The Interactive Fly

Genes involved in tissue and organ development

Midgut


Intestinal stem cells
  • Origin of endoderm and the midgut
  • Physiological and stem cell compartmentalization within the Drosophila midgut
  • Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult Drosophila midgut
  • The adult Drosophila gastric and stomach organs are maintained by a multipotent stem cell pool at the foregut/midgut junction in the cardia (proventriculus)
  • A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila
  • EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells
  • A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis
  • Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway
  • Transcriptional control of stem cell maintenance in the Drosophila intestine
  • Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut
  • Drosophila midgut homeostasis involves neutral competition between symmetrically dividing intestinal stem cells
  • Niche appropriation by Drosophila intestinal stem cell tumours
  • Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila
  • Rac1 drives intestinal stem cell proliferation and regeneration
  • Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling
  • CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo
  • The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila
  • Regional control of Drosophila gut stem cell proliferation: EGF establishes GSSC proliferative set point & controls emergence from quiescence
  • EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors
  • Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia
  • Bunched and Madm function downstream of Tuberous Sclerosis Complex to regulate the growth of intestinal stem cells in Drosophila
  • Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis
  • Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila
  • Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells
  • Enteroendocrine cells support intestinal stem-cell-mediated homeostasis in Drosophila
  • Haemocytes control stem cell activity in the Drosophila intestine
  • Drosophila Pez acts in Hippo signaling to restrict intestinal stem cell proliferation
  • Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies
  • Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells
  • Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila
  • Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in Drosophila
  • Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration
  • Diversity of fate outcomes in cell pairs under lateral inhibition
  • Tis11 mediated mRNA decay promotes the reacquisition of Drosophila intestinal stem cell quiescence
  • Drosophila dyskerin is required for somatic stem cell homeostasis
  • Intestinal stem cell pool regulation in Drosophila
  • Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism
  • Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut
  • Loss of the mucosal barrier alters the progenitor cell niche via JAK/STAT signaling
  • Nutritional control of stem cell division through S-Adenosylmethionine in Drosophila intestine
  • Autophagy maintains stem cells and intestinal homeostasis in Drosophila

    Biology of the Midgut
  • Ecdysone-regulated genomic networks in Drosophila: Midgut gene expression during metamorphosis
  • Ecdysone-induced receptor tyrosine phosphatase PTP52F regulates Drosophila midgut histolysis by enhancement of autophagy and apoptosis
  • Dpp regulates autophagy-dependent midgut removal and signals to block ecdysone production
  • Apoptosis restores cellular density by eliminating a physiologically or genetically induced excess of enterocytes in the Drosophila midgut
  • Suppression of insulin production and secretion by a Decretin hormone
  • A subset of enteroendocrine cells is activated by amino acids in the Drosophila midgut
  • A subset of neurons controls the permeability of the peritrophic matrix and midgut structure in Drosophila adults
  • Metamorphosis of the Drosophila visceral musculature and its role in intestinal morphogenesis and stem cell formation
  • Regional cell-specific transcriptome mapping reveals regulatory complexity in the adult Drosophila midgut
  • Misregulation of an adaptive metabolic response contributes to the age-related disruption of lipid homeostasis in Drosophila
  • High sugar diet disrupts gut homeostasis though JNK and STAT pathways in Drosophila
  • The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila
  • Hs3st-A and Hs3st-B regulate intestinal homeostasis in Drosophila adult midgut
  • Reduced gut acidity induces an obese-like phenotype in Drosophila melanogaster and in mice
  • More Drosophila enteroendocrine peptides: Orcokinin B and the CCHamides 1 and 2
  • Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch
  • miR-263a regulates ENaC to maintain osmotic and intestinal stem cell homeostasis in Drosophila
  • Control of lipid metabolism by Tachykinin in Drosophila
  • Transforming growth factor beta/Activin signaling functions as a sugar-sensing feedback loop to regulate digestive enzyme expression
  • Suppressor of Deltex mediates Pez degradation and modulates Drosophila midgut homeostasis
  • A tetraspanin regulates septate junction formation in Drosophila midgut
  • A systematic analysis of Drosophila regulatory peptide expression in enteroendocrine cells
  • A role for the Drosophila zinc transporter Zip88E in protecting against dietary zinc toxicity
  • Pleiotropic and novel phenotypes in the Drosophila gut caused by mutation of drop-dead
  • The cis-regulatory dynamics of embryonic development at single-cell resolution
  • Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster

    Gut and ageing, lifespan, microbiome, immunocompetence, disease
  • Age-associated loss of lamin-B leads to systemic inflammation and gut hyperplasia
  • Genetic, molecular and physiological basis of variation in Drosophila gut immunocompetence
  • Distinct shifts in microbiota composition during Drosophila aging impair intestinal function and drive mortality
  • Preventing age-related decline of gut compartmentalization limits microbiota dysbiosis and extends lifespan
  • Gut microbiota in Drosophila melanogaster interacts with Wolbachia but does not contribute to Wolbachia-mediated antiviral protection
  • Stable host gene expression in the gut of adult Drosophila melanogaster with different bacterial mono-associations
  • Drosophila melanogaster establishes a species-specific mutualistic interaction with stable gut-colonizing bacteria
  • The Drosophila microbiome has a limited influence on sleep, activity, and courtship behaviors
  • A Mesh-Duox pathway regulates homeostasis in the insect gut
  • Probabilistic invasion underlies natural gut microbiome stability
  • Gut microbiota modifies olfactory-guided microbial preferences and foraging decisions in Drosophila
  • How gut transcriptional function of Drosophila melanogaster varies with the presence and composition of the gut microbiota
  • Misato underlies visceral myopathy in Drosophila
  • RNA polymerase III limits longevity downstream of TORC1
  • Inflammation-modulated metabolic reprogramming is Required for DUOX-dependent gut immunity in Drosophila
  • Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila
  • The Drosophila melanogaster gut microbiota provisions thiamine to its host
    Genes active in the midgut


    Anterior midgut
    Posterior midgut


    Midgut

    Origin of endoderm and the midgut

    The midgut is derived from the anterior and posterior midgut primordia during the process of gastrulation [Images]. It should be kept in mind that the most terminal aspects of the embryo are fated to become gut endoderm. The terminal system (torso), regulating tailless and huckebein are responsible for this fate determination. Gastrulation is the defining event of gut morphogenesis. The anterior midgut is formed from the anterior midgut primordium; the posterior midgut is derived from the posterior midgut primordium, and the midgut proper is derived from endodermal cells that migrate from both anterior and posterior primordia. The gut is enshrouded in mesoderm which forms vascular musculature around the gut, and is also responsible for creating the gastric ceca. Overlying mesoderm communicates with the gut by secreted factors and through contact.

    Dpp has a prime function during endoderm induction in Drosophila. Dpp is secreted from the outer cell layer of the embryonic midgut (the visceral mesoderm) where its main source of expression in parasegment ps7 depends directly on the homeotic gene Ultrabithorax. In the same cell layer, Dpp stimulates expression of another extracellular signal, Wingless (Wg), in a neighboring parasegment that, in turn, feeds back to ps7 to stimulate Ubx expression. Thus, Dpp is part of a "parautocrine" feedback loop for Ubx (i.e., an autocrine feedback loop based partly on paracrine action that sustains its own expression through Dpp and Wg). Dpp also spreads to the inner layer of the embryonic midgut, the endoderm, where it synergizes with Wg to induce expression of the homeotic gene labial (lab). To achieve this, Dpp locally elevates the endodermal expression levels of Drosophila D-Fos with which it cooperates to induce lab. Differentiation of various cell types in the larval gut depends on these inductive effects of Dpp and Wg (Bienz, 1997 and references).

    A secondary signal has been discovered with a permissive role in this process; it comes from Vein, a neuregulin-like ligand that stimulates the Epidermal growth factor receptor (Egfr) and Ras signaling. Dpp and Wg up-regulate vein expression in the midgut mesoderm in two regions overlapping the Dpp sources. Experiments based on lack of function and ectopic stimulation of Dpp and Egfr signaling show that these two pathways are functionally interdependent and that they synergize with one another other, revealing functional intertwining. The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras. Since up-regulation of vein requires dpp and wg, Vein is considered a secondary signal of Dpp and Wg. Vein stimulates homeotic gene expression in both cell layers of the midgut (Szüts, 1998).

    Physiological and stem cell compartmentalization within the Drosophila midgut

    The Drosophila midgut is maintained throughout its length by superficially similar, multipotent intestinal stem cells that generate new enterocytes and enteroendocrine cells in response to tissue requirements. This study found that the midgut shows striking regional differentiation along its anterior-posterior axis. At least ten distinct subregions differ in cell morphology, physiology and the expression of hundreds of genes with likely tissue functions. Stem cells also vary regionally in behavior and gene expression, suggesting that they contribute to midgut sub-specialization. Clonal analyses showed that stem cells generate progeny located outside their own subregion at only one of six borders tested, suggesting that midgut subregions resemble cellular compartments involved in tissue development. Tumors generated by disrupting Notch signaling arose preferentially in three subregions and tumor cells also appeared to respect regional borders. Thus, apparently similar intestinal stem cells differ regionally in cell production, gene expression and in the ability to spawn tumors (Marianes, 2013).

    These experiments significantly expand previous knowledge of regional variation within the Drosophila midgut. At the levels of cell morphology, cell behavior and gene expression, the midgut is much more highly organized than a uniform cellular tube containing an acidic middle region or 'stomach'. Regionalization likely supports the complex metabolic tasks carried out by the midgut. Ingested food goes through multiple intermediate stages during digestion and these steps may be most efficient if carried out in a controlled sequence. Some of these steps are associated with an array of bacterial species that constitute the normal intestinal microbiome. This work will allow the function of genes, pathways, cells and regions within the midgut to be tested in digestion, tissue maintenance, microbiome function and immunity (Marianes, 2013).

    After this work was submitted for publication (Buchon et al. (2013) also described midgut regionalization; they classified five major midgut divisions (R1–R5), 13 total subregions (e.g., R1a and R1b), and 2 microregions (BR2–3, BR3–4). Converting counts of cell number within the 10 regions described in this to the fractional length coordinates used by (Buchon, 2013) suggests a close correspondence between the studies: A1 = R1a + R1b; A2 = R2a + R2b; A3 = R2c; Cu = R3a + R3b; LFC = R3c; Fe = BR3–R4, P1 = R4a + R4b; P2 = R4c; P3 = R5a; P4 = R5b. In the case of single regions in the current system that Buchon split in two, differences in gene expression were also usually noted; for example, between the anterior and posterior Cu region Buchon (2013) or the subregions of A2 and P1 that differ in lipid accumulation. Enterocyte morphology and qualitative gene expression differences were noted in defining subregions. In contrast to the current study, Buchon (2013) views constrictions to be of primary importance and define two constrictions as micro-regions in their own right. This study found that enterocyte subregions abut directly, and favors the view that constrictions are mesodermal features, not microregions (Marianes, 2013).

    The results argue strongly that the striking regionalization of structure and gene expression within the midgut is maintained at least in part by regional differences between their resident stem cells. In the midgut subregions surrounding five different boundaries, no single stem cell was found that produced differentiated cells on the opposite side of the boundary, that is from a region different from the one in which it resided. All the ‘non-crossing’ clones contacted the regional boundary, and in 78% the founder stem cell was located on or within one cell of the boundary, such that progeny cells could have reached the adjacent region prior to differentiation. In contrast, boundary clones with the same general properties almost always crossed the LFC/Fe boundary, showing that ‘non-crossing’ behavior would not occur by chance. Consistent with the existence of epigenetic differences in stem cells that limit trans-regional differentiation, clones frequently pushed into adjacent regions at the boundary, but retained their autonomous identity based on marker expression. Mechanical and/or adhesive forces may also contribute to maintaining some regional boundaries. Indeed, the tendency of tumor cells to respect regional boundaries suggests that cell-cell interactions at boundaries are likely to be important as well as stem cell programming (Marianes, 2013).

    The behavior of ISC clones at regional boundaries is reminiscent of the behavior of clones in developing imaginal discs at 'compartment' boundaries. In the developing Drosophila embryo and imaginal discs, the engrailed gene and hedgehog signaling play important roles in defining posterior compartments. No expression of engrailed or the closely related gene invected was seen in any midgut region, and expression of hedgehog pathway components was similar on both sides of non-crossing boundaries such as Fe/P1. Dorsal ventral compartments in the developing wing are mediated by apterous and by Notch signaling. Apterous was expressed only at very low levels throughout all regions and Serrate was found at significant levels only in posterior regions 2–4 where the gene is dispensable for cell differentiation. In the developing vertebrate brain, Hox genes are important in specifying developmental compartments. However, Hox genes are only expressed at very low levels in endodermal cells during embryogenesis and these genes were very weakly expressed in the RNAseq studies, perhaps due to expression in non-endodermal cells within these samples. Consequently, the genetic basis for adult midgut compartmentalization probably differs from previously studied examples of tissue regionalization (Marianes, 2013).

    The homeotic transcription factor labial (lab) is an outstanding candidate for a regional regulatory factor. In the embryonic and larval gut, lab is required for Cu cell specification, differentiation and maintenance. The gene is expressed in copper cells, but not elsewhere in the larval midgut, and similar specificity of lab expression was seen in the adult. When lab is mis-expressed during embryonic development in other midgut regions, the copper region can expand. Endodermal cell identity along the a/p axis may be determined by signals from adjacent mesoderm during embryogenesis, and then fixed by the induction of secondary factors such as lab. Gene expression within the midgut muscles might play a similar role in the adult midgut, however, expression boundaries of muscle genes were frequently offset with respect to endodermal regions. Whether this bears any relationship to the documented offset in homeotic gene expression between the ectoderm and visceral endoderm in embryonic development remains unclear. A key question is whether individual or combinations of differentiation regulators analogous to lab specify other midgut subregions in which the ISCs fail to generate cells across regional boundaries. The RNAseq data should provide a valuable resource in identifying such factors. For example, one potential candidate, the homeotic gene defective proventriculus (dve), functions in copper cells and its expression was observed to fall sixfold between Fe and P1 (Marianes, 2013).

    One remaining question is whether a pre-existing pattern of larval midgut subdivision plays any role in the origin of adult midgut organization. The larval gut has a middle acidic region containing copper cells and an iron region like the adult tissue, and EM studies show additional morphological differences. However, it is not known whether regions analogous to other eight midgut domains described in this study exist in larvae. The larval midgut contains nests of diploid intestinal precursors that proliferate following pupariation to build the adult gut and establish its ISCs. Larval midgut domains might serve as a template for adult regionalization if gut precursor cells within each region already differ autonomously and do not mix during pupal development. However, cells do cross boundaries between the hindgut and midgut during pupal gut development. Identical regionalization within larval and adult guts might be disadvantageous to species with very different larval and adult diets, hence many adult midgut regions are likely to be established de novo or to be re-specified during pupal development (Marianes, 2013).

