This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The knowledge of complete genomic sequences has made possible the task of compiling the genome-wide expression profiles of cells and tissues. At the moment, this 'transcriptome' data is available only through the use of DNA microarrays, which provide expression data devoid of spatial information and with very limited temporal resolution. However, the spatio-temporal richness of gene expression patterns exhibited by developing embryos demands that genome-scale in-situ detection techniques be developed, if a meaningful description of the embryonic transcriptome is to be attained. Toward this end, we have started a collaboration with Molecular Probes, Inc., to develop protocols for the in-situ fluorescent detection of multiple mRNAs, and our ultimate hope is to be able to visualize 8-10 different expression patterns, using confocal microscopy, in single specimens. We are currently exploring various labeling strategies, and believe that with the right combination of available fluorophores, excitation lines, and emission filters, we can achieve this number in multiplex staining experiments. The resources of the NCMIR is invaluable to us in the design and execution of the microscopy required for this project. Whole-mount in-situ hybridization of early Drosophila melanogaster embryo displaying the expression patterns of three genes essential for the specification of cell fates. In this view of a field of cells on the ventral surface of the embryo, cell nuclei turn up as dark holes surrounded by the pseudo-colored cytoplasmic accumulation of mRNA, and in some nuclei the primary sites of transcription are visible. A central, broad expression domain of the gene snail (sna, in blue) is symmetrically flanked by a single row of cells expressing the gene single-minded (sim, in red) and striped patches of rhomboid (rho, in green) expression. In the center of the sna domain, the cells have begun to fold inward, representing the first movements of gastrulation which will ultimately bring into the juxtaposition the two rows of sim expressing cells. Sna, sim, and rho are crucial for the proper determination of the embryo's mesoderm, nervous system midline, and ventral ectoderm, respectively. Separate detection of the three gene products was accomplished with the following methods. First, digoxigenin (sna), biotin (sim), and fluorescein (rho) conjugated nucleotides were used to synthesize antisense riboprobes, which were then hybridized to fixed embryos. Next, after washing, antibodies against digoxigenin and fluorescein, and streptavidin-HRP(which binds to biotin) were added. Finally, secondary antibodies were used to detect the anti-fluorescein (Alexa 488 conjugate) and the anti-digoxigenin (Alexa 647 conjugate), while the HRP was used to deposit a fluorescent tyramide (Alexa 568 conjugate). Images were collected on the Radiance confocal at NCMIR using the 20x objective and all three color channels.
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