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Analysis of Developmental Gene Regulation via Quantitative Imaging

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Proper spatiotemporal expression of genes is essential during development. One method of regulation of signaling-responsive genes is at the level of transcription. In this work, I present the adaptation of single molecule fluorescent in situ hybridization for use in Drosophila imaginal disc tissues in order to more precisely quantify transcript levels in these tissues. I show the detection of nascent and mature mRNA molecules. I also present the development of robust automated image analysis in order to identify transcripts and localize them to the nearest nuclei in a 3D image volume. Using these methodologies, it is now possible to count individual transcripts in Drosophila imaginal disc tissues and to perform spatial analysis of gene expression using this method. Single-cell quantitative studies of transcription have revealed that transcription occurs intermittently, in bursts. I utilized smFISH in the wing imaginal disc in order to quantify mRNA of genes downstream of the evolutionarily conserved Wg and Dpp morphogen gradients. I compared these experimental results with predicted results from in silico modeling of transcription in order to predict outcomes when transcriptional burst parameters are varied. My results indicate that the transcription levels of these genes appear to share a common method of control by burst frequency modulation. Additionally, I utilized quantitative analysis of fluorescent proteins in the eye imaginal disc in order to explore the regulation of a key regulator in neuronal fate transition in this tissue, Yan. I show that in the absence of micro-RNA miR-7 regulation, Yan protein levels are mildly derepressed in undifferentiation precursor cells in the eye disc. This is consistent with the known role of miRNAs as weak repressors of gene expression during development. I also present evidence that slowing metabolism makes miR-7 repression of Yan unnecessary, supporting the hypothesis that weak repressors are required for gene expression during rapid growth.

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