Multiplex in Situ Visualization of the Drosophila Transcriptome in Blastoderm Embryos

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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