    Many mammalian tissues such as skin, muscle, lung, liver, and intestine contain thousands of spatially dispersed stem cells, like the Drosophila midgut. The current studies raise the question of whether these tissues exhibit finer grained regional patterns of gene expression than has been previously recognized, patterns that might be supported by small autonomous differences in their stem cells. Currently, the strongest indication for such regionalization comes from studies of the intestine. Lineage labeling shows that similar stem cells expressing Lgr5 exist along the mammalian gut despite the fact that enterocytes, enteroendocrine cells and bacterial symbionts differ regionally. For example, iron absorption in mammals takes place primarily in the duodenum, a specialized subregion of the small intestine located just downstream from the acidic stomach. This is similar to the position of the midgut iron region just downstream from the acid-producing parietal cells of the Cu and LFC regions. The antibacterial lectin RegIIIγ, which like Drosophila PGRPs recognizes bacterial peptidoglycans, is expressed most prominently in the distal region of the small intestine. The existence of tissue and stem cell regionalization in other mammalian tissues deserves further detailed investigation (Marianes, 2013).

    The human large intestine is much more prone to cancer than the small intestine. The current studies suggest that regional differences in the properties of apparently similar stem cells and tissue cells contribute to such differences. The midgut zones most favorable for the expansion of Notch-deficient cells showed pre-existing differences in Notch signaling within the early enterocyte lineage. Delta expression did not decrease shortly after ISC division, as in other regions, and Notch signaling persisted throughout enterocyte development. Curiously, same tumor-prone regions with persistent Notch signaling also were enriched in lipid droplets. At present it is not clear how the altered signaling, regional metabolic activity and tumor susceptibility are related. Additionally, regional differences in the microbiome, as suggested by the observation of domain-specific expression of PGRP proteins, may also influence the occurrence of cancer. Gastric bacteria such as Helicobacter pylori contribute to stomach cancer, while colonic Bacteroides fragilis likely promote gut cell DNA damage and colon cancer. Regional tissue differences likely also affect rates of tumor progression and metastasis. These observations emphasize the importance of understanding tissues region by region (Marianes, 2013).

    In sum, the Drosophila midgut provides an outstanding tissue in which to explore and understand the significance of intrinsic stem cell differences. GAL4 drivers were identified that allow gene expression to be manipulated in all intestinal cell types, including cells such as circular muscle and enteric neurons that are thought to contribute to niche function. Will altering the expression of genes that normally differ between regions cause ISCs to generate cells with heterotypic characteristics? Such studies might eventually make it possible to stimulate medically useful responses from the endogenous stem cells that remain within a diseased tissue (Marianes, 2013).

    Local control of intestinal stem cell homeostasis by enteroendocrine cells in the adult Drosophila midgut

    Enteroendocrine cells populate gastrointestinal tissues and are known to translate local cues into systemic responses through the release of hormones into the bloodstream. This study reports a novel function of enteroendocrine cells acting as local regulators of intestinal stem cell (ISC) proliferation through modulation of the mesenchymal stem cell niche in the Drosophila midgut. This paracrine signaling acts to constrain ISC proliferation within the epithelial compartment. Mechanistically, midgut enteroendocrine cells secrete the neuroendocrine hormone Bursicon, which acts (beyond its known roles in development) as a paracrine factor on the visceral muscle (VM). Bursicon binding to its receptor, DLGR2 (Rickets), the ortholog of mammalian leucine-rich repeat-containing G protein-coupled receptors (LGR4-6), represses the production of the VM-derived EGF-like growth factor Vein through activation of cAMP. This study has therefore identified a novel paradigm in the regulation of ISC quiescence involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells (Scopelliti, 2014).

    Bursicon, also known as the tanning hormone, has been studied for decades due its essential role as the last hormone in the cascade of Ecdysis. In all invertebrate metazoa, this endocrine cascade is fundamental to coordinate molting events during animal lifetime, and in holometabolous insects, such as Drosophila, it control metamorphosis. Fly gene expression data suggest that the endocrine hormones and their cognate receptors involved in key stages of development may have other roles during adult animal life. However, these functional roles are largely unknown. This study is the first to demonstrate a role of Bursicon beyond development (Scopelliti, 2014).

    A model is suggested in which Bursicon from enteroendocrine cells in the posterior midgut acts through DLGR2 to increase the production of cAMP within the VM, a mesenchymal ISC niche. This signaling limits the production of niche-derived, EGF-like Vein, leading to ISC quiescence. Burs protein expression was detected via immunolabeling in approximately 50% of the enteroendocrine cells of the posterior midgut, which appeared in stochastic spatial distribution within the most posterior segment of the adult midgut. Given that the percentage of enteroendocrine cells expressing Burs remained constant, it is likely that Burs expression might label a subtype of enteroendocrine cells within the midgut (Scopelliti, 2014).

    The evidence indicates that burs mRNA levels are upregulated in the midgut during the phase of relative ISC quiescence in mature animals under homeostatic conditions. Conversely, during the phase of growth of the young immature gut or the dysplastic phase of the aging gut (both characterized by relative high rates of ISC proliferation) burs levels were relatively low, and burs overexpression was sufficient to suppress ISC proliferation. Therefore, the results provide the first demonstration of a tissue-intrinsic role of enteroendocrine cells, which drives homeostatic stem cell quiescence in the adult Drosophila midgut. Future studies should further characterize the upstream mechanisms controlling Burs production in the midgut, which might be linked to the yet undefined events involved in the regulation of overall tissue size and proliferation (Scopelliti, 2014).

    Enteroendocrine cells are well known for their ability to mediate interorgan communication via hormone secretion into the bloodstream. The results demonstrate a novel, local role for enteroendocrine cells as paracrine regulators of stem cell proliferation. Such a mechanism could be phylogenetically conserved and take place in the mammalian intestine and other tissues of the gastrointestinal tract. This may therefore represent an unappreciated but yet important function of these cells beyond their conventional endocrine role (Scopelliti, 2014).

    Mammalian LGRs are thought to drive ISC proliferation acting as receptors for the Wnt agonists R-spondins, which are unrelated to Bursicon and absent in the fly genome. Accordingly, no changes were detected in either Wg levels or signaling in burs or rk mutant midguts (Scopelliti, 2014).

    Unexpectedly for a Wnt agonists and positive regulators of ISC proliferation, recent studies suggest that LGRs can act as tumor suppressors in colorectal cancer. Moreover, mammalian LGRs have also been shown to be activated by alternative ligands and promote cAMP signaling after the binding of yet unknown ligand(s). Therefore, it is likely that an unidentified functional homolog of Bursicon may act as an additional LGR ligand in mammals, driving ISC quiescence by regulating mitogenic signals from the surrounding niche as described here. Remarkably, DLGR2 shows closer sequence homology to the still poorly characterized LGR4, which (consistent with the Drosophila data) is expressed by the murine intestinal smooth-muscle layers and can signal via cAMP production. Consistent with the model, a recent study correlates loss-of-function mutations in LGR4 with multiple types of human epithelial carcinomas. Therefore, the results uncovered a novel biological role for LGRs, which is likely to impact mammalian stem cell research by providing a mechanistic framework for the so far correlative mammalian evidence toward a potential role of LGRs as tumor suppressor genes (Scopelliti, 2014).

    Altogether, these results demonstrate a novel paradigm in the regulation of intestinal homeostasis involving the conserved ligand/receptor Bursicon/DLGR2 and a previously unrecognized tissue-intrinsic role of enteroendocrine cells, which may provide insights into other stem cell based systems (Scopelliti, 2014).

    The adult Drosophila gastric and stomach organs are maintained by a multipotent stem cell pool at the foregut/midgut junction in the cardia (proventriculus)

    Stomach cancer is the second most frequent cause of cancer-related death worldwide. Thus, it is important to elucidate the properties of gastric stem cells, including their regulation and transformation. To date, such stem cells have not been identified in Drosophila. Using clonal analysis and molecular marker labeling, this study has identified a multipotent stem-cell pool at the foregut/midgut junction in the cardia (proventriculus). Daughter cells migrate upward either to anterior midgut or downward to esophagus and crop. The cardia functions as a gastric valve and the anterior midgut and crop together function as a stomach in Drosophila; therefore, the foregut/midgut stem cells have been named gastric stem cells (GaSC). JAK-STAT signaling regulates GaSC proliferation, Wingless signaling regulates GaSC self-renewal, and hedgehog signaling regulates GaSC differentiation. The differentiation pattern and genetic control of the Drosophila GaSCs suggest the possible similarity to mouse gastric stem cells. The identification of the multipotent stem cell pool in the gastric gland in Drosophila will facilitate studies of gastric stem cell regulation and transformation in mammals (Singh, 2011).

    This study has identified multipotent gastric stem cells at the junction of the adult Drosophila foregut and midgut. The GaSCs express the Stat92E-GFP reporter, wg-Gal4 UAS-GFP, and Ptc, and are slowly proliferating. The GaSCs first give rise to the fast proliferative progenitors in both foregut and anterior midgut. The foregut progenitors migrate downward and differentiate into crop cells. The anterior midgut progenitors migrate upward and differentiate into midgut cells. However, at this stage because of limited markers availability and complex tissues systems at cardia location, it is uncertain how many types of cells are produced and how many progenitor cells are in the cardia. Clonal and molecular markers analysis suggest that cardia cells are populated from gastric stem cells at the foregut/midgut (F/M) junction; however, it cannot be ruled out that there may be other progenitor cells with locally or limited differential potential that may also take part in cell replacement of cardia cells. Nevertheless, the observed differentiation pattern of GaSCs in Drosophila may be similar to that of the mouse gastric stem cells. Gastric stem cells in the mouse are located at the neck-isthmus region of the tubular unit. They produce several terminally differentiated cells with bidirectional migration, in which upward migration towards lumen become pit cells and downward migration results in fundic gland cells (Singh, 2011).

    Three signal transduction pathways differentially regulate the GaSC self-renewal or differentiation. The loss of JAK-STAT signaling resulted in quiescent GaSCs; that is, the stem cells remained but did not incorporate BrdU or rarely incorporated BrdU. In contrast, the amplification of JAK-STAT signaling resulted in GaSC expansion (Singh, 2011).

    These observations indicate that JAK-STAT signaling regulates GaSC proliferation. In contrast, the loss of Wg signaling resulted in GaSC loss, while the amplification of Wg resulted in GaSC expansion, indicating that Wg signaling regulates GaSC self-renewal and maintenance. Finally, the loss of Hh signaling resulted in GaSC expansion at the expense of differentiated cells, indicating that Hh signaling regulates GaSC differentiation. The JAK-STAT signaling has not been directly connected to gastric stem cell regulation in mammal. However, the quiescent gastric stem cells/progenitors are activated by interferon γ (an activator of the JAK-STAT signal transduction pathway), indicating that JAK-STAT pathways may also regulate gastric stem cell activity in mammals. Amplification of JAK-STAT signaling resulted in expansion of stem cells in germline, posterior midgut and malpighian tubules of adult Drosophila. In the mammalian system, it has been reported that activated STAT contributes to gastric hyperplasia and that STAT signaling regulates gastric cancer development and progression. Wnt signaling has an important function in the maintenance of intestinal stem cells and progenitor cells in mice and hindgut stem cells in Drosophila, and its activation results in gastrointestinal tumor development. Tcf plays a critical role in the maintenance of the epithelial stem cell. Mice lacking Tcf resulted in depletion of epithelial stem-cell compartments in the small intestine as well as being unable to maintain long-term homeostasis of skin epithelia. A recent study even demonstrates that the Wnt target gene Lgr5 is a stem cell marker in the pyloric region and at the esophagus border of the mouse stomach. Further, it has been found that overactivation of the Wnt signaling can transform the adult Lgr5+ve stem cells in the distal stomach, indicating that Wnt signaling may also regulate gastric stem cell self-renewal and maintenance in the mammal. Sonic Hedgehog (Shh) and its target genes are expressed in the human and rodent stomach. Blocking Shh signaling with cyclopamine in mice results in an increase in the cell proliferation of gastric gland, suggesting that Shh may also regulate the gastric stem cell differentiation in mice. These data together suggest that the genetic control of the Drosophila GaSC may be similar to that of the mammalian gastric stem cells (Singh, 2011).

    The potential GaSCs niche. In most stem cell systems that have been well characterized to date, the stem cells reside in a specialized microenvironment, called a niche.66 A niche is a subset of neighboring stromal cells and has a fixed anatomical location. The niche stromal cells often secrete growth factors to regulate stem cell behavior, and the stem cell niche plays an essential role in maintaining the stem cells, which lose their stem-cell status once they are detached from the niche (Singh, 2011).

    Loss of the JAK-STAT signaling results in the GaSCs being quiescent; the stem cells remain but do not proliferate or rarely proliferate. The Dome receptor is expressed in GaSCs, while the ligand Upd is expressed in adjacent cells. Upd-positive hub cells function as a germline stem cell niche in the Drosophila testis. Further, thia study demonstrated that overexpression of upd results in GaSC expansion, suggesting that the Upd-positive cells may function as a GaSC niche. Furthermore, while Stat92E-GFP expression is regulated by the JAK-STAT signaling in other systems, its expression at the F/M junction seems independent of the JAK-STAT signaling because Stat92E-GFP expression is not significantly disrupted in the Stat92Ets mutant flies, suggesting that the GaSCs may have unique properties (Singh, 2011).

    The stomach epithelium undergoes continuous renewal by gastric stem cells throughout adulthood. Disruption of the renewal process may be a major cause of gastric cancer, the second leading cause of cancer-related death worldwide, yet the gastric stem cells and their regulations have not been fully characterized. A more detailed characterization of markers and understanding of the molecular mechanisms control gastric stem cell behavior will have a major impact on future strategies for gastric cancer prevention and therapy. The information gained from this report may facilitate studies of gastric stem cell regulation and transformation in mammals (Singh, 2011).

    A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila

    The speed of stem cell differentiation has to be properly coupled with self-renewal, both under basal conditions for tissue maintenance and during regeneration for tissue repair. Using the Drosophila midgut model, this study analyzed at the cellular and molecular levels the differentiation program required for robust regeneration. The intestinal stem cell (ISC) and its differentiating daughter, the enteroblast (EB), were observed to form extended cell-cell contacts in regenerating intestines. The contact between progenitors is stabilized by cell adhesion molecules, and can be dynamically remodeled to elicit optimal juxtacrine Notch signaling to determine the speed of progenitor differentiation. Notably, increasing the adhesion property of progenitors by expressing Connectin is sufficient to induce rapid progenitor differentiation. It was further demonstrated that JAK/STAT signaling, Sox21a and GATAe form a functional relay to orchestrate EB differentiation. Thus, this study provides new insights into the complex and sequential events that are required for rapid differentiation following stem cell division during tissue replenishment (Zhai, 2017).

    EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells

    Tissue-specific adult stem cells are commonly associated with local niche for their maintenance and function. In the adult Drosophila midgut, the surrounding visceral muscle maintains intestinal stem cells (ISCs) by stimulating Wingless (Wg) and JAK/STAT pathway activities, whereas cytokine production in mature enterocytes also induces ISC division and epithelial regeneration, especially in response to stress. This study shows that EGFR/Ras/ERK signaling is another important participant in promoting ISC maintenance and division in healthy intestine. The EGFR ligand Vein is specifically expressed in muscle cells and is important for ISC maintenance and proliferation. Two additional EGFR ligands, Spitz and Keren, function redundantly as possible autocrine signals to promote ISC maintenance and proliferation. Notably, over-activated EGFR signaling could partially replace Wg or JAK/STAT signaling for ISC maintenance and division, and vice versa. Moreover, although disrupting any single one of the three signaling pathways shows mild and progressive ISC loss over time, simultaneous disruption of them all leads to rapid and complete ISC elimination. Taken together, these data suggest that Drosophila midgut ISCs are maintained cooperatively by multiple signaling pathway activities and reinforce the notion that visceral muscle is a critical component of the ISC niche (Xu, 2011).

    Adult stem cells commonly interact with special microenvironment for their maintenance and function. Many adult stem cells, best represented by germline stem cells in Drosophila and C. elegans, require one primary maintenance signal from the niche while additional signals may contribute to niche integrity. ISCs in the Drosophila midgut do not seem to fit into this model. Instead, they require cooperative interactions of three major signaling pathways, including EGFR, Wg and JAK/STAT signaling, for long-term maintenance. Importantly, Wg or JAK/STAT signaling over-activation is able to compensate for ISC maintenance and proliferation defects caused by EGFR signaling disruption, and vice versa. Therefore, ISCs could be governed by a robust mechanism, signaling pathways could compensate with each other to safeguard ISC maintenance. The mechanisms of the molecular interactions among these pathways in ISC maintenance remains to be investigated. In mammals, ISCs in the small intestine are primarily controlled by Wnt signaling pathways, and there are other ISC specific markers not controlled by Wnt signaling. In addition, mammalian ISCs in vitro strictly depend on both EGFR and Wnt signals, indicating that EGFR and Wnt signaling may also cooperatively control mammalian ISC fate. It is suggested that combinatory signaling control of stem cell maintenance could be a general mechanism for ISCs throughout evolution (Xu, 2011).

    The involvement of EGFR signaling in Drosophila ISC regulation may bring out important implications to understanding of intestinal diseases, in which multiple signaling events could be involved. For example, in addition to Wnt signaling mutation, gain-of-function K-Ras mutations are frequently associated with colorectal cancers in humans. Moreover, activation of Wnt signaling caused by the loss of adenomatous polyposis coli (APC) in humans initiates intestinal adenoma, but its progression to carcinoma may require additional mutations. Interestingly, albeit controversial, Ras signaling activation is suggested to be essential for nuclear β-catenin localization, and for promoting adenoma to carcinoma transition. In the Drosophila midgut, loss of APC1/2 genes also leads to intestinal hyperplasia because of ISC overproliferation. Given that EGFR signaling is generally activated in ISCs, it would be interesting to determine the requirements of EGFR signaling activation in APC-loss-induced intestinal hyperplasia in Drosophila, which might provide insights into disease mechanisms in mammals and humans (Xu, 2011).

    Previous studies suggest that intestinal VM structures the microenvironment for ISCs by producing Wg and Upd maintenance signals. This study identified Vn, an EGFR ligand, as another important ISC maintenance signal produced from the muscular niche. Therefore, ISCs are maintained by multiple signals produced from the muscular niche. In addition, Spi and Krn, two additional EGFR ligands, were identified that function redundantly as possible autocrine signals to regulate ISCs. These observations are consistent with a previous observation that paracrine and autocrine EGFR signaling regulates the proliferation of AMPs during larval stages, suggesting that this mechanism is continuously utilized to regulate adult ISCs for their maintenance and proliferation. The only difference is that the proliferation of AMP cells is unaffected when without autocrine Spi and Krn, due to redundant Vn signal from the VM, whereas autocrine Spi/Krn and paracrine Vn signals are all essential in adult intestine for normal ISC maintenance and proliferation. It was found that Vn and secreted form of Spi have similar roles in promoting ISC maintenance and activation, but additional regulatory or functional relationships among these ligands require further investigation, as the necessity of multiple EGFR ligands is still not completely understood. It is known that secreted/activated Spi and Krn are diffusible signals, but clonal analysis data show that Spi and Krn can display autonomous phenotypes. This observation indicates that these two ligands could behave as very short range signals in the intestinal epithelium, or they could diffuse over long distance but the effective levels of EGFR activation could only be achieved in cells where the ligands are produced. Interestingly, palmitoylation of Spi is shown to be important for restricting Spi diffusion in order to increase its local concentration required for its biological function. Whether such modification occurs in intestine is unknown, but it is speculated that Vn, Spi and Krn, along with the possibly modified forms, may have different EGFR activation levels or kinetics, and only with them together effective activation threshold could be reached and sustained in ISCs to control ISC behavior. Therefore, a working model is proposed that ISCs may require both paracrine and autocrine mechanisms in order to achieve appropriate EGFR signaling activation for ISC maintenance and proliferation.

    Mechanisms of JAK/STAT signaling activation is rather complex. In addition to Upd expression from the VM, its expression could also be detected in epithelial cells with great variability in different reports, possibly due to variable culture conditions. Upon injury or pathogenic bacterial infection, damaged ECs and pre-ECs are able to produce extra cytokine signals, including Upd, Upd2 and Upd3, to activate JAK/STAT pathway in ISCs to promote ISC division and tissue regeneration. Several very recent studies suggest that EGFR signaling also mediates intestinal regeneration under those stress conditions in addition to its requirement for normal ISC proliferation. Therefore, in addition to basal paracrine and autocrine signaling mechanisms that maintain intestinal homeostasis under normal conditions, feedback regulations could be employed or enhanced under stress conditions to accelerate ISC division and epithelial regeneration (Xu, 2011).

    Evidence so far has indicated a central role of N signaling in controlling ISC self-renewal. N is necessary and sufficient for ISC differentiation. In addition, the downstream transcriptional repressor Hairless is also necessary and sufficient for ISC self-renewal by preventing transcription of N targeting genes in ISCs. Therefore, N inhibition could be a central mechanism for ISC fate maintenance in Drosophila. High Dl expression in ISCs may lead to N inhibition, though how Dl expression is maintained in ISCs at the transcriptional level is not clear yet. Hyperactivation of EGFR, Wg or JAK/STAT signaling is able to induce extra Dl+ cells, suggesting that these three pathways might cooperatively promote Dl expression in ISCs. It is also possible that these pathways regulate Dl expression indirectly. As Dl-N could have an intrinsically regulatory loop for maintaining Dl expression and suppressing N activation, these pathways could indirectly regulate Dl expression by targeting any component within the regulatory loop. Identifying their respective target genes by these signaling pathways in ISCs would be an important starting point to address this question (Xu, 2011).

    A feedback amplification loop between stem cells and their progeny promotes tissue regeneration and tumorigenesis

    Homeostatic renewal of many adult tissues requires balanced self-renewal and differentiation of local stem cells, but the underlying mechanisms are poorly understood. This study identified a novel feedback mechanism in controlling intestinal regeneration and tumorigenesis in Drosophila. Sox21a, a group B Sox protein, is preferentially expressed in the committed progenitor named enteroblast (EB) to promote enterocyte differentiation. In Sox21a mutants, EBs do not divide, but cannot differentiate properly and have increased expression of mitogens, which then act as paracrine signals to promote intestinal stem cell (ISC) proliferation. This leads to a feedback amplification loop for rapid production of differentiation-defective EBs and tumorigenesis. Notably, in normal intestine following damage, Sox21a is temporally downregulated in EBs to allow the activation of the ISC-EB amplification loop for epithelial repair. It is proposed that executing a feedback amplification loop between stem cells and their progeny could be a common mechanism underlying tissue regeneration and tumorigenesis (Chen, 2016b).

    Nonautonomous regulation of Drosophila midgut stem cell proliferation by the insulin-signaling pathway

    Drosophila adult midgut intestinal stem cells (ISCs) maintain tissue homeostasis by producing progeny that replace dying enterocytes and enteroendocrine cells. ISCs adjust their rates of proliferation in response to enterocyte turnover through a positive feedback loop initiated by secreted enterocyte-derived ligands. However, less is known about whether ISC proliferation is affected by growth of the progeny as they differentiate. This study shows that nutrient deprivation and reduced insulin signaling results in production of growth-delayed enterocytes and prolonged contact between ISCs and newly formed daughters. Premature disruption of cell contact between ISCs and their progeny leads to increased ISC proliferation and rescues proliferation defects in insulin receptor mutants and nutrient-deprived animals. These results suggest that ISCs can indirectly sense changes in nutrient and insulin levels through contact with their daughters and reveal a mechanism that could link physiological changes in tissue growth to stem cell proliferation (Choi, 2011).

    Previous studies have focused on responses of ISC proliferation to enterocyte death, delineating a positive feedback mechanism by which ligands secreted from dying enterocytes activate ISC proliferation. The data propose a model of additional regulation where cell contact between ISCs and newly formed enteroblasts acts to inhibit ISC proliferation through a negative feedback loop (see Cell contact regulates ISC proliferation) (Choi, 2011).

    Nutrient deprivation leads to decreased ISC proliferation rates and clones containing fewer cells than clones made in animals fed a rich diet. However, it is unclear why these clones fail to eventually reach the same size as wild-type clones. One possibility is that nutrient-deprived midguts contain fewer cells. Therefore, the number of cells that each ISC needs to generate to maintain tissue homeostasis would be smaller. A second possibility is built on the observation that turnover and production of 8n and 16n enterocytes is reduced in animals fed a poor diet, and this could result in the depletion of a source of promitotic ligands, thereby decreasing the need for a stem cell to divide (Choi, 2011).

    Protein deprivation and reduced insulin signaling leads to an increase in the number of lower ploidy enterocyte daughters per midgut, suggesting that endoreduplication in the midgut is regulated by nutrition. Because enterocyte turnover is reduced in nutrition-deprived animals, it raises the intriguing possibility that 8n cells act to inhibit the growth and endoreduplication of 4n cells into mature enterocytes through an as-yet-unidentified signal. These similarities between nutrient-deprived clones and dInR mutant clones suggest that the effects of nutrition may be mediated in part through the insulin-signaling pathway. Consistent with a role for nutrition and the insulin-signaling pathway in growth and endoreduplication, constitutive activation of dInR in ISC clones led to enterocytes with significantly higher ploidy than normal. Interestingly, these clones were smaller than wild-type, suggesting that excessive or prolonged contact between enterocytes and ISCs may also play a role in the regulation of ISC proliferation (Choi, 2011).

    The findings raise the as-yet-unexplored possibility that germ-line stem cell and neuroblast stem cell daughters might also nonautonomously regulate stem cell proliferation. When both the ISC and the enteroblast were mutant for dInR, a further increase in cell cycle arrest was observed, suggesting an autonomous role for insulin signaling in the regulation of ISC proliferation (Choi, 2011).

    Significantly higher levels of DE-cadherin were found between both dInR mutant enteroblast and wild-type ISCs and dInR mutant enteroblasts and dInR mutant ISCs, demonstrating that the insulin-signaling pathway regulates the stability of the adherens junction. The results are striking because, in the ovary and testis, loss of dInR signaling in the germ-line stem cell niche leads to a decrease rather than an increase in DE–cadherin at the adherens junction (Choi, 2011).

    The data presented in this study demonstrate that the enteroblast can nonautonomously regulate the rate of ISC proliferation. How might this be achieved? One possibility is that the enteroblast inhibits ISC proliferation by providing a short-range inhibitory signal whose effect is removed as the ISC and enteroblast separate. A second possibility is that separation of ISCs and enteroblasts leads to the release from a cellular compartment of a factor that can drive proliferation. The ideal candidate is β-catenin, which is not only a member of the adherens junction but also a transcriptional activator, which is required for ISC proliferation (Choi, 2011).

    Recently, ISCs and enteroblast number were examined under protein-poor conditions in old animals expressing green fluorescent protein driven by the escargot promoter (esg-GFP), which is thought to be specific to ISCs and enteroblasts. A decrease in esg-GFP–positive cells was observed in 16- to 17- and 20- to 21-d-old animals fed a poor diet, leading to the conclusion that ISC maintenance is regulated by a protein-poor diet. In contrast, this study did not observe a decrease in ISC number in females fed a protein-poor diet. Presumably, the modest decrease in GFP-positive cells observed by the previous study was due to loss of the excess enteroblasts seen in aging midguts, which is consistent with recently published work showing that insulin-signaling mutants can suppress this aging phenotype (Choi, 2011).

    Transcriptional control of stem cell maintenance in the Drosophila intestine

    Adult stem cells maintain tissue homeostasis by controlling the proper balance of stem cell self-renewal and differentiation. The adult midgut of Drosophila contains multipotent intestinal stem cells (ISCs) that self-renew and produce differentiated progeny. Control of ISC identity and maintenance is poorly understood. This paper describes how transcriptional repression of Notch target genes by a Hairless-Suppressor of Hairless complex is required for ISC maintenance; genes of the Enhancer of split complex [E(spl)-C] are identified as the major targets of this repression. In addition, the bHLH transcription factor Daughterless was found to be essential to maintain ISC identity, and bHLH binding sites promote ISC-specific enhancer activity. It is proposed that Daughterless-dependent bHLH activity is important for the ISC fate and that E(spl)-C factors inhibit this activity to promote differentiation (Bardin, 2010).

    Adult stem cells self-renew and, at the same time, give rise to progeny that eventually differentiate. This work provides evidence that one of the strategies used to maintain the identity of ISCs in Drosophila is to repress the expression of Notch target genes. Consistent with this finding, the loss of a general regulator of transcriptional repression, the Histone H2B ubiquitin protease Scrawny, gives a similar phenotype to Hairless. Additionally, several recent studies indicate that transcriptional repression of differentiation genes may be a central hallmark of stem cells in general (Bardin, 2010).

