Genetic interactions among targets of master regulator genes as drivers of complex behavior in Drosophila intestinal stem cells

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PROJECT SUMMARY Several master regulator (MR) genes have been characterized in tissue stem cells across organs and species. However, they remain poor candidates for therapeutic manipulation because they are highly pleiotropic, affecting hundreds of targets and operating across many organ systems. Therefore, only a better understanding of how less pleiotropic downstream MR targets coordinate stem cell proliferation, self-renewal and differentiation will unlock the full potential of stem cells in regenerative medicine. The long-term goal of this project is to map the regulatory landscape established by MR genes and their targets in intestinal stem cells (ISCs), using the fruit fly Drosophila melanogaster as a model system. Drosophila ISCs divide asymmetrically, giving rise to a new ISC and a sister that will become an absorptive enterocyte (EC) or a hormone-secreting enteroendocrine cell (EE). The specific hypothesis driving this proposal is that cross-regulatory interactions between MR target pathways can lead to non- linear, unpredictable outcomes on ISC behavior when they are manipulated simultaneously. To test this hypothesis, CAP, Klaroid and Indy, three experimentally validated targets of the ISC MR genes Escargot and STAT, will be manipulated alone or in combination within ISCs using an inducible Gal4/UAS system. The effect of their individual vs. combined manipulations on intestinal homeostasis will be assessed via three separate but complementary approaches. In Aim 1, immunofluorescence microscopy will be used to compare ISC number, morphology, mitotic rate, and differentiation potential, based on well-established cell type markers (esg-GFP for ISCs, Su(H) activation for EBs, Pdm1 and Pros staining for ECs and EEs, respectively). Automated image analysis through ImageJ and CellProfiler will be used to analyze multiple images per group, allowing a robust statistical analysis of the data. In Aim 2, fluorescent activity reporters will be used to compare the effect of single vs. dual MR target manipulations on key ISC signal transduction pathways (EGFR, Notch, Wnt, STAT and JNK). In Aim 3, lifespan and intestinal barrier integrity (Smurf) assays will be used to compare the effect that individual vs. combined MR target manipulations have on the regenerative capacity of intestinal tissue following chemical injury or pathogenic infection. These research aims may generate evidence that challenges the widely held premise that combination therapies can only improve outcome due to additive complementation of positive effects. If so, this project will have a significant impact on our conceptual approach to stem cell manipulation for regenerative medicine. In addition, this project was specifically designed to engage a large number of students from underrepresented backgrounds in biomedical research, satisfying another important mission of the NIH: to diversify the scientific workforce, and thus foster innovation, improve research quality and enhance the public trust and investment in science.