Craig A. Micchelli

Evidence that stem cells reside in the adult Drosophila midgut epithelium

Adult stem cells maintain organ systems throughout the course of life and facilitate repair after injury or disease. A fundamental property of stem and progenitor cell division is the capacity to retain a proliferative state or generate differentiated daughter cells; however, little is currently known about signals that regulate the balance between these processes. Here, we characterize a proliferating cellular compartment in the adult Drosophila midgut. Using genetic mosaic analysis we demonstrate that differentiated cells in the epithelium arise from a common lineage. Furthermore, we show that reduction of Notch signalling leads to an increase in the number of midgut progenitor cells, whereas activation of the Notch pathway leads to a decrease in proliferation. Thus, the midgut progenitors default state is proliferation, which is inhibited through the Notch signalling pathway. The ability to identify, manipulate and genetically trace cell lineages in the midgut should lead to the discovery of additional genes that regulate stem and progenitor cell biology in the gastrointestinal tract.

γ-Secretase/presenilin inhibitors for Alzheimer’s disease phenocopy Notch mutations in Drosophila

Signaling from the Notch (N) receptor is essential for proper cell‐fate determinations and tissue patterning in all metazoans. N signaling requires a presenilin (PS)‐dependent transmembrane‐cleaving activity that is closely related or identical to the γ‐secretase proteolysis of the amyloid‐β precursor protein (APP) involved in Alzheimers disease pathogenesis. Here, we show that N‐[N‐(3,5‐difluorophenacetyl)‐L‐alanyl]‐(S)‐phenylglycine t‐butyl ester, a potent γ‐secretase inhibitor reported to reduce amyloid‐β levels in transgenic mice, prevents N processing, translocation, and signaling in cell culture. This compound also induces developmental defects in Drosophila remarkably similar to those caused by genetic reduction of N. The appearance of this phenocopy depends on the timing and dose of compound exposure, and effects on N‐dependent signaling molecules established its biochemical mechanism of action in vivo. Other γ‐secretase inhibitors caused similar effects. Thus, the three‐dimensional structure of the drug‐binding site(s) in Drosophila γ‐secretase is remarkably conserved vis‐à‐vis the same site(s) in the mammalian enzyme. These results show that genetics and developmental biology can help elucidate the in vivo site of action of pharmacological agents and suggest that organisms such as Drosophila may be used as simple models for in vivo prescreening of drug candidates.

Adenomatous polyposis coli regulates Drosophila intestinal stem cell proliferation.

Adult stem cells define a cellular reserve with the unique capacity to replenish differentiated cells of a tissue throughout an organisms lifetime. Previous analysis has demonstrated that the adult Drosophila midgut is maintained by a population of multipotent intestinal stem cells (ISCs) that resides in epithelial niches. Adenomatous polyposis coli (Apc), a tumor suppressor gene conserved in both invertebrates and vertebrates, is known to play a role in multiple developmental processes in Drosophila. Here, we examine the consequences of eliminating Apc function on adult midgut homeostasis. Our analysis shows that loss of Apc results in the disruption of midgut homeostasis and is associated with hyperplasia and multilayering of the midgut epithelium. A mosaic analysis of marked ISC cell lineages demonstrates that Apc is required specifically in ISCs to regulate proliferation, but is not required for ISC self-renewal or the specification of cell fate within the lineage. Cell autonomous activation of Wnt signaling in the ISC lineage phenocopied Apc loss and Apc mutants were suppressed in an allele-specific manner by abrogating Wnt signaling, suggesting that the effects of Apc are mediated in part by the Wnt pathway. Together, these data underscore the essential requirement of Apc in exerting regulatory control over stem cell activity, as well as the consequences that disrupting this regulation can have on tissue homeostasis.

JAK/STAT signaling coordinates stem cell proliferation and multilineage differentiation in the Drosophila intestinal stem cell lineage

Adult stem cells are the most primitive cells of a lineage and are distinguished by the properties of self-renewal and multipotency. Coordinated control of stem cell proliferation and multilineage differentiation is essential to ensure a steady output of differentiated daughter cells necessary to maintain tissue homeostasis. However, little is known about the signals that coordinate stem cell proliferation and daughter cell differentiation. Here we investigate the role of the conserved JAK/STAT signaling pathway in the Drosophila intestinal stem cell (ISC) lineage. We show first, that JAK/STAT signaling is normally active in both ISCs and their newly formed daughters, but not in terminally differentiated enteroendocrine (ee) cells or enterocyte (EC) cells. Second, analysis of ISC lineages shows that JAK/STAT signaling is necessary but not sufficient for daughter cell differentiation, indicating that competence to undergo multilineage differentiation depends upon JAK/STAT. Finally, our analysis reveals JAK/STAT signaling to be a potent regulator of ISC proliferation, but not ISC self-renewal. On the basis of these findings, we suggest a model in which JAK/STAT signaling coordinates the processes of stem cell proliferation with the competence of daughter cells to undergo multilineage differentiation, ensuring a robust cellular output in the lineage.

Dorsoventral lineage restriction in wing imaginal discs requires Notch

The formation of boundaries that prevent the intermixing of cells is an important developmental patterning mechanism. The compartmental lineage restrictions that appear in the developing imaginal discs of Drosophila are striking examples of such boundaries. However, little is known about the cellular mechanism underlying compartmental lineage restrictions. The dorsoventral (D/V) lineage restriction that arises late in the developing wing imaginal disc requires the dorsal expression of the transcription factor Apterous and it has been hypothesized that apterous (ap) maintains compartmentalization by directly regulating the expression of molecules that modify cell adhesion or affinity. However, ap expression also regulates signalling between dorsal and ventral compartments, resulting in high levels of Notch signalling at the D/V boundary. Here we show that the formation of Notch-dependent boundary cells is required for the D/V lineage restriction.

