Multiplex in Situ Visualization of the Drosophila Transcriptome in Blastoderm Embryos

[120728]基因组测序完成后，biera面临的主要挑战是确定发育期间和成年期所有基因的表达模式。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表达，但这种方法缺乏空间区分，而且在时间和量级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的平均基因表达水平。为了理解基因之间的相互调节关系，其中许多基因以高度动态和空间受限的模式进行调节，人们必须最终了解基因组在整个发育过程中是如何以细胞为基础表达的。在单细胞分辨率下获得精细尺度基因表达数据的最明显方法是进行基因组尺度原位杂交实验。Bier博士、McGinnis博士和Pevzner博士将共同开发一种多重原位杂交方法来解决这个问题，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，这个协作团队将通过将其应用于两个定义良好的假设驱动问题来验证该方法。本课题的具体目标是：1)开发一种多重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)鉴定介导信号通路间串扰的基因。由于相同的遗传系统在不同的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时空尺度分析将为分析这些核心遗传途径如何在进化过程中作为自然选择修改的基础，以定制不同环境和生态位的身体计划提供一个重要的框架。这一知识对于解决后生动物系统发育的深层结构至关重要，并可能揭示多细胞后生动物是否采用了兼性殖民地单细胞生物中存在的极性产生机制，以沿着a /P轴创造异聚模式。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构成创建细胞状态详细数学模型的基础，并将对评估成年生物体如何反应至关重要

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