Investigating the function of Hindsight/RREB-1 in the intestinal stem cell lineage of Drosophila

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Stem cells are undifferentiated cells that have the ability of self-renewal as well as the capacity to differentiate into specialized cell types. Understanding the regulation of adult stem cells is a very active area of research, and the possibility of using stem cells to treat injury or disease represents the foundation of the new concept of “regenerative medicine”. It has only recently been established that the fruit fly (Drosophila), despite having a lifespan of only 45-60 days, houses stem cells within differentiated adult tissues.  For example, as in mammals, old or damaged differentiated cells in the gut epithelium are constantly shed and lost, and the replacement of these cells depends on the activity of a population of intestinal stem cells (ISCs) that reside throughout the gut epithelium. Interestingly, many genes and mechanisms regulating ISCs are conserved between Drosophila and mammals. Research using Drosophila has several advantages, including powerful and sophisticated genetic techniques, and these tools can now be applied to fundamental questions regarding the function and behaviour of stem cells. In the Drosophila midgut, ISCs divide to produce one daughter cell that retains the ISC identity and a second committed daughter cell known as an enteroblast (EB). EB differentiation occurs without further cell division, and EBs may become either absorptive enterocytes (ECs) or secretory enteroendocrine cells (EEs). My NSERC-funded research has involved investigating the function of the C2H2 type Zinc-finger transcription factor encoded by the gene hindsight. We recently found that hindsight is expressed in all midgut cell types. ISCs, EBs, and EEs, however, are associated with lower levels of Hindsight than differentiated ECs. In addition, we have found that RNAi-mediated depletion of Hindsight results in the loss of all ISCs, and, strikingly, that increasing the level of Hindsight is both necessary and sufficient for EC differentiation. This proposal outlines experiments to elucidate the role of Hindsight in ISCs and differentiated ECs. For example, why is Hindsight required for ISC maintenance? What happens to ISCs as they are “lost”? And what is the function of Hindsight in mature differentiated ECs? In addition to addressing these questions, we also propose experiments to better understand the function of Hindsight, which is the Drosophila homologue of human Ras Responsive Element Binding protein-1 (RREB-1). There is some evidence that Hindsight is modified by phosphorylation, and we propose investigating how Hindsight function might be affected by such modification. RREB-1 has been implicated in pancreatic, thyroid, and colon cancer – and basic research of its homologue in the Drosophila will not only advance the understanding of RREB-1 function in humans, but may ultimately contribute to developing novel approaches for treating these forms of cancer.