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.