    Two models have been proposed for Hairless activity. One proposes that Hairless competes with NICD for interaction with Su(H), thereby preventing transcriptional activation of Notch target genes by low-level Notch receptor activation. A second, non-exclusive, model proposes that Hairless antagonizes the transcriptional activation of Notch target genes by tissue-specific transcription factors other than Notch. Since the loss of Su(H) can suppress the phenotype of Hairless on ISC clone growth, it is proposed that Hairless promotes ISC maintenance by repressing the transcription of genes that would otherwise be activated by Notch signaling in ISCs. Thus, Hairless appears to set a threshold level to buffer Notch signaling in ISCs. In the absence of this repression, the expression of E(spl)-C genes and other Notch targets would lead to loss of the ISC fate. Importantly, the current findings suggest a mechanism for how the transcriptionally repressed state is turned off and activation of the differentiation program is initiated: high activation of Notch in enteroblasts (EBs) displaces Hairless from Su(H) and leads to expression of the E(spl)-C genes (Bardin, 2010).

    E(spl)-C bHLH repressors act in part through their ability to inhibit bHLH activators. The data demonstrate that Da is also essential to maintain ISC fate and that E-box Da-binding sites are required to promote ISC-specific enhancer activity. Thus, it is proposed that activation of E(spl)-C genes by Notch in EBs downregulates Da bHLH activity and thereby contributes to turning off ISC identity in the differentiating cell. The specificity of ISC-specific E-box expression might be due to the ISC-specific expression of a bHLH family member. Although array analysis raised the possibility that Scute may be specifically expressed in ISCs, genetic analysis indicates that scute function is not essential for ISC maintenance. Alternatively, specificity of gene expression might result from inhibition of bHLH activity in the EB and differentiating daughters, possibly by E(spl)-bHLH factors, rather than by the ISC-specific expression of a Da partner. It is also possible that a non-bHLH, ISC-specific factor restricts the Da-dependent bHLH activity to ISCs in a manner similar to the synergism observed in wing margin sensory organ precursors (SOPs) between the Zn-finger transcription factor Senseless and Da (Bardin, 2010).

    Recently, a role for the Da homologs E2A (Tcf3) and HEB (Tcf12) has been found in mammalian ISCs marked by the expression of Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (Lgr5) and, in this context, E2A and HEB are thought to heterodimerize with achaete-scute like 2 (Ascl2), which is essential for the maintenance and/or identity of Lgr5+ ISCs (van der Flier, 2009). In Drosophila, however, AS-C genes are not essential for ISC maintenance, but appear to play a role in enteroendocrine fate specification. The observation that Da bHLH activity is required for the identity of both Drosophila ISCs and mammalian Lgr5+ ISCs suggests that there might be conservation at the level of the gene expression program. Additionally, the bHLH genes Atoh1 (Math1) and Neurog3 are both important for differentiation of secretory cells in the mammalian intestine. Clearly, further analysis of the control of Da/E2A bHLH activity, as well as of the gene networks downstream of Da/E2A, will be of great interest (Bardin, 2010).

    The data suggest that ISC fate is promoted both by inhibition of Notch target genes through Hairless/Su(H) repression and by activation of ISC-specific genes through bHLH activity. How then is asymmetry in Notch activity eventually established between the two ISC daughters to allow one cell to remain an ISC and one cell to differentiate? Three types of mechanism can be envisioned that would allow for asymmetry of Notch signaling (Bardin, 2010).

    First, the binary decision between the ISC and EB fates might result from a competition process akin to lateral inhibition for the selection of SOPs. In this process, feedback loops establish directionality by amplifying stochastic fluctuations in signaling between equivalent cells into a robust unidirectional signal. The finding that the Da activator and E(spl)-bHLH repressors are important to properly resolve ISC/EB fate is consistent with this type of model. Activation of the Notch pathway in one of the daughter cells could then lead to the changes in nuclear position previously noted (Bardin, 2010).

    Second, the asymmetric segregation of determinants could bias Notch-mediated cell fate decisions. The cell fate determinants Numb and Neur are asymmetrically segregated in neural progenitor cells to control Notch signaling. However, this study found no evidence for the asymmetric segregation of these proteins in dividing ISCs. Additionally, the data indicate that Numb is not important to maintain ISC fate. It cannot be excluded, however, that another, unknown Notch regulator is asymmetrically segregated to regulate the fate of the two ISC daughters (Bardin, 2010).

    A third possibility is that after ISC division, one of the two daughter cells receives a signal that promotes differential regulation of Notch. Indeed, it has been noted that the axis of ISC division is tilted relative to the basement membrane, resulting in one of the progeny maintaining greater basal contact than the other. An extracellular signal coming either basally or apically could bias the Notch-mediated ISC versus EB fate decision. For instance, Wg secreted by muscle cells could act as a basal signal to counteract Notch receptor signaling activity in presumptive ISCs. This could be accomplished by Wg promoting bHLH activity or gene expression. Indeed, Wg has been demonstrated to promote proneural bHLH activity in Drosophila (Bardin, 2010).

    These models are not mutually exclusive, however, and proper control of ISC and differentiated cell fates during tissue homeostasis might involve multiple mechanisms (Bardin, 2010).

    Coordination of insulin and Notch pathway activities by microRNA miR-305 mediates adaptive homeostasis in the intestinal stem cells of the Drosophila gut

    Homeostasis of the intestine is maintained by dynamic regulation of a pool of intestinal stem cells. The balance between stem cell self-renewal and differentiation is regulated by the Notch and insulin signaling pathways. Dependence on the insulin pathway places the stem cell pool under nutritional control, allowing gut homeostasis to adapt to environmental conditions. This study presents evidence that miR-305 is required for adaptive homeostasis of the gut. miR-305 regulates the Notch and insulin pathways in the intestinal stem cells. Notably, miR-305 expression in the stem cells is itself under nutritional control via the insulin pathway. This link places regulation of Notch pathway activity under nutritional control. These findings provide a mechanism through which the insulin pathway controls the balance between stem cell self-renewal and differentiation that is required for adaptive homeostasis in the gut in response to changing environmental conditions (Foronda, 2014).

    Drosophila midgut homeostasis involves neutral competition between symmetrically dividing intestinal stem cells

    The Drosophila adult posterior midgut has been identified as a powerful system in which to study mechanisms that control intestinal maintenance, in normal conditions as well as during injury or infection. Early work on this system has established a model of tissue turnover based on the asymmetric division of intestinal stem cells (ISC). From the quantitative analysis of clonal fate data, this study shows that tissue turnover involves the neutral competition of symmetrically dividing stem cells, mediated by Dl/N signaling. This competition leads to stem-cell loss and replacement, resulting in neutral drift dynamics of the clonal population. As well as providing new insight into the mechanisms regulating tissue self-renewal, these findings establish intriguing parallels with the mammalian system, and confirm Drosophila as a useful model for studying adult intestinal maintenance (de Navascues, 2012).

    This study has analysed long- and short-term lineage progression by size, survival and composition, and shows that Drosophila midgut is maintained by population asymmetry. This contrasts with a general notion in the field that homeostasis is based on the fate asymmetry of the ISC offpsring. The results reveal that ISCs divide symmetrically in response to the differentiation and subsequent loss of a neighbouring ISC (or vice versa), which leads to neutral drift of the clonal population. It is estimated that, in a context of fast turnover (two or three divisions per day), 2 in 10 divisions result in stem-cell loss and replacement. The results further indicate that the fate of the ISC daughters might not be specified on division, but rather resolved through competition between proximate cells following division (de Navascues, 2012).

    The observation of neutral competition calls for the elucidation of its underlying molecular mechanism. By the nature of the process of neutral competition in this system, the associated mechanism must play a fundamental role in ISC self-renewal and enteroblast (EB) commitment and be able to implement, non-cell autonomously, the stochastic resolution of binary fate decisions. Dl-N signalling fulfils both requirements: numerous experiments underpin its central role in the choice of commitment versus self-renewal in the midgut, and studies of its function in neurogenesis have established an intrinsic ability to resolve stochastically binary choices of cell fate through the process of 'lateral inhibition', which involves reciprocal signalling between equivalent cells (de Navascues, 2012).

    The model of population asymmetry involves neutral competition between proximate cells (therefore based on extrinsic signals), which implies that ISC daughters are intrinsically equivalent at birth, and that fate is resolved after ISC division. Therefore, the ISC daughters are functionally equivalent and a good substrate for lateral inhibition, This is compatible with the observations that ISC daughter cells express N and they segregate Dl symmetrically. Furthermore, evidence is provided that this situation leads to mutual signalling. The excess of ISCs found in null and hypomorphic conditions for N could be interpreted, in the framework of a unidirectional Dl signal from the ISC towards the EB, as a failure to implement the EB fate and a lapse into the 'default' ISC fate. However, this interpretation cannot account for the increased number of cells committing to EB fate in +/l(1)NB and +/NMCD1 midguts. These alleles exhibit Dl-dependent increased N signalling which, in a scenario of unidirectional signalling, should not affect the balance of EB commitment. Rather, their phenotype suggests that both ISC daughters can receive Dl signal and reach the threshold of N activity for commitment. This is further highlighted by the parallel between the effects of these alleles on the ISC/EB ratio and on the development of peripheral nervous system (PNS) of Drosophila, a classical model for lateral inhibition. In the PNS, a reduction of N activity, as in +/N55e11, leads to neurogenic phenotypes with supernumerary sensory bristles, whereas excess of N activity, as in +l(1)NB and +/NMCD1, results in antineurogenic phenotypes, with reduced number of bristles and more cells adopting the epidermal fate. It is noteworthy that the molecular machinery that participates in Dl/N-mediated lateral inhibition in other contexts is also involved in ISC/EB fate decision. With these elements, it is proposed that ISCs divide symmetrically, and the fate of the progeny is resolved through lateral inhibition mediated by Dl/N signalling (de Navascues, 2012).

    Lateral inhibition thus provides a straightforward way of implementing neutral competition. In many cases, Dl/N interaction will be restricted to sibling cells, the progeny of a single ISC division, and these cases will resolve into an ISC/EB asymmetric pair. However, if in the course of tissue turnover the division of two ISCs occurred in close proximity, non-sibling cells could interact via Dl/N and engage in competition for the ISC fate through lateral inhibition. This in turn may lead to sibling cells adopting identical fates and therefore in ISC loss and replacement. Such behaviour would translate precisely to the coupled events of loss and replacement implicit in the model, with the value of 2r weighting, combined, the chance of this contact and of its resolution into ISC loss and replacement. In this regard, the rich variety of esg+ cell nest composition in EB and ISC cells, as well as the frequent proximity of ISC pairs (19% of nearest pairs), suggests that such encounters are possible in space. This may require nests to drift in position, but it is significant that, unlike other Drosophila adult stem cells, ISCs are not associated with niches having hub-like anatomical properties (de Navascues, 2012).

    Although the current observations can, in principle, be explained solely as a result of lateral inhibition, other mechanisms cannot be ruled out. In particular, the tissue could allow for a combination of either symmetric or intrinsically asymmetric divisions such that part of the ISC divisions that result in asymmetric fate could derive from either neutral competition or an intrinsic regulatory process. However, to conform with the observation that ISCs are equipotent, the stem-cell progeny of an intrinsically determined asymmetric cell division will have the same chance for loss and replacement in subsequent divisions as any other ISC. In other words, when facing their next division, all ISCs irrespective of their lineage history, would have to decide, stochastically, whether to undergo intrinsically asymmetric division, and, if they do not, the fate outcome of the division would be resolved again, stochastically, by extrinsically driven neutral competition. It is difficult to conceive of a scenario for the molecular regulation of such a system (de Navascues, 2012).

    Although the model provides an excellent fit to the early time course, the departure of the model at day 16 is significant. Although the clone size distribution conforms closely with the predicted scaling form, consistent with the same underlying pattern of ISC fate, the overall average size of the surviving clones is smaller than predicted by a simple extrapolation of the fits to the earlier time point data with the same ISC loss/replacement rate. The departure of theory and experiment may reflect a breakdown of homeostasis due to ageing. In particular, at this point of the clone chase the flies are 21-23 days old, an age at which ageing-related non-homeostatic phenotypes can be detected in the midgut. Indeed, such behaviour might be explained by a shift towards uncompensated loss of terminally differentiated cells, consistent with the fact that the clone density continues to fall, and in a manner consistent with theory. Alternatively, it is also possible that the heat shock, which does not produce damage leading to detectable alteration of the tissue size or cell density, instead triggers the acceleration of tissue turnover, an effect that would last at least 8 days, but less than 16. This effect could be similar, but milder, to that observed in the recently described Drosophila gastric adult stem cells, which show a sharp activation of homeostatic turnover in response to heat shock. This view is supported by the observation that the mitotic index in the posterior midgut is, after heat-based clonal induction, higher than in untreated organs (de Navascues, 2012).

    There are similarities between the Drosophila midgut and mammalian intestine at the levels of cell biology and genetics. This study adds a new parallel from the perspective of their strategy for homeostatic maintenance. In humans, studies of methylation patterns point at stem-cell loss and replacement in the intestinal crypt and in the mouse, using an approach similar to that employed in this study, recent studies have shown that the intestinal crypt is maintained by an equipotent Lgr5+ stem-cell population in which the loss of cells from the stem-cell compartment is compensated by the symmetric multiplication of neighbours. Further evidence suggests that stem-cell competence in the small intestine is ensured by proximity to Paneth cells, which aggregate throughout the crypt base. As tissue is turned over, Lgr5+ cells undergo neutral competition resulting in a progression towards crypt monoclonality. At longer timescales, crypts undergo fission, leading to a further 'coarsening' of the clonal population. It is speculated that in Drosophila, the esg+ nests fulfil a function analogous to intestinal crypts, playing host to a much smaller equipotent cell population, and undergoing fission with a far greater frequency. However, in contrast with intestinal crypt, it appears that ISC competence in Drosophila does not require a separate niche-supporting cell (de Navascues, 2012).

    In summary, these studies show that, in Drosophila, adult midgut homeostasis follows a pattern of population asymmetry involving an equipotent population of ISCs. ISC division may result in any of all three possible fate outcomes leading to asymmetric fate (ISC/EB), symmetric duplication (two ISCs), or symmetric differentiation (two EBs), the latter two balanced in frequency. These findings point at a mechanism involving lateral inhibition and provide a natural framework to explain regeneration following injury or infection (de Navascues, 2012).

    Niche appropriation by Drosophila intestinal stem cell tumours

    Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. This study investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch(N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis (Patel, 2015).