Quiescent gastric stem cells maintain the adult Drosophila stomach

The adult Drosophila copper cell region or “stomach” is a highly acidic compartment of the midgut with pH < 3. In this region, a specialized group of acid-secreting cells similar to mammalian gastric parietal cells has been identified by a unique ultrastructure and by copper-metallothionein fluorescence. However, the homeostatic mechanism maintaining the acid-secreting “copper cells” of the adult midgut has not been examined. Here, we combine cell lineage tracing and genetic analysis to investigate the mechanism by which the gastric epithelium is maintained. Our investigation shows that a molecularly identifiable population of multipotent, self-renewing gastric stem cells (GSSCs) produces the acid-secreting copper cells, interstitial cells, and enteroendocrine cells of the stomach. Our assays demonstrate that GSSCs are largely quiescent but can be induced to regenerate the gastric epithelium in response to environmental challenge. Finally, genetic analysis reveals that adult GSSC maintenance depends on Wnt signaling. Characterization of the GSSC lineage in Drosophila, with striking similarities to mammals, will advance the study of both homeostatic and pathogenic processes in the stomach.

Generation of enteroendocrine cell diversity in midgut stem cell lineages.

The endocrine system mediates long-range peptide hormone signaling to broadcast changes in metabolic status to distant target tissues via the circulatory system. In many animals, the diffuse endocrine system of the gut is the largest endocrine tissue, with the full spectrum of endocrine cell subtypes not yet fully characterized. Here, we combine molecular mapping, lineage tracing and genetic analysis in the adult fruit fly to gain new insight into the cellular and molecular mechanisms governing enteroendocrine cell diversity. Neuropeptide hormone distribution was used as a basis to generate a high-resolution cellular map of the diffuse endocrine system. Our studies show that cell diversity is seen at two distinct levels: regional and local. We find that class I and class II enteroendocrine cells can be distinguished locally by combinatorial expression of secreted neuropeptide hormones. Cell lineage tracing studies demonstrate that class I and class II cells arise from a common stem cell lineage and that peptide profiles are a stable feature of enteroendocrine cell identity during homeostasis and following challenge with the enteric pathogen Pseudomonas entomophila. Genetic analysis shows that Notch signaling controls the establishment of class II cells in the lineage, but is insufficient to reprogram extant class I cells into class II enteroendocrine cells. Thus, one mechanism by which secretory cell diversity is achieved in the diffuse endocrine system is through cell-cell signaling interactions within individual adult stem cell lineages. Summary: Molecular mapping, lineage tracing and genetic analyses provide insight into the mechanisms governing enteroendocrine cell diversity in the adult Drosphila gastrointestinal tract.

Development and Characterization of a Chemically Defined Food for Drosophila

Diet can affect a spectrum of biological processes ranging from behavior to cellular metabolism. Yet, the precise role of an individual dietary constituent can be a difficult variable to isolate experimentally. A chemically defined food (CDF) permits the systematic evaluation of individual macro- and micronutrients. In addition, CDF facilitates the direct comparison of data obtained independently from different laboratories. Here, we report the development and characterization of a CDF for Drosophila. We show that CDF can support the long-term culture of laboratory strains and demonstrate that this formulation has utility in isolating macronutrient from caloric density requirements in studies of development, longevity and reproduction.

The origin of intestinal stem cells in Drosophila

Renewing tissues in the adult organism such as the gastrointestinal (GI) epithelium depend on stem cells for epithelial maintenance and repair. Yet, little is known about the developmental origins of adult stem cells and their niches. Studies of Drosophila adult midgut precursors (AMPs), a population of endodermal progenitors, demonstrate that adult intestinal stem cells (ISCs) arise from the AMP lineage and provide insight into the stepwise process by which the adult midgut epithelium is established during development. Here, I review the current literature on AMPs, where local, inductive and long‐range humoral signals have been found to control progenitor cell behavior. Future studies will be necessary to determine the precise mechanism by which adult intestinal stem cells are established in the endodermal lineage. Developmental Dynamics 241:85–91, 2012.

Regional Control of Drosophila Gut Stem Cell Proliferation: EGF Establishes GSSC Proliferative Set Point & Controls Emergence from Quiescence

Adult stem cells vary widely in their rates of proliferation. Some stem cells are constitutively active, while others divide only in response to injury. The mechanism controlling this differential proliferative set point is not well understood. The anterior-posterior (A/P) axis of the adult Drosophila midgut has a segmental organization, displaying physiological compartmentalization and region-specific epithelia. These distinct midgut regions are maintained by defined stem cell populations with unique division schedules, providing an excellent experimental model with which to investigate this question. Here, we focus on the quiescent gastric stem cells (GSSCs) of the acidic copper cell region (CCR), which exhibit the greatest period of latency between divisions of all characterized gut stem cells, to define the molecular basis of differential stem cell activity. Our molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. We find that under baseline conditions, when GSSCs are largely quiescent, the lowest levels of EGF ligands in the midgut are found in the CCR. However, acute epithelial injury by enteric pathogens leads to an increase in EGF ligand expression in the CCR and rapid expansion of the GSSC lineage. Thus, the unique proliferative set points for gut stem cells residing in physiologically distinct compartments are governed by regional control of niche signals along the A/P axis.