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.

嗨，2002年，内胚层GRN的第一个临时版本是 出版（Davidson等，2002a； Davidson等，2002b），在过去的一年中，Ectoderm GRN的初稿发表了，其中包括McClay Lab的贡献（Bradham和McClay，2006年）；参见http://sugp.caltech.edu/endomes/#ectonetworkdiagram（图LA，LB）。埃里克·戴维森（Eric Davidson）基于来自许多实验室的数据来巩固网络模型，并从戴维森（Davidson）和麦克莱（McClay）实验室（McClay Labs）进行了重大贡献。我于2002年在戴维森实验室度过了休假一年，这一停留时间导致了与埃里克·戴维森（Eric Davidson）和他的实验室成员进行长期合作。迄今为止9篇论文来了 通过与该提案有关的许多其他论文的合作以及正在进行的研究（Amore等，2003; Davidson等，2003; Davidson等，2002a; Davidson等，2002b; Oliveri等，2003; Oliveri等，2003; Oliveri等，2006; 2006; 2006; Otim等，2004; otim等，2004; sodergren et al and，2006）。最初的目标是巩固一个反映在前30个小时内反映内胚层规范顺序的GRN（直到开始 gastmlation）。网络组件仅限于转录因子和信号转导。这是因为我们知道，当时包括所有细胞生物学以及发展生理学的发展是无法实现的。我们还想以网络的每个边缘和每个节点的方式来缩合GRNS 可以通过实验进行身份验证。开发了技术，以使当前GRN的每个组成部分通常具有至少三个独立的实验支持来源。在某些情况下，在顺式调节水平上验证了预测的三名输入，这种连接“硬化”了先前对网络的预测（以这种方式验证的那些顺序调节的结合次数由图LA中的较厚边缘表示）。正如该计划项目的戴维森（Davidson）组成部分所见，努力继续增加 更多的顺式调节性确认，并制定新策略，从而可以对网络解决方案的这种乏味但脆性的组成部分进行更快速的分析。该项目的McClay组成部分是将信号转导设备协调到转录网络组件，并提供多种实验性胚胎学方法来验证网络预测，并将GRN连接到形态发生。这项工作需要许多新的测定法，并导致了本提案中概述的网络的当前观点。 可能会问，为什么要详细了解如何指定胚胎？毕竟，当向学生展示GRN时，听到复杂性的可听见。尽管有这种反应，但现实是所有细胞中规范的机制都是许多转录因子和许多信号传导设备的高度复杂的相互作用。在每个细胞的每个核内部都有一个运作网络的转录因子网络，该网络涉及发育进展和该功能细胞的生理学。 每次两个子细胞都采用不同的身份时，单个网络状态必须分为两个不同的网络状态。细胞网络在胚胎中的布置和分布必须不断协调，并且已经发现许多信号输入以适应这些要求。简而言之，要了解发展，必须探索基因调节网络组件，差异机制，通过细胞之间的信号传导的互连以及控制一个控制一个进展的亚电路 朝着成年的胚胎和一个新的生殖周期。这是一个挑战，但是如果人们试图真正知道该系统的运作方式，那么对基因调节网络的详细分析至关重要。接受这一点，下一个问题是“如何最好地分析网络电路？”目前，许多实验室都问这个问题。一些人利用微阵列分析，蛋白质组学测定，CHLP-CHIP分析和其他高吞吐量方法来识别网络的候选分子。尽管这些方法为网络提供了候选人，而 它们产生的图表看起来很复杂，通常不是认证的cormections。挑战和目标必须是对每个连接实际在生物体中起作用。只有在有机体中可以在有机体中对网络进行身份验证，并且信号输入合理化时，才能开始了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节网络的复杂性和动态如何编程胚胎的早期细胞，然后将这些细胞通过 Gastmlation的形态发生运动。 到胃开始时，至少部分指定了每个氘代表中的外胚层，中胚层和内胚层。该项目的长期目标是建立和了解基因调节网络（GRNS）如何使用海胆胚作为模型来管理细菌层的规范。 在此应用程序中，我们扩展了该目标，以了解外胚层和内胚层GRNS如何cormect到并控制Archenteron Invagination和Ectodermecteming的事件。海胆被用作这种努力的模型，因为它非常适合审问规范机制，而这种胚胎中加斯特姆化的相对简单性是氘代表早期发育的原型。在最初的七年中，随着该项目在戴维森（Davidson）和麦克莱（McClay Labs）中展开，并提供了Sea Hilchin Commxmity的额外贡献，超过80个转录因素（也许还有80多个订单）和一个 鉴定了信号转导输入的数量，并将其纳入该生物体中三重细胞规范的核视图中。当前建模的GRN（下图LA，B）为该计划项目中提出的下一代研究提供了模板。在这里，在此子项目中，三个目标将把网络研究推向新的领域，以建立“下一代”方法。首先，当前GRN的进程的变化基于以间隔收集的数据。一个重要的目标 建议是建立用于在单个细胞中收集GRN状态的工具。这个目标将更好地使我们能够启动内胚层和中胚层细胞如何为形态发生准备然后执行形态发生。正如Davidson项目基因组学家在其目的（VFA）中所详述的那样，而发展生物学家则更喜欢查看随着开发的进展，该网络在每个细胞中运行的网络的子电路（VFN）。这是因为与发育生物学家相关的信息是朝向和控制形态发生的每个核中的GRN状态。 大量的Pubhcations提供了有关Archenteron Invagination如何工作的轶事信息（始终采用黑框方法）。在这里，令人兴奋的挑战是发现如何在网络级别控制这些属性。这项工作仅仅是许多人的重大努力的启动，以了解细胞的复杂和动态重排如何在编写和信号传导水平上控制。此外，我们将将GRN探索扩展到patteming的cormection中。外胚层 用睫状带分隔成口腔和一半分成口服和原住民。在2006年的基因组注释项目中，我们领导了注释所有已知的信号分子的努力。第三个目标将利用这项工作和在功能上识别信号输入的能力。我们的努力以及戴维森实验室在理解外胚层GRN方面的进步，将提供有关如何在CA之间生产和分发的信息。 500个外胚层的细胞。