    This paper described a step-wise series of events during the earliest stage of tumour development in a stem cell niche. First, the combination of environmentally triggered mitogenic signalling and a mutation that compromises differentiation generates small clusters of differentiation-defective stem-like cells. Autocrine (Spi/EGFR) signalling between these cells then promotes their expansion into clusters, which quickly reach a size capable of physically disrupting the surrounding epithelium and driving the detachment and apical extrusion of surrounding epithelial cells (that is, ECs). This loss of normal cells seems to involve tumour cell/epithelial cell competition through integrin-mediated adhesion. Subsequently, the loss of epithelial integrity (specifically, EC detachment) triggers stress signalling (JNK, Yki/YAP) in the surrounding epithelium and underlying VM, and these stressed tissues respond by producing cytokines (Upd2,3) and growth factors (Vn, Pvf, Wg, dILP3). These signals are normally used within the niche to activate stem cells for epithelial repair, but in this context they further stimulate tumour growth in a positive feedback loop. It is noteworthy that in this example a single mutation that blocks differentiation is sufficient to drive early tumour development, even without secondary mutations in growth signalling pathways that might make the tumour-initiating cells growth factor- and niche-independent (for example, Ras, PTEN). Thus, tumour cell-niche interactions can be sufficient to allow tumour-initiating cells to rapidly expand, increasing their chance to acquire secondary mutations that might enhance their growth or allow them to survive outside their normal niche. This study highlights the importance of investigating the factors that control paracrine stem cell mitogens and survival signals in the niche environment. Tumour-niche interactions may be important to acquire a sizable tumour mass before the recruitment of a tumour-specific microenvironment that supports further tumour progression. A careful analysis of similar interactions in other epithelia, such as in the lung, skin or intestine could yield insights relevant to the early detection, treatment and prevention of cancers in such tissues (Patel, 2015).

    Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila

    Tissue-specific stem cells are maintained by both local secreted signals and cell adhesion molecules that position the stem cells in the niche microenvironment. In the Drosophila midgut, multipotent intestinal stem cells (ISCs) are located basally along a thin layer of basement membrane that composed of extracellular matrix (ECM), which separates ISCs from the surrounding visceral musculature: the muscle cells constitute a regulatory niche for ISCs by producing multiple secreted signals that directly regulate ISC maintenance and proliferation. This study shows that integrin-mediated cell adhesion, which connects the ECM and intracellular cytoskeleton, is required for ISC anchorage to the basement membrane. Specifically, the alpha-integrin subunits including alphaPS1 encoded by mew and alphaPS3 encoded by scb, and the beta-integrin subunit encoded by mys are richly expressed in ISCs and are required for the maintenance, rather than their survival or multiple lineage differentiation. Furthermore, ISC maintenance also requires the intercellular and intracellular integrin signaling components including Talin, Integrin-linked kinase (Ilk), and the ligand, Laminin A. Notably, integrin mutant ISCs are also less proliferative, and genetic interaction studies suggest that proper integrin signaling is a prerequisite for ISC proliferation in response to various proliferative signals and for the initiation of intestinal hyperplasia after loss of adenomatous polyposis coli (Apc). These studies suggest that integrin not only functions to anchor ISCs to the basement membrane, but also serves as an essential element for ISC proliferation during normal homeostasis and in response to oncogenic mutations (Lin, 2013).

    Rac1 drives intestinal stem cell proliferation and regeneration

    Adult stem cells are responsible for maintaining the balance between cell proliferation and differentiation within self-renewing tissues. The molecular and cellular mechanisms mediating such balance are poorly understood. The production of reactive oxygen species (ROS) has emerged as an important mediator of stem cell homeostasis in various systems. Recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). This study used the adult Drosophila midgut and the mouse small intestine to directly address the role of Rac1 in ISC proliferation and tissue regeneration in response to damage. The results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. The data point to an evolutionarily conserved role of Rac1 in intestinal homeostasis and highlight the value of combining work in the mammalian and Drosophila intestine as paradigms to study stem cell biology (Myant, 2013b).

    The epithelium of the posterior adult Drosophila midgut is replenished by ISCs. Each ISC proliferates to give rise to an uncommitted enteroblast (EB), which will differentiate into either an enterocyte (EC) or an enteroendocrine cell (ee). ISCs are the only proliferative cells within the adult fly posterior midgut (Myant, 2013b).

    Recent work shows that deletion of Rac1 suppresses intestinal hyperproliferation and ROS production in Apc-deficient mice (Myant, 2013a). It was therefore first asked whether Rac1 is sufficient to drive ROS production within ISCs in the Drosophila midgut. The UAS/Gal4 system was used to specifically overexpress Drosophila Rac1 in ISCs/EBs (progenitor cells) using the temperature-controlled escargot-gal4, UAS-gfp; tubulin-gal80ts driver (esgts > gfp). Overexpression of Rac1 resulted in a dramatic expansion of the esg > gfp cell population and increased ROS production in the midgut. These results suggest that Rac1 overexpression in progenitor cells is sufficient to drive ROS production within the intestinal epithelium (Myant, 2013b).

    The epithelium of the adult posterior Drosophila midgut has a remarkable regenerative capacity. Damage induced by agents such as bacterial infection, Bleomycin, or dextran sodium sulfate (DSS) treatment leads to activation of ISC proliferation to regenerate the damaged intestinal epithelium. Previous work demonstrated that ROS production is essential for damaged-induced ISC proliferation in the fly midgut (Buchon, 2009). It was therefore asked whether ROS upregulation was important for the phenotype resulting from Rac1 overexpression in the midgut. Consistent, with the previous report preventing ROS production by NAC impaired ISC proliferation in posterior midguts from flies infected with the pathogenic bacteria Pseudomonas entomophila (Pe). Importantly, NAC treatment strongly suppressed ISC hyperproliferation in Rac1-overexpressing midguts (). These results suggest that ROS production is essential for Rac1-dependent ISC hyperproliferation in the intestine (Myant, 2013b).

    It was finally asked whether Rac1 was necessary to drive ISC proliferation in response to damage. This is a question, which also derives from previous work in the mammalian intestine. A genetic approach was used to knockdown Rac1 within progenitors cells of the Drosophila midgut by RNA interference (RNAi) (esgts > Rac1-IR). Knockdown of Rac1 by 2 independent RNAi lines resulted in almost complete suppression of ISC proliferation in regenerating posterior midguts subject to Pe infection. Similar to the Drosophila midgut, the mammalian intestine displays a remarkable regenerative capacity following damage. Therefore the conservation of the requirement for Rac1 during intestinal regeneration across these species was addressed. Rac1 was conditionally deleted from the mouse intestinal epithelium using the vil-Cre-ERT2, and the effect of Rac1 loss on tissue regeneration upon DNA damage was tested. Consistent with the results in the fly midgut, Rac1 deletion significantly suppressed regeneration in the mouse intestinal epithelium (Myant, 2013b).

    Altogether, this work results suggest a central conserved role for the small GTPase RAC1 as a driver of ISC proliferation through the production of ROS. These data highlight RAC1 as key player and potential therapeutic target for conditions linked to oxidative stress such as cancer and aging (Myant, 2013b).

    Brahma is essential for Drosophila intestinal stem cell proliferation and regulated by Hippo signaling

    Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. This study shows that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped-Yorkie transcriptional complex, and the Hippo (Hpo) signaling pathway directly restricts ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promotes ISC proliferation. These findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation (Jin, 2013).

    SWI/SNF complex subunits regulate the chromatin structure by shutting off or turning on the gene expression during differentiation. Recently, the findings from several research reports based on the stem cell system reveal important roles of chromatin remodeling complex in stem cell state maintenance. The current study suggests that the chromatin remodeling activity of Brm complex is required for the proliferation and differentiation of Drosophila ISCs. Based on these findings, it is proposed that Brm is critical for maintaining Drosophila intestinal homeostasis. High levels of Brm in the ISC nucleus represent high proliferative ability and are essential for EC differentiation; low levels of Brm in the EC nucleus may be a response for homeostasis. Changes in Brm protein levels result in the disruption of differentiation and deregulation of cell proliferation. In line with previous findings in human, the cell-type-specific expression of Drosophila homologs BRG1 and BRM are also detected in adult tissues. BRG1 is mainly expressed in cell types that constantly undergo proliferation or self-renewal, whereas BRM is expressed in other cell types. These observations indicate that Brm may act similarly to BRG1 and BRM in controlling proliferation and differentiation (Jin, 2013).

    The Hpo pathway restricts cell proliferation and promotes cell death at least in two ways: inhibiting the transcriptional co-activator Yki and inducing activation of pro-apoptotic genes such as caspases directly. This study has identified a novel regulatory mechanism of the Hpo pathway in maintaining intestinal homeostasis. In this scenario, Brm activity is regulated by the Hpo pathway. In normal physiological conditions, under the control of Hpo signaling, the function of Yki–Sd to promote ISC proliferation is restricted and the pro-proliferation of target genes such as diap1 that inhibits Hpo-induced caspase activity cannot be further activated. Therefore, Hpo signaling normally functions to restrict cell numbers in the midgut by keeping ISC proliferation at low levels. Yki is enriched in ISCs, but predominantly inactivated in cytoplasm by the Hpo pathway. The knockdown of Yki in ISCs did not cause any phenotype in the midgut, suggesting that Yki is inactivated in ISCs under normal homeostasis. During an injury, Hpo signaling is suppressed or disrupted, Yki translocates into the nuclei to form a complex with Sd, which may allow Yki–Sd to interact with Brm complex in the nucleus to activate transcriptional targets. Of note, the loss-of-function of Brm resulted in growth defect of ISCs, suggesting that Brm is required for ISC homeostasis and possessing a different role of Brm from Yki in the regulation of ISCs. It is possible that the function of Brm on ISC homeostasis is regulated via other signaling pathways by recruiting other factors. Therefore, different phenotypes induced by the loss-of-function of Brm and Yki in midgut might be due to different regulatory mechanisms. Despite its unique function cooperating with Yki in midgut, that Brm complex is essential for Yki-mediated transcription might be a general requirement for cell proliferation. While this manuscript was under preparation, Irvine lab reported a genome-wide association of Yki with chromatin and chromatin-remodeling complexes (Oh, 2013). These results support the model developed in this paper (Jin, 2013).

    The current results also suggest that the interaction between Brm and Yki–Sd transcriptional complex is under tight regulation. The loss of Hpo signaling stabilizes Brm protein, whereas the active Hpo pathway restricts Brm levels by activating Drosophila caspases to cleave Brm at the D718 site and inhibiting downstream target gene diap1 transcription simultaneously. In addition, overexpression of Brm complex components induces only a mild enhancement on midgut proliferation. One possibility is that overexpressing only one of the Brm complex components does not provide full activation of the whole complex; the other possibility is that due to the restriction of the Hpo signaling, as overexpressing BrmD718A mutant protein in ISCs/EBs exhibits a stronger phenotype than expressing the wild-type Brm and coexpression of BrmD718A completely rescues the impairment of Hpo-induced ISC proliferation. D718A mutation blocks the caspase-dependent Brm cleavage and exhibits high activity in promoting ISC proliferation. This study has defined a previously unknown, yet essential epigenetic mechanism underlying the role of the Hpo pathway in regulating Brm activity (Jin, 2013).

    It is a novel finding that Brm protein level is regulated by the caspase-dependent cleavage. To focus on the function of Brm cleavage in the presence of cell death signals, attempts were made to examine the activities of the cleaved Brm fragments. Although in vivo experiments did not show strong activity of Brm N- and C-cleavage products in promoting proliferation of ISCs, the C-terminal fragment of Brm that contains the ATPase domain exhibits a relative higher activity than the N-terminal fragment in ISCs. The cleavage might induce faster degradation of Brm N- and C-terminus, since it was difficult to detect N- or C-fragments of Brm by Western blot analysis without MG132 treatment. It reveals that the degradation events of Brm including both ubiquitination and cleavage at D718 site can be important for Brm functional regulation under different conditions. To this end, the intrinsic signaling(s) may balance the activity of Brm complex through degradation of some important components, such as Brm, to maintain tissue homeostasis. Of note, the cleavage of Brm at D718 is occurred at a novel DATD sequence that is not conserved in human Brm. It has been reported that Cathepsin G, not caspase, cut hBrm during apoptosis, suggesting that the cleavage regulatory mechanism of Brm is relatively conserved between Drosophila and mammals (Jin, 2013).

    This study provides evidence that the Brm complex plays an important role in Drosophila ISC proliferation and differentiation and is regulated by multi-levels of Hpo signaling. The findings indicate that Hpo signaling not only exhibits regulatory roles in organ size control during development but also directly regulates epigenetics through a control of the protein level of epigenetic regulatory component Brm. In mammals, it is known that Hpo signaling and SWI/SNF complex-mediated chromatin remodeling processes play critical roles in tissue development. Malfunction of the Hpo signaling pathway and aberrant expressions of SWI/SNF chromatin-remodeling proteins BRM and BRG1 have been documented in a wide variety of human cancers including colorectal carcinoma. Thus, this study that has implicated a functional link between Hpo signaling pathway and SWI/SNF activity may provide new strategies to develop biomarkers or therapeutic targets (Jin, 2013).

    CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

    During development eukaryotic gene expression is coordinated by dynamic changes in chromatin structure. Measurements of accessible chromatin are used extensively to identify genomic regulatory elements. Whilst chromatin landscapes of pluripotent stem cells are well characterised, chromatin accessibility changes in the development of somatic lineages are not well defined. This study shows that cell-specific chromatin accessibility data can be produced via ectopic expression of E. coli Dam methylase in vivo, without the requirement for cell-sorting (CATaDa). Chromatin accessibility was profiled in individual cell-types of Drosophila neural and midgut lineages. Functional cell-type-specific enhancers were identified, as well as novel motifs enriched at different stages of development. Finally, global changes were shown in the accessibility of chromatin between stem-cells and their differentiated progeny. These results demonstrate the dynamic nature of chromatin accessibility in somatic tissues during stem cell differentiation and provide a novel approach to understanding gene regulatory mechanisms underlying development (Aughey, 2018).

    The conserved Misshapen-Warts-Yorkie pathway acts in enteroblasts to regulate intestinal stem cells in Drosophila

    Similar to the mammalian intestine, the Drosophila adult midgut has resident stem cells that support growth and regeneration. How the niche regulates intestinal stem cell activity in both mammals and flies is not well understood. This study shows that the conserved germinal center protein kinase Misshapen restricts intestinal stem cell division by repressing the expression of the JAK-STAT pathway ligand Upd3 in differentiating enteroblasts. Misshapen, a distant relative to the prototypic Warts activating kinase Hippo, interacts with and activates Warts to negatively regulate the activity of Yorkie and the expression of Upd3. The mammalian Misshapen homolog MAP4K4 similarly interacts with LATS (Warts homolog) and promotes inhibition of YAP (Yorkie homolog). Together, this work reveals that the Misshapen-Warts-Yorkie pathway acts in enteroblasts to control niche signaling to intestinal stem cells. These findings also provide a model in which to study requirements for MAP4K4-related kinases in MST1/2-independent regulation of LATS and YAP (Li, 2014).

