Title: Establishment and Maintenance of Adult Stem Cell Identity
Description: Adult stem cells maintain tissue integrity by dividing and producing progeny that differentiate to replace damaged and old cells, as they are lost. Both division and differentiation must be tailored to the number and type of cells lost for homeostasis to be maintained. In this thesis I address how adult stem cell identity is established and maintained in the Drosophila midgut. The Drosophila midgut, like the intestine of mammals, is composed of multiple regions that contain cells of distinct morphology and function. In this study I focus on the acidified region of the midgut, referred to as the copper cell region (CCR), in order to understand how regional stem cells are established and maintain their identity. I show that intestinal stem cells (ISCs) are specified during metamorphosis. Stem cell number increases by symmetric division of pupal escargot (esg) expressing cells. By altering the expression of the Notch ligand Delta in the muscle of pupae I show that Delta from the muscle is involved in maintaining undifferentiated pupal ISCs.

Next I investigate the origin of the adult CCR and the pathways that regulate copper cell and copper cell identity. Despite the fact that both the larval and the adult CCR cells function as acid secreting cells and express the homeodomain protein Labial, the adult CCR is distinct from the larval copper cells: it arises from a distinct set of cells and does not express the enhancers that have been shown to regulate larval copper cell expression of Labial. I identify a new enhancer in the first intron of labial that accurately reflects expression of Labial protein. I show that the BMP pathway is expressed from the visceral muscle above the adult CCR and that the Dpp ligand is necessary for copper cell differentiation. I then show that the ISCs of the CCR are normally slowly dividing (once every 4-5 days), but will divide and differentiate with damage or cell death. CCR ISCs are stimulated to divide by the JAK/STAT pathway, the same pathway that regulates proliferation in response to damage in the rest of the midgut.

CCR ISC differentiation is also Notch dependent just as the rest of the midgut is. I also show that the quiescence of CCR ISCs is dependent on acidification of that region, suggesting that acidification is responsible for a decrease in damage and subsequent low turnover. I then investigate when regional ISC identity is established and show that both Labial and BMP signaling are present in one region of the pupal midgut beginning at 20 hours after pupal formation. Then I express dpp from all of the muscle cells to show that pupal cells can be specified into copper cells during pupation but not afterwards. BMP activation in pupal EC cells is capable of transforming them to copper cells, but those cells are not maintained in the adult. BMP activation in pupal ISCs also alters their identity, as they begin producing ectopic copper cells after several days in the adult. I show that copper cell ISC identity can be altered only during a window of pupation and that those cells can then respond to BMP signals in the adult to produce new copper cells.

This demonstrates that adult stem cell identity in this region is the result of intrinsic or remembered signals that restrict or alter the ability to receive local environmental cues. Finally I investigate the role of chromatin modifying genes in establishment and maintenance of ISC identity. I carried out an RNAi screen and identified a number of genes whose knockdown alters ISC identity. I also demonstrate that when ISCs are heritably marked during pupation rather than in adulthood, a distinct set of quiescent ISCs can be identified. This evidence indicates that our current understanding of midgut ISC proliferation is incomplete and that a novel quiescent stem cell might exist in the Drosophila midgut.