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

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 cell that will become an absorptive enterocyte (EC) or a hormone-secreting enteroendocrine (EE) cell. The driving hypothesis in this proposal is that hidden genetic interactions among MR target genes lead to complex ISC behavior when they are manipulated simultaneously. To test this hypothesis, CAP, Klaroid and Indy, three experimentally validated targets of the MR genes Escargot and Stat92E, 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 counts, 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 with ImageJ and CellProfiler will be used for a robust and unbiased 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 (“Smurf”) assays will be used to compare the effect that individual vs. combined MR target manipulations have on the regenerative capacity of intestinal tissue during aging and following chemical injury or pathogenic infection. Combination therapies are usually explored later and reactively in pre-clinical research, only after mono-targeted therapies have reached their limit in therapeutic potential. The research described here is highly innovative in that it proposes to implement combinatorial interventions much earlier in the therapeutic space search, which could be of great significance for future studies on stem cells in the context of regenerative medicine. In addition, this project was specifically designed to engage undergraduate students in a highly impactful biomedical research experience, fulfilling another important mission of the NIH; i.e., to train a skilled and motivated workforce that will foster innovation, improve research capacity and guarantee the highest research quality for the nation.