    Previous studies have shown that endothelial cells (ECs) produce regulatory factors in response to infection and damage and function as part of the niche to regulate intestinal stem cell (ISC)-mediated regeneration. Meanwhile, recent reports show that enteroblasts (EBs) can also produce growth factors including EGF receptor ligands, Wingless and Upd3, although the pathways that regulate their production are not known. The current results demonstrate that differentiating EBs also function as an important part of the niche to regulate ISC division via the Msn pathway. EB-specific knockdown of msn leads to highly increased Upd3 expression and midgut proliferation. A previous report suggests that undifferentiated EBs if remain in contact with the mother ISC can inhibit proliferation. Although the hyperproliferating midguts after loss of Msn contain many EBs, these EBs do go into normal differentiation and express high level of Upd3, which may overcome any inhibitory effect of undifferentiated EBs on ISC proliferation (Li, 2014).

    Msn is known to regulate a number of biological processes. During embryonic dorsal closure the MAP kinase pathway Slipper-Hemipterous-JNK is downstream of Msn, and Slipper is able to bind to Msn in vitro. In the adult midgut, JNK is a mediator of aging-related intestinal dysplasia and is a stress-activated kinase in ECs to positively regulate ISC division. While the current RNAi experiments show that JNK has a function in EBs to negatively regulate ISC proliferation, this phenotype is not dependent on Upd3 or Yki. No change of JNK phosphorylation was detected after loss of Msn. Mammalian MAP4K4 has also been shown to function independently of JNK in some biological contexts. Therefore, Msn and JNK probably have independent functions in the midgut (Li, 2014).

    This study has instead uncovered an interaction of Msn with Wts and subsequently regulation of Yki. Hpo-Wts-Yki has been demonstrated to have a function in ECs for stress and damage-induced response. Gal4 driven experiments have many caveats including cell-type specificity, differences in promoter strengths, and knockdown efficiency in different cell types. Nonetheless, the results of many parallel experiments that this study conducted strongly suggest that Msn and Hpo independently regulate Wts-Yki in EBs and ECs, respectively. How the Msn and Hpo pathways in the two cell types are coordinately regulated to produce an appropriate amount of Upd3 to achieve desirable intestinal growth under different circumstances remains an important question to be answered (Li, 2014).

    Previous experiments in developing discs suggest that Wts and Yki but not Hpo act downstream of cytoskeleton regulators. Similarly, the mammalian Hpo homologs MST1/2 appear not to be involved in LATS regulation after cytoskeletal perturbation in some cell types. In vivo assay in midgut suggests a function for Msn, Yki and Upd3 downstream of actin capping proteins in EBs. Similarly, the Latrunculin B effect on MEFs suggests that MAP4K4 is required for cytoskeleton-regulated LATS and YAP phosphorylation. The situation in mammalian cells may be more complicated because the Msn/MAP4K4 subfamily also includes two other closely related kinases TNIK and MINK1. Proper regulation of Wts by the cytoskeleton may require both positive and negative regulators, because recent work in flies identified the LIM-domain protein Jub as a negative regulator of Wts in response to cytoskeletal tension. It will be interesting in future studies to determine how positive and negative regulators of Wts act in a coordinated manner to regulate cell fate and proliferation in response to cytoskeletal tension (Li, 2014).

    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. This study has focused 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. Molecular genetic analysis demonstrates that the mitogenic EGF signaling pathway is a limiting factor controlling GSSC proliferation. 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, specifically Spitz and Vein, 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 (Strand, 2013).

    The CCR epithelium is the exclusive site of large acid-secreting copper cells responsible for generating a low pH compartment in the midgut. Gastric stem cells in the CCR are normally quiescent but are robustly stimulated to replenish the unique differentiated cells of the gastric epithelium in response to injury by enteric pathogens or heat stress. This study resolvse outstanding issues related to the GSSC lineage, demonstrating the presence of tripotent GSSC lineages in the CCR. In addition, wa central role is demonstrated for the conserved EGF signaling pathway in controlling the emergence of gastric stem cells from quiescence. Taken together, two key differences between GSSCs and intestinal stem cells (ISCs) are now evident: the unique region specific cell lineages that they support (copper, interstitial, enteroendocrine vs. enterocyte and enteroendocrine) and their activity levels (quiescent vs. active). Thus, maintenance of physiologically and functionally distinct compartments of the adult midgut depends upon the activity of distinct stem cell lineages (Strand, 2013).

    What is the nature of the unique molecular program that governs the observed differences in GSSC and ISC proliferative behavior? This study indicates that regional differences in gut stem cell proliferation are controlled by regional differences in EGF ligand availability. First, reporters of EGF pathway activity are normally very low in the CCR under baseline conditions, when GSSCs are quiescent. However, damage to the gastric epithelium by enteric infection increases local EGF ligand expression and Erk phosphorylation. This EGF activation directly correlates with an observed increase in proliferating GSSCs. Second, ectopic activation of the EGF pathway is sufficient to cell-autonomously promote GSSC proliferation in the absence of environmental challenge. Finally, functional EGF signaling is required for GSSC proliferation following enteric infection and for GSSC lineage expansion. Importantly, these studies of GSSCs in the CCR are similar to previous studies demonstrating that EGF signals are an essential part of the core niche program controlling the ISC lineage. Thus, regional control of EGF ligands, and perhaps other regulators of EGF pathway activity, are essential in generating gastrointestinal stem cell niches with distinct proliferative set points (Strand, 2013).

    In this light, it is worth noting that over-expression of epidermal growth factors and their receptors are associated with human gastric cancer, the second leading cause of cancer-related deaths worldwide. In addition, Ménétrier’s disease is a hyperproliferative disorder of the stomach caused by over-expression of the EGF ligand TGF-α. Over production of TGF-α and increased EGF signaling is associated with an expansion of surface mucous cells and a reduction in parietal and chief cells. Gastric stem cells are the proposed cell-of-origin in Ménétrier’s disease, but this has not been directly tested due to a lack of gastric stem cell specific markers in the murine system. Advances in understanding how EGF ligand availability controls activity of the acid-secreting gastric stem cell lineage in Drosophila raises the possibility that hyperplastic conditions associated with the human stomach might arise when ectopic EGF ligands draw resident stem cells out of their quiescent state (Strand, 2013).

    EGF signaling appears to be only one aspect of the region specific program controlling gastric stem cells in the adult copper cell region. Previous studies have shown that a Delta-lacZ enhancer trap line was not present in GSSCs under baseline conditions. In the course of this study, it was observed that Pseudomonas entomophila challenge also leads to an increase in Delta ligand expression in dividing cells, suggesting a role for Delta/Notch signaling in the GSSC lineage. In addition, elegant studies of GSSCs under baseline conditions have recently shown that the secreted BMP/Dpp signaling pathway is both necessary and sufficient to specify copper cells in the adult midgut and acts via the labial transcription factor. Interestingly, while the highest levels of Dpp pathway reporters are detected in the CCR, manipulation of the BMP/Dpp pathway did not affect GSSC proliferation. Thus, the GSSC lineage is influenced by secreted niche factors, which independently control both GSSC proliferation and cell fate specification (Strand, 2013).

    In conclusion, understanding GI regionality and homeostatic diversity along the A/P axis is important for several reasons. It is now possible to gain insight into how the modification of a core GI niche program, which adapts each stem cell to its compartment specific physiology, leads to difference in lineage output. Second, disruption of regional identity along the GI tract is associated with a class of precancerous conditions called metaplasias, in which one region of the GI tract takes on the attributes of another. Finally, both the establishment and maintenance of tumorigenic lineages exhibit marked preferences along the A/P axis of the gut. The striking similarities between vertebrate and invertebrate GI biology, suggest that delving deeper into the mechanisms underlying Drosophila midgut regionalization will continue to provide important insights into these fundamental biological problems (Strand, 2013).

    EGFR signaling regulates the proliferation of Drosophila adult midgut progenitors

    In holometabolous insects, the adult appendages and internal organs form anew from larval progenitor cells during metamorphosis. The adult Drosophila midgut, including intestinal stem cells (ISCs), develops from adult midgut progenitor cells (AMPs) that proliferate during larval development in two phases. Dividing AMPs, as visualized using esgGal4-driven GFP expression, first disperse, but later proliferate within distinct islands, forming large cell clusters that eventually fuse during metamorphosis to make the adult midgut epithelium. Signaling through the EGFR/RAS/MAPK pathway is necessary and limiting for AMP proliferation. Midgut visceral muscle produces a weak EGFR ligand, Vein, which is required for early AMP proliferation. Two stronger EGFR ligands, Spitz and Keren, are expressed by the AMPs themselves and provide an additional, autocrine mitogenic stimulus to the AMPs during late larval stages (Jiang, 2009).

    Drosophila AMPs were previously thought to be relatively quiescent during larval development, dividing just once or twice, and not initiating rapid proliferation until the onset of metamorphosis. This is the case for several other larval progenitor/imaginal cell types, such as the abdominal histoblasts and cells in the salivary gland, foregut and hindgut imaginal rings. Studies have suggested that AMP proliferation might precede the onset of metamorphosis. However, the extensive proliferation of the AMPs that is seen in this study has not been reported and the early larval proliferative phase when the AMPs divide and disperse has not been reported. The extensive proliferation of the AMPs is similar to that of the larval imaginal disc cells, which also proliferate throughout larval development, dividing about ten times (Jiang, 2009).

    AMPs occurs in two distinct phases. In early larvae, the AMPs divide and disperse throughout the midgut to form individual islets. During later larval development, the AMPs continue to divide but do so within these islets, forming large cell clusters. It is speculated that in the early larva, secretion of Vn from the midgut visceral muscle (VM) cells results in low-level activation of EGFR signaling in the AMPs, which is sufficient for their proliferation and might also promote their dispersal. No proliferation defects were seen in AMPs defective in shot function, suggesting that the mechanism of EGFR activation used by tendon cells during muscle/tendon development is probably not the same as in the larval midgut. Specifically, it is unlikely that the Shot-mediated concentration of Vn on AMPs activates EGFR signaling in the AMPs during early larval development. Consistent with this, dpERK staining is only seen in AMP clusters and not in the isolated AMPs present at early larval stages (Jiang, 2009).

    The mechanisms that regulate the transition between these two proliferation phases remain unclear. Fewer AMP clusters are seen when sSpi, sKrn, lambdaTOP (activated Egfr) or RasV12 were induced in the AMPs starting from early larval stages, suggesting that EGFR signaling, in addition to its crucial role as an AMP mitogen, might also play a role in AMP cluster formation. In the late larval midgut (96-120 hours AED), high-level EGFR activation, resulting from expression of spi and Krn in the AMPs themselves, might not only promote AMP proliferation, but might also suppress AMP dispersal and thus promote formation of the AMP clusters. How the timing and location of Spi- or Krn-mediated EGFR activation are regulated during larval development is also unclear. It is noted, however, that the pro-ligand form of Krn acted similarly to sKrn, and that no functions were uncovered for the Rho-like gene products that regulate Spi and Krn function by proteolytic cleavage in other tissues. This suggests that the localized expression of these ligands in the AMP clusters might be the critical parameter that controls their effects. Consistent with this, Rho-independent cleavage and function of Krn have been documented (Reich, 2002; Jiang, 2009).

    In the developing Drosophila wing, EGFR/RAS/MAPK signaling promotes the expression and controls the localization of the cell adhesion molecule Shotgun (Shg, Drosophila DE-cadherin). RasV12-expressing clones generated in the wing imaginal disc are round, much like the AMP clusters described in this study, owing to increased adhesive junctions. In developing Drosophila trachea, EGFR activity upregulates shg expression to maintain epithelial integrity in the elongating tracheal tubes. In the eye, EGFR activity leads to increased levels of Shg and adhesion between photoreceptors. Given these precedents, it seems reasonable to suggest that high-level EGFR activity in the AMP islets upregulates Shg and promotes the homotypic adhesion of the AMPs. Alternatively, changes in the differentiated cells of the midgut epithelium might promote AMP clustering. In either case, the dispersal of early AMPs and subsequent formation of late AMP clusters facilitate the formation of the adult midgut epithelium during metamorphosis (Jiang, 2009).

    This study confirms previous reports that Drosophila AMPs replace larval midgut epithelial cells to form the adult midgut epithelium during metamorphosis. Furthermore, it was shown that the majority of AMPs lose esgGal4-driven GFP expression as they differentiate to form the new adult midgut epithelium. These cells lacked Prospero, which marks enteroendocrine cells in both the larval and adult midgut. They went through several rounds of endoreplication during late pupal development, and thus probably all differentiated into adult enterocytes (ECs). During early metamorphosis, some cells in the new midgut epithelium remained small and diploid and maintained strong esgGal4 expression. For several reasons, it is thought that these esg-positive cells are the future adult intestinal stem cells (ISCs). (1) esgGal4 expression marks AMPs, including adult ISCs and enteroblasts. (2) Mitoses in the adult midgut are only observed in ISCs, and this study observed mitoses only in the esg-positive cells during metamorphosis. (3) esg-positive cells migrated to the basal side of the midgut epithelium, the location of adult ISCs. (4) AMP clones generated during early larval development contained just a few esg-positive cells when the new adult midgut first formed (24 hours APF), but when such clones were scored in newly eclosed adults, they contained large numbers of ECs, as well as cells positive for the enteroendocrine marker Prospero and the ISC marker Delta. This suggests that a small fraction of AMPs differentiate into adult ISCs. However, esg-positive cells in the new pupal midgut lacked Delta expression until eclosion, suggesting that they are probably not mature adult ISCs (Jiang, 2009).

    How a small fraction of AMPs are selected to become adult ISCs in the newly formed pupal midgut epithelium is not known. One possibility is that the adult ISCs are determined during larval development, long before the formation of the adult midgut. Another is that they are specified during early metamorphosis. This second hypothesis is preferred for several reasons. First, in the lineage analysis, it was found that all AMP clones induced during early larval stages formed multiple clusters. This suggests that there are no quiescent AMPs in the larval midgut. Second, when AMP clones were induced at mid-third instar, the mosaic clusters always contained multiple GFP-positive cells, suggesting that all AMPs in the mid-third instar midgut remain equally proliferative. Third, during larval development, differentiation of the AMPs were never observed, as judged by their ploidy (diploid) and lack of expression of the enteroendocrine marker Prospero. Fourth, all AMPs appeared to express esgGal4 throughout larval development. Given the crucial role that Notch signaling plays in regulating AMPs during embryonic midgut development and ISCs in adult midgut homeostasis, it is edexpect that Notch might also function to specify adult ISCs during metamorphosis (Jiang, 2009).

    EGFR signaling is both required and sufficient to promote AMP proliferation. Hyperactivation of EGFR signaling, such as by expression of activated Ras (RasV12), promoted massive AMP overproliferation and generated hyperplastic midguts that were clearly dysfunctional. In contrast, inhibiting EGFR/RAS/MAPK signaling dramatically reduced AMP proliferation. Furthermore, the ability of EGFR signaling to induce ectopic AMP proliferation is almost unique. With the exception of larval hemocytes, activated EGFR signaling does not promote cell proliferation in the imaginal discs, salivary gland imaginal rings, abdominal histoblasts, foregut and hindgut imaginal rings. This suggests that the regulation of AMP proliferation is different from that in other imaginal cells (Jiang, 2009).

    Despite the obvious differences between adult ISCs and their larval progenitors, the AMPs, there are also similarities. (1) When the new adult midgut epithelium forms, larval AMPs give rise to the new adult midgut including the adult ISCs. Many genes, such as esg, that are specifically expressed in the larval AMPs are also expressed in the adult ISCs. (2) The structure of the midgut epithelium with basal AMPs or ISCs is similar in larval and adult stages. (3) vn expression in larval VM persists in the adult midgut, suggesting that Vn from the adult VM might also regulate the ISCs (Jiang, 2009).

    In two Drosophila stem cell models, the testis and ovary, stem cells reside in special niches comprising other supporting cell types. These niches maintain the stem cells and provide them with proliferative cues. For example, in the testis, germ stem cells attach to the niche that comprises cap cells. The cap cells release Jak/Stat and BMP ligands [Upd (Os) and Gbb/Dpp], which maintain the stem cells and induce their proliferation. Whether Drosophila ISCs utilize supporting cells that constitute a niche remains unclear. This study shows that multiple EGFR ligands are involved in the regulation of Drosophila AMP proliferation. During early larval development, the midgut VM expresses the EGFR ligand vn, which is required for AMP proliferation. Thus, the early AMPs might be considered to require a niche comprising non-epithelial VM. Later in larval development, however, the AMPs express two other EGFR ligands, spi and Krn, which are capable of autonomously promoting their proliferation and may render vn dispensable. This study found, however, that depleting spi and Krn in the AMPs did not affect AMP proliferation, suggesting that vn or another trigger of EGFR/RAS/MAPK activity might complement spi and Krn in late-stage larvae (Jiang, 2009).

    Bunched and Madm function downstream of Tuberous Sclerosis Complex to regulate the growth of intestinal stem cells in Drosophila

    The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. This study shows that the leucine zipper protein Bunched and the adaptor protein MLF1-adaptor molecule (Madm) are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, these data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells (Nie, 2015).

    Homeostasis and regeneration of an adult tissue is normally supported by resident stem cells. Elucidation of the mechanisms that regulate stem cell-mediated homeostasis is important for the development of therapeutics for various diseases. The intestine with fast cell turnover rate supported by actively proliferating stem cells is a robust system to study tissue homeostasis. In the mouse intestine, two interconverting intestinal stem cell (ISC) populations marked by Bmi1 and Lgr5 located near the crypt base can replenish cells of various lineages along the crypt-villus axis Furthermore, recent data suggest that Lgr5+ cells are the main stem cell population and that immediate progeny destined for the secretory lineage can revert to Lgr5+ stem cells under certain conditions [6, 7]. Together, the results suggest previously unexpected plasticity in stem cell maintenance and differentiation in the adult mammalian intestine (Nie, 2015).

    In the adult Drosophila midgut, which is equivalent to the mammalian stomach and small intestine, ISCs are distributed evenly along the basal side of the monolayered epithelium to support repair. The maintenance and regulation of Drosophila midgut ISCs depend on both intrinsic and extrinsic factors. When a midgut ISC divides, it generates a renewed ISC and an enteroblast (EB) that ceases to divide and starts to differentiate. The ISC-EB asymmetry is established by the Delta-Notch signaling, with Delta in the renewed ISC activating Notch signaling in the newly formed neighboring EB . Growth factors such as Wingless/ Wnt, insulin-like peptides, Decapentaplegic/BMP, Hedgehog and ligands for the EGF receptor and JAK-STAT pathways are secreted from surrounding cells and constitute the niche signals that regulate both ISC division and EB differentiation. ISC-intrinsic factors including Myc, Target of Rapamycin (TOR) and Tuberous Sclerosis Complex act to coordinate the growth and division of ISCs. Furthermore, chromatin modifiers such as Osa, Brahma and Scrawny function within ISCs to regulate Delta expression or ISC proliferation (Nie, 2015).

    This study reports the identification of the leucine zipper protein Bunched (Bun) and the adaptor protein myeloid leukemia factor 1 adaptor molecule (Madm) as intrinsic factors for ISC proliferation. A single bun genomic locus generates multiple predicted transcripts that encode 4 long isoforms, BunA, F, G and P, and 5 short isoforms, BunB, C, D, E, H and O. The first identified mammalian homolog of Bun is TGF-β1 stimulated clone-22 (TSC-22). In the mouse genome four different TSC- 22 domain genes also encode multiple short and long isoforms. All isoforms of Bun and TSC-22 contain an approximately 200 amino acids C-terminal domain where the conserved TSC-box and leucine zippers are located. The originally identified TSC-22 is a short isoform and various assays suggest that it suppresses cancer cell proliferation and may function as a transcriptional regulator. Meanwhile, in Drosophila, the long Bun isoforms positively regulate growth, while the short isoforms may antagonize the function of long isoforms. Transgenic fly assays also demonstrate that the long TSC-22 can rescue the bun mutant phenotypes, whereas short isoforms cannot. These results suggest an alternative model that the long Bun isoforms positively regulate proliferation, while the short isoforms may dimerize with and inhibit the functions of long isoforms (Nie, 2015).

    Madm also can promote growth. The long isoform BunA binds to Madm via a conserved motif located in the N- terminus that is not present in the short Bun isoforms. The molecular function of this novel BunA- Madm complex, nonetheless, remains to be elucidated. The results in this report demonstrate that Bun and Madm modulate the Tuberous Sclerosis Complex-target of Rapamycin (TOR)-eIF4E binding protein (4EBP) pathway to regulate the growth and division of ISCs in the adult midgut (Nie, 2015).

    This report shows that Bun and Madm are intrinsically required for ISC growth and division. The results suggest a model that Bun and Madm form a complex in the cytoplasm to promote cellular growth and proliferation. The evidence that support this model includes the observation that transgenic expressed Bun localizes in the cytoplasm of midgut precursor cells, similar to the results from transfection in S2 cells and immune-staining in eye discs. Bun physically and functionally interacts with Madm, which has also been proposed as a cytoplasmic adaptor protein. Adding a nuclear localization signal to Bun reduced the growth promoting ability of Bun. Although there is a possibility this signal peptide changes the functionality in an unpredicted way, the interpretation is favored that Bun normally acts in the cytoplasm and with Madm to regulate the proliferation of ISCs. This is in contrast to mammalian TSC-22, which was reported to function in the nucleus (Nie, 2015).

    The results seem to contradict a previous publication reporting that TSC-22 arrests proliferation during human colon epithelial cell differentiation. However, this apparent contradiction is resolved when the growing evidence for distinct functions for large and small Bun/ TSC-22 isoforms is considered. The Bun/TSC-22 proteins have short and long isoforms that contain the conserved TSC-box and leucine zippers in the C-terminal domain. The prototypical TSC-22 protein, TSC22D1-001, may act as a transcriptional regulator and repress cancer cell proliferation, particularly for blood lineages. Another recent model suggests that in Drosophila the long Bun isoforms interact with Madm and have a growth promoting activity, which is inhibited by the short Bun isoforms. Similarly, the long isoform, TSC22D1-002, enhances proliferation in mouse mammary glands, whereas the short isoform promotes apoptosis. Unpublished result that transgenic expression of BunB also has lower function than BunA in fly intestinal progenitor cells is consistent with this model where large isoforms have a distinct function, namely in growth promotion (Nie, 2015).

    Loss of either Bun or Madm can potently suppress all the growth stimulation by multiple pathways in the midgut as shown in this report. These results are intrepeted to indicate that Bun and Madm do not act specifically in one of the signaling pathways tested but instead function in a fundamental process required for cell growth, such as protein synthesis or protein turnover. It is therefore speculated that Bun and Madm may regulate the TOR pathway. In support of this idea, it was shown that bunRNAi or MadmRNAi efficiently suppresses the Tuberous Sclerosis Complex 2RNAi-induced cell growth and p4EBP phenotypes. A recent study of genetic suppression of TOR complex 1-S6K function in S2 cells also suggests that Bun and Madm can interact with this pathway. Furthermore, proteomic analyses of Bun and Madm interacting proteins in S2 cells have shown interactions with ribosomal proteins and translation initiation factors. Therefore, a model is proposed that Bun and Madm function in the Tuberous Sclerosis Complex-TOR- 4EBP pathway to regulate protein synthesis in ISCs for their growth, which is a prerequisite for ISC proliferation. Suppression of Tuberous Sclerosis Complex mutant cell growth phenotype by bun or Madm RNAi was substantial but not complete. Earlier papers demonstrated that Bun also interacts with Notch and EGF pathway in ovary follicle cells. Therefore by definition Bun and Madm are neither 100% essential nor restricted to the TOR pathway. The genetic data suggest that Bun and Madm work downstream of Tuberous Sclerosis Complex and upstream of 4EBP, but they could also work in parallel to the TOR pathway components (Nie, 2015).

    ISCs with loss of Tuberous Sclerosis Complex function have substantial cell size increase. Meanwhile, the Bun/ Madm overexpression caused increased ISC division but not cell hypertrophy. Both loss of Tuberous Sclerosis Complex and overexpression of Bun/Madm should promote cell growth but the phenotypes at the end are different. It is speculated that the reason is the Bun/Madm overexpressing ISCs are still capable of mitosis, while the Tuberous Sclerosis Complex mutant ISCs do not divide anymore thereby resulting in the very big cells. In Bun and Madm overexpressing mid- guts, the p-H3+ and GFP+ cell count showed a significant increase, indicating increased mitosis. Therefore, an explanation is that Bun and Madm overexpression may increase cell size/cell growth, but when they grow to certain size they divide, resulting in rather normal cell size (Nie, 2015). The knockout of the Madm mammalian homolog, NRBP1, can cause accumulation of the short isoform TSC22D2. Up-regulation of Madm/NRBP1 has been associated with poor clinical outcome and increased growth of prostate cancer. Further analysis based on this model may reveal whether high ratio of long Bun/TSC22 isoforms over short isoforms may associate with high Madm activity and poor clinical outcomes (Nie, 2015).

    Ret receptor tyrosine kinase sustains proliferation and tissue maturation in intestinal epithelia

    Expression of the Ret receptor tyrosine kinase is a defining feature of enteric neurons. Its importance is underscored by the effects of its mutation in Hirschsprung disease, leading to absence of gut innervation and severe gastrointestinal symptoms. This study reports a new and physiologically significant site of Ret expression in the intestine: the intestinal epithelium. Experiments in Drosophila indicate that Ret is expressed both by enteric neurons and adult intestinal epithelial progenitors, which require Ret to sustain their proliferation. Mechanistically, Ret is engaged in a positive feedback loop with Wnt/Wingless signalling, modulated by Src and Fak kinases. Ret is also expressed by the developing intestinal epithelium of mice, where its expression is maintained into the adult stage in a subset of enteroendocrine/enterochromaffin cells. Mouse organoid experiments point to an intrinsic role for Ret in promoting epithelial maturation and regulating Wnt signalling. These findings reveal evolutionary conservation of the positive Ret/Wnt signalling feedback in both developmental and homoeostatic contexts. They also suggest an epithelial contribution to Ret loss-of-function disorders such as Hirschsprung disease (Perea, 2017).

    These findings in Drosophila indicate that Ret is expressed not only by enteric neurons, but also by the adult somatic stem cells of the intestinal epithelium. In contrast to known Ret functions in other progenitor cell types -- for example, in spermatogonia or the hematopoietic system -- Ret is not required for the survival of adult somatic stem cells in the intestine, but sustains both their homeostatic and regenerative proliferative capacity. Gain- and loss-of-function experiments point to the existence of positive feedback between Ret and Wg signalling. Despite abundant genetic evidence that Wg signalling promotes stem cell proliferation in flies, the source of Wg has remained unclear. Using new, improved tools to visualise Wg expression, the current findings lend further support to recent data (Tian, 2016) indicating that the source of Wg ligand is not the stem cells themselves, despite the striking Ret-driven upregulation of Wg on their surface. How might Ret signalling in adult intestinal progenitors lead to Wg protein upregulation in these cells without affecting its transcript? Two possible ways in which it might do so is by upregulating the expression of Wg receptor(s) on their surface, and/or by promoting signalling from stem cells to the Wg-producing cells at the intestinal boundaries and/or the visceral muscles (Buchon, 2013; Tian, 2016) to increase Wg release/trafficking (Perea, 2017).

    Epithelial Ret is not a peculiarity of the fly intestine; Ret is also expressed in the developing intestinal epithelium of mice, prior to the maturation of enteroendocrine or Lgr5-positive stem cells. Although immunohistochemical analyses have not revealed a specific Ret-positive cell population at this stage, ex vivo experiments using epithelial cultures devoid of enteric neuron or mesenchyme point to an intrinsic role for Ret at this stage in promoting epithelial maturation. The mechanism underlying the maturation-promoting effects of Ret may involve positive feedback between Ret and Wnt signalling similar to those found in flies. Indeed, the Wnt pathway target Axin2 is reduced in epithelial cultures derived from Ret51 mice and upregulated when wild-type FEnS are treated with the Ret ligand GDNF: a treatment that also promotes their branching. These data are consistent with the previous finding that elevated Wnt signalling promotes FEnS to organoid maturation and is reminiscent of the Wnt11/Ret autoregulatory loop promoting ureteric branching during kidney development. The relevant source of Wnt driving tissue maturation is currently unknown and is most likely not epithelial. The Drosophila finding that Wg ligand upregulation is not transcriptional underscores the importance of considering tissue crosstalk and non-autonomous signalling in any future studies addressing Wnt contributions to epithelial maturation in mice (Perea, 2017).

