口部两半，并由纤毛带将它们分开。在 2006 年基因组注释项目中，我们领导了对所有已知信号分子进行注释的工作。第三个目标将利用这一努力以及功能性识别信号输入的能力。我们的努力与戴维森实验室在理解外胚层 GRN 方面取得的进展相结合，将深入了解模式信息如何在 ca 之间产生和分布。 500 个外胚层细胞。