Multiplex in Situ Visualization of the Drosophila Transcriptome in Blastoderm Embryos

囊胚胚胎中果蝇转录组的多重原位可视化

• 批准号: 0120728
• 负责人: Ethan Bier
• 金额: $ 135万
• 依托单位: University of California-San Diego
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-10-01 至 2007-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0120728&HistoricalAwards=false
• 关键词: Multiplex; Situ; Visualization; Drosophila; Transcriptome

0120728BierA major challenge following the completion of genome sequencing is to determine the expression patterns of all genes during development and in the adult. Obtaining this data is critical if we are to unravel the complex regulatory networks that regulate genome expression. Although whole genome gene expression can be analyzed by current microarray techniques, this method lacks spacial discrimination and is relatively low resolution in time and magnitude, since by its nature, it measures average gene expression levels over large heterogeneous cell populations. To understand the regulatory interrelationships between genes, many of which are regulated in highly dynamic and spatially restricted patterns, one must ultimately know how the genome is expressed on a cell-by cell basis throughout development. The most obvious way to obtain fine scale gene expression data at single cell resolution is by performing genome scale in situ hybridization experiments. Dr. Bier, Dr. McGinnis, and Dr. Pevzner will address this problem jointly by developing a multiplex in situ hybridization method that will greatly facilitate and enable the acquisition of genome expression data at single cell resolution. In addition, this collaborative team will validate the method by applying it to two well defined hypothesis driven questions. The specific goals of this proposal are to: 1) Develop a multiplex RNA in situ hybridization labeling technique, 2) Analyze Hox gene regulatory networks repressing limb development, and 3) Identify genes mediating cross-talk between signaling pathways. Impact Statement: Because the same genetic systems create pattern during development in diverse metazoans, fine spatial and temporal scale analysis of these regulatory relationships in Drosophila will provide an essential framework for analyzing how these core genetic pathways have served as substrates for modification by natural selection during evolution to tailor body plans to different environments and ecological niches. This knowledge is essential for resolving deep structures of metazoan phylogeny and may reveal whether multicellular metazoans co-opted a polarity generating mechanism present in facultative colonial unicellular organisms to create metameric pattern along the A/P axis. In addition, the methodologies we develop and the understanding we gain of cellular responses to developmental signals will form the basis for creating detailed mathematical models of cellular states and will be critical for evaluating how adult organisms respond

基因组测序完成后的一个主要挑战是确定所有基因在发育过程中和在成人中的表达模式。如果我们要解开调控基因组表达的复杂调控网络，获得这些数据是至关重要的。虽然目前的微阵列技术可以分析全基因组基因的表达，但这种方法缺乏空间分辨率，在时间和大小上的分辨率相对较低，因为它的本质是测量大型异质细胞群体的平均基因表达水平。为了了解基因之间的调控相互关系，其中许多基因是以高度动态和空间受限的模式进行调控的，人们最终必须知道基因组在整个发育过程中是如何在逐个细胞的基础上表达的。在单细胞分辨率下获得精细的基因表达数据的最明显的方法是进行基因组规模的原位杂交实验。比尔博士、麦金尼斯博士和佩夫兹纳博士将共同解决这一问题，开发一种多重原位杂交方法，这种方法将极大地促进并使获得单细胞分辨率的基因组表达数据成为可能。此外，这个合作团队将通过将其应用于两个定义明确的假设驱动的问题来验证该方法。这一建议的具体目标是：1)发展一种多重RNA原位杂交标记技术，2)分析抑制肢体发育的HOX基因调控网络，3)识别介导信号通路之间串扰的基因。影响陈述：由于相同的遗传系统在不同后生动物的发育过程中创造了模式，对果蝇这些调控关系的精细空间和时间尺度分析将提供一个必要的框架，用于分析这些核心遗传途径如何作为进化过程中自然选择修改的底物，以适应不同的环境和生态位。这一知识对于解决后生动物系统发育的深层结构是至关重要的，并可能揭示多细胞后生动物是否利用兼性殖民地单细胞生物中存在的一种极性产生机制来沿A/P轴创建异构体模式。此外，我们开发的方法和我们对细胞对发育信号的反应的理解将形成建立细胞状态的详细数学模型的基础，并将对评估成年生物体的反应至关重要。