    At first sight, the Ret effects on developmental maturation in mice appear to be different from its homeostatic role in flies. However, this study found that Ret continues to be expressed in the adult small intestine, where Ret expression is prominent in a subset of enteroendocrine cells positive for the secretory marker chromogranin-A. Based on their position, these cells may correspond to enterochromaffin cells: intriguing cells that contribute 90% of the serotonin in circulation, control gastrointestinal motility and secretions and have recently been shown to be chemosensory. A very recent study has blurred the distinction between enteroendocrine cells and their precursors by revealing expression overlaps between markers of enteroendocrine precursor identity and differentiated fate (including chromogranin-A), and by suggesting that differentiated enteroendocrine cells can have stem cell-like properties (Yan, 2017). This is exciting because it suggests that, whilst the cellular classification of Ret-positive cells based on known markers may differ between flies (ISCs) and mice (enteroendocrine), Ret-enabled stem cell functionality may contribute to regeneration in both epithelia. Intriguingly, endocrine tumours derived from the small intestine, ileal carcinoids, secrete serotonin and have been reported to express high levels of Ret. Conditional deletion of Ret in adult intestinal epithelium will, in future, determine its contribution to enteroendocrine fate and will help establish possible enteroendocrine contributions to intestinal homeostasis and tumour formation (Perea, 2017).

    Consistent with ex vivo transfection studies in mammalian cells, pointing to physical association between Ret and c-Src, this study found that Src kinase Src42A is required downstream of Ret to activate Wg signalling. These findings therefore strengthen the link between two pathways previously known to control ISC proliferation in flies-Src and Wg, and may provide a physiological context for the previously reported, Src-dependent mitogenic effects of mutated, oncogenic Ret in the Drosophila developing retina. The finding that, in the Drosophila intestine, Src kinases control expression of a mitogenic module consisting of String/Cdc25 and cyclin E provides a simple link between Ret activation and its pro-proliferative effects. Overactive Src kinases do, however, lead to intestinal tumours so mechanisms must be in place to limit the positive Ret/Wg feedback loop so that it sustains homeostatic proliferation, but does not result in tumour formation. Availability of Wg ligand may be an extrinsic mechanism, but the focal adhesion kinase Fak may provide a cell-intrinsic break downstream of Ret/Src activation. Consistent with this idea, Ret expression leads to both Src42A and Fak phosphorylation, but this study found that the two kinases have opposing effects on proliferation: Src42A promotes proliferation downstream of Ret, whereas Fak blocks it. Hence, despite the fact that blocking Fak function may represent a therapeutic opportunity in some cancers, the current findings are more aligned with a previous study (Macagno, 2014) that suggested that, at least in the context of Ret-driven tumorigenesis, Fak can act as a tumour suppressor. In future, it will also be of interest to explore how the Ras/Raf/Erk pathway, activated by Ret in other contexts and previously shown to affect ISC proliferation in flies, intersects with Src/Fak/Wg signalling in response to Ret activation (Perea, 2017).

    Both Wnt and Src pathways can have strong effects on proliferation, differentiation and/or tumorigenesis in the murine intestine. Src is required for mouse intestinal tumourigenesis following upregulation of Wnt signalling. Based on functional findings in Drosophila and expression data in mice, a possible contribution of the Ret- and CgA-positive cells to this process deserves further investigation. It will also be of interest to investigate how Src/Fak kinase signalling contributes to the maturation of foetal intestinal epithelial cells and whether this is important in the development of intestinal disorders (Perea, 2017).

    Ret expression is one of the defining features of enteric neurons. This study has found another evolutionarily conserved and physiologically significant site of Ret expression: the intestinal epithelium. The presence of Ret in these two gastrointestinal cell types of very different developmental origin raises the possibility that the development and/or physiology of enteric neurons, intestinal epithelial progenitors and, in mammals, Ret-expressing intestinal lymphoid cells is coordinated. Such coordination may, for example, help ensure a match between the size of the intestinal epithelium, the number of innervating neurons during development and the transition from an immature foetal epithelium into a functional epithelium involved in nutrient uptake and interorgan signalling. In mammals, Ret ligands of the glial cell line derived neurotrophic factor (Gdnf) family may orchestrate Ret signalling in these three tissues. In flies (which lack these Ret ligands), integrins have been shown to interact with Ret in sensory neurons. Interfering with integrin expression in the Drosophila intestine can have different effects on intestinal progenitor proliferation, survival and/or orientation depending on whether the integrins are removed from the progenitors or their niche-the visceral muscles. Intriguingly, integrin downregulation in adult intestinal progenitors reduces their normal proliferation and can suppress their overproliferation in response to overactive Wingless signalling: phenotypes strikingly similar to those resulting from Ret downregulation. In the light of the known links between integrins and Fak/Src signalling in both normal and cancer cells and the effects that this study has found for Src and Fak downstream of Ret activation, Ret could provide a new route for the integrin activation of the Src/Fak complex. Alternatively, the recent finding that GDF15, a divergent member of the TGF-β superfamily, signals through a GDNF family receptor α-like in a Ret-dependent way also raises the intriguing possibility that TGF-β-like ligands modulate Ret signalling in the intestinal epithelium, potentially linking intestinal regeneration with the known GDF15 roles in food intake/body weight (Perea, 2017).

    The crucial requirement for Ret in enteric nervous system development is underscored by disorders such as HSCR, in which Ret loss of function leads to almost complete absence of enteric innervation in varying lengths of the distal gut. Whilst the contribution of enteric aganglionosis to HSCR is unquestionable, the current findings raise the possibility that, if the epithelial expression of Ret is conserved in humans, dysregulation of epithelial signalling may contribute to disorders that, like HSCR, result from Ret mutation. Epithelial Ret signalling might also contribute to other aspects of gastrointestinal physiology previously shown to be affected by reduced Ret function, such as intestinal motility, gut-microbiota interactions and the compensatory response to massive small bowel resection. Interestingly, HSCR is typically diagnosed around birth due to defects in gastrointestinal functions. This coincides with the first demands on intestinal function, which could reflect not only neuronal defects related to peristalsis, but also defects associated with the transition from a foetal into a functional adult epithelium. Given that many of the pathways that drive tissue expansion and the maintenance of non-differentiated progenitor populations during foetal development are deregulated in cancer, a possible contribution of Ret signalling to colorectal tumours also deserves further investigation (Perea, 2017).

    Accumulation of differentiating intestinal stem cell progenies drives tumorigenesis

    Stem cell self-renewal and differentiation are coordinated to maintain tissue homeostasis and prevent cancer. Mutations causing stem cell proliferation are traditionally the focus of cancer studies. However, the contribution of the differentiating stem cell progenies in tumorigenesis is poorly characterized. This study reports that loss of the SOX transcription factor, Sox21a, blocks the differentiation programme of enteroblast (EB), the intestinal stem cell progeny in the adult Drosophila midgut. This results in EB accumulation and formation of tumours. Sox21a tumour initiation and growth involve stem cell proliferation induced by the Unpaired 2 mitogen released from accumulating EBs generating a feed-forward loop. EBs found in the tumours are heterogeneous and grow towards the intestinal lumen. Sox21a tumours modulate their environment by secreting matrix metalloproteinase and reactive oxygen species. Enterocytes surrounding the tumours are eliminated through delamination allowing tumour progression, a process requiring JNK activation. These data highlight the tumorigenic properties of transit differentiating cells (Zhai, 2015).

    Wildtype adult stem cells, unlike tumor cells, are resistant to cellular damages in Drosophila

    Adult stem cells or residential progenitor cells are critical to maintain the structure and function of adult tissues (homeostasis) throughout the lifetime of an individual. Mis-regulation of stem cell proliferation and differentiation often leads to diseases including cancer, however, how wildtype adult stem cells and cancer cells respond to cellular damages remains unclear. This study found that in the adult Drosophila midgut, intestinal stem cells (ISCs), unlike tumor intestinal cells, are resistant to various cellular damages. Tumor intestinal cells, unlike wildtype ISCs, are easily eliminated by apoptosis. Further, their proliferation is inhibited upon autophagy induction, and autophagy-mediated tumor inhibition is independent of caspase-dependent apoptosis. Interestingly, inhibition of tumorigenesis by autophagy is likely through the sequestration and degradation of mitochondria, as compromising mitochondria activity in these tumor models mimics the induction of autophagy and increasing the production of mitochondria alleviates the tumor-suppression capacity of autophagy. Together, these data demonstrate that wildtype adult stem cells and tumor cells show dramatic differences in sensitivity to cellular damages, thus providing potential therapeutic implications targeting tumorigenesis (Ma, 2016).

    Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells

    Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). Loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1 (Korzelis, 2014).

    Stem cell identity is controlled by both extrinsic cues from the niche and cell-intrinsic transcriptional programs. Thus far, most studies of the Drosophila midgut have focused on the niche-derived signals that control midgut stem cell self-renewal. This study demonstrates a cell-intrinsic role for the Snail family transcription factor, Escargot, in controlling ISC self-renewal and differentiation. Loss of Esg leads to a rapid loss of all stem/progenitor cells in the midgut, due to their differentiation, whereas Esg overexpression keeps these cells permanently in an undifferentiated state. The dramatic effects of manipulating Esg levels support a central role for this Snail family member in controlling stem cell identity in the fly intestine (Korzelis, 2014).

    A transcriptomics analysis indicated that Esg acts as a transcriptional repressor of a large diverse set of differentiation genes. These targets include transcription factors specific to ECs and EEs (Pdm1, Prospero) and genes used in digestion, immunity and cytoarchitectural specialization. Interestingly, one of these transcription factors, Pdm1, plays an important role in EC differentiation: ectopic expression of Pdm1 in progenitor cells was sufficient to trigger EC differentiation, partially mimicking the esg loss of function phenotype. The rapid loss of the Esg-expressing cell population upon Pdm1 overexpression suggests that Pdm1 might repress Esg expression, perhaps directly. In this case, Esg and Pdm1 together would constitute a negative feedback switch that governs EC differentiation (Korzelis, 2014).

    Expression analysis also raised the possibility that Esg activates progenitor cell-specific genes in ISCs and EBs. These include the EGF signaling components Cbl, spitz, argos and Egfr as well as the Jak/Stat receptor domeless. Both EGFR and Jak/STAT pathways are crucial for ISC growth and maintenance, and receptivity to these signals is downregulated in differentiated ECs and EEs. While Snail family members are best understood as repressors, the Esg paralog Snail has been reported to function as a context-dependent transcriptional activator (Rembold, 2014), suggesting that an activating role for Esg is also plausible. The function of Esg as either an activator or repressor is likely determined by co-factors and/or other transcription factors acting on the same promoters that are expressed in the ISC and EB population. In the Drosophila embryo, Snail cooperates with Twist at distinct promoters to activate EMT gene expression during mesoderm formation (Rembold, 2014). Snail2 can bind to Sox9 to activate expression from its own promoter during chick neural crest formation. In its role as a repressor, Esg binds the co-repressor CtBP to maintain somatic Cyst stem cells and hub cells in the Drosophila male testis. Future work to unravel the complete transcriptional network within which Esg functions to maintain the stem/progenitor state should prove to be very interesting (Korzelis, 2014).

    The data support a model in which Esg acts in a circuit with Delta-Notch signaling to control the switch from stem/progenitor identity to differentiated cell identities. In its simplest form, this circuit might be a bistable switch in which Esg and Notch mutually inhibited each other, with Esg being 'on' and dominant in progenitor cells and Notch signaling 'on' and dominant in their differentiated progeny, the enterocytes. However, the constant presence of a substantial population of intermediate progenitor cells, the enteroblasts (EBs), which express both Esg and Notch reporter genes, indicates that a simple bistable switch is not an accurate conception. Indeed, EBs, defined here as cells positive for both Esg and the Notch reporter Su(H)GBE-LacZ, can persist for many days in the absence of ISC division. Thus, the EB transition state is metastable. In this transition state, Notch is apparently active, but secondary downstream targets that directly affect differentiation, such as Pdm1, brush border Myosin and smooth septate junction proteins, remain repressed. Since these genes are rapidly activated following depletion of Esg, it is suggested that their repression is most likely mediated by Esg binding (Korzelis, 2014).

    Two potential explanations are provided for the longevity of the EB transition state. First, it is suggested that the repression of esg transcription by Notch is indirect and that this delays esg silencing. Silencing of Esg is not likely to be mediated by the Notch-regulated transcription factor Su(H) (a transcriptional activator) but by downstream repressors that act only after enterocyte or endocrine differentiation has begun. Pdm1 in ECs and Prospero in EEs are presently the most obvious candidates. Both are specifically induced coincident with Esg silencing, in ECs and EEs, respectively, and Dam-ID assays suggest that Pros has binding sites in the esg locus. The finding that overexpression of Pdm1 caused the rapid differentiation of Esg+ stem/progenitor cells supports the notion that Pdm1 could directly repress Esg expression to control EC differentiation. Furthermore, nubbin/Pdm1 was found to restrict expression of Notch target genes in the Drosophila larval wing disc. Hence, Pdm1 likely triggers EC differentiation by downregulating both Esg and the expression of Notch target genes in the EB. Therefore, Notch is only transiently active in EBs but fully off in mature ECs with high levels of Pdm1 (Korzelis, 2014).

    While a delay circuit that controls the silencing of Esg is likely, theoretically it cannot explain how Esg+ EBs can persist for such long periods during times of low gut epithelial turnover and then rapidly differentiate during gut regeneration. Hence, it is speculated that a second input signal acts in combination with Notch-dependent factor(s) to silence Esg. This second signal is likely to be a downstream effector of the growth factor signaling network that also drives ISC division and gut epithelial renewal. Of the transcriptional effectors involved in maintaining gut homeostasis, the most obvious candidate as an indirect mediator of esg repression is Stat92E, which is activated by the highly stress-dependent cytokines, Upd2 and Upd3. Tellingly, the cytokine receptor, Dome, Janus Kinase (hop) and Stat92E are all required for EB maturation into ECs. If the silencing of esg was dependent upon both Notch and Stat92E, and Delta-Notch signaling was irreversible once resolved; then, the Notch+ Esg+ EB transition state should in principle be stable in conditions of low Jak/Stat signaling, as is observed during periods of midgut quiescence. It needs to be noted, however, that ISCs and EBs maintain appreciable levels of Stat-reporter gene expression even during relative quiescence, and so, in this model, it would be Stat activity above some threshold that would combine with Notch signaling to trigger differentiation. Since Jak/Stat signaling also triggers ISC division, a surge in cytokine signaling could coordinately trigger both the differentiation of older EBs and the production of new ones in this model , thus explaining how a significant EB population is maintained even as stem cell activity waxes and wanes (Korzelis, 2014).

    Snail family transcription factors have been described as regulators of epithelial-to-mesenchyme transitions (EMT) that occur during development, wound healing and cancer metastasis. In some contexts, notably metastasis, EMT is believed to accompany the acquisition of stem-like properties. Although Esg itself has not been reported to regulate EMT, its paralog in flies (Sna) and homologs in mammals (Snai1, Snai2) do promote EMT. Interestingly, RNA-seq experiments showed that not only Esg, but Snail, Worