Egg to Embryo: Gene Regulatory Circuitry in Development

说明（由申请人提供）：这是一份申请，申请延续和扩大目前已进入第9年的计划项目。在这里，我们建议在许多方向上建立我们在解决和验证基因调控网络（GRN）的发展方面取得的巨大成功。grn从基因组调控代码的角度为发育过程提供了因果解释，其中所有物种特异性发育过程最终都被编程。一个发展中的GRN作为一个概念性的、系统级的逻辑图，具有直接的预测能力。因此，grn在具有调控意义的功能基因组DNA序列与胚胎发生和体表形成生物学之间架起了桥梁。他们通过指定调控相互作用来做到这一点，这些相互作用导致了不同细胞区域中调控状态的进展。近年来，该计划一直负责为任何正在发育的动物有机体提供最先进的发育GRN的实验解决方案。这是海胆胚胎内胚层区域的GRN。最近在这项工作中获得了一个原理的证明，即随着GRN接近完成，它确实提供了所有观察到的生物学功能的解释。我们现在打算利用我们开发的越来越多的成功的技术方法来分析GRN，以应对迄今为止无法实现的挑战，或者甚至无法定义。目前的海胆胚胎GRN关注的是从最早的合子基因组活动（卵裂开始）到原肠胚形成之前，最终形成肠道、成骨细胞系和非成骨中胚层的一半胚胎。口腔和流产外胚层grn也已开始。我们打算以质量不同的方式扩展这个GRN，如果成功，这将代表根本性的进步。在DAVIDSON组件中，GRN将扩展到包括基因组分析预测的所有调节基因，并观察到在内胚层区域特异性表达，以及通过原肠形成的口腔和体外外胚层，直至中胚层细胞类型出现，以及内胚层和外胚层细分（例如口腔，胃，后肠）已被指定区域。这将相当于GRN的几倍扩张，但也会产生质的新变化。因此，除了顶端神经发生区域，它将首次代表一个全球的（几乎）完整的胚胎GRN，包括几乎所有胚胎区域的规范机制：它将使我们能够看到大多数整个发育中的胚胎的基因组调控程序的维度和组织，这是直到最近才有人想象到的。在McCLAY组件中，GRN将向不同的方向扩展：下游分化和形态发生基因，它们完成构建组织和表达其特定功能的实际工作：这将使我们能够看到在规范过程中建立的转录空间调节状态如何被用来控制发育的实际“工作”，这是我们目前只能瞥见的东西。我们相信当前POI支持的GRN分析的概念和技术进步现在已经足够成熟，因此将这些想法和方法转移到开发羊膜系统的时机已经成熟：因此，在BRONNER-FRASER组件中，将为小鸡神经嵴构建系统级GRN。这不仅本身就是一个迷人的、脊椎动物特有的发育特征，而且由于广泛的前期工作，海胆GRN项目中出现的许多方法的应用似乎已经成熟。

DESCRIPTION (provided by applicant): This is an application for continuation and broadening of a Program Project now in its 9* year. Here we propose to build in many directions on the large success we have had in solving and authenticating a gene regulatory network (GRN) for development. GRNs provide causal explanations for developmental processes in the terms of the genomic regulatory code, where all species-specific developmental processes are ultimately programmed. A developmental GRN serves as a conceptual, system-level logic map, of direct predictive power. Thus GRNs bridge between functional genomic DNA sequence of regulatory significance and the biology of embryogenesis and body plan formation. They do this by specifying the regulatory interactions which causally drive the progression of regulatory states in diverse cellular territories. During recent years, this Program has been responsible for the experimental solution of the most advanced developmental GRN yet available for any developing animal organism. This is the GRN underlying the specification of the endomesodermal territories of the sea urchin embryo. Recently proof of the principle that as a GRN approaches completion it indeed provides explanation of all the observed biological functions has been obtained in this work. We now intend to capitalize on the growing suite of successful technological approaches to GRN analysis that we have developed, to confront challenges that heretofore were inaccessible, or could not even have been defined. The current sea urchin embryo GRN concerns about half of the embryo, that ultimately forming the gut, the skeletogenic cell lineages and the non-skeletogenic mesoderm, from the earliest zygotic genomic activity (at the beginning of cleavage) to just before gastrulation. A start on the oral and aboral ectodermal GRNs has also been made. We intend to expand this GRN in qualitatively distinct ways which will represent radical advances if successful. In the DAVIDSON COMPONENT the GRN will be expanded to include all regulatory genes predicted by genomic analysis, and observed to be expressed specifically in the endomesodermal territories, as well as in the oral and aboral ectoderm through gastrulation, to the point where the mesodermal cell types have appeared, and endodermal and ectodermal subdivisions (e.g. mouth, stomach, hindgut) have been territorially specified. This will amount to a several fold expansion of the GRN, but also produce a qualitatively new departure. Thus, except for the apical neurogenic domain, it would represent for the first time ever a global, (almost) whole embryo GRN that includes the mechanism for specification of almost all embryonic territories: it would enable us to see the dimensions and the organization of the genomic regulatory program for most of a whole developing embryo, something no one could have imagined until very recently. In the McCLAY COMPONENT the GRN will be extended in a different direction: to the downstream differentiation and morphogenesis genes that do the actual work of building tissues and expressing their particular functions: this will enable us to see how the transcriptional spatial regulatory states established during specification are used to control the actual "jobs" of development, something of which we have but glimpses at present. We believe the conceptual and technological advances of the GRN analysis supported by the current POI are now sufficiently mature so that the time is ripe for transfer of these ideas and methods to developing amniote systems: thus in the BRONNER-FRASER COMPONENT a system-level GRN is to be constructed for chick neural crest. This is not only in its own right a fascinating, vertebrate specific, developmental feature, but due to extensive prior work, would appear ripe for application of many of the approaches emerging from the sea urchin GRN project. This Program has been designed in a heavily interdependent way so that each Component will enjoy and indeed will require close scientific interactions with other Components. These interactions are detailed in the following. No less crucial will be the general reliance of all components on two Core Units. These are the SPECIALIZED RESEARCH SUPPORT (SRC) CORE and the SCIENTIFIC AND ADMINISTRATIVE COORDINATION (SAC) CORE. The functions of the SRC CORE will be to provide special high tech services that will be essential to all three P01 Components, i.e., procedural and instrumental functions that would require a vast outlay of equipment and a vast effort to develop know-how were these functions to be duplicated in each independent lab. Among these, as detailed below, are: (1) construction of cis-regulatory expression vectors by BAC recombineering; (2) accurate amplification of ng quantity RNA preparations; (3) large scale arraying services; (4) bioinformatics services; (5) year round supply of sea urchins; (6) use of a newly on-line instrument, the NanoString nCounter System for direct measurement of large numbers of specific transcripts simultaneously and very quantitatively on small amounts of material. The SAC CORE will constitute the central managerial oversight of the whole project; will interface with individual University grants managers; will coordinate personnel, fiscal, and publication policies; will ensure maximally economic supply acquisitions; and will arrange for webcast conferences among Components as well as biannual meetings of POI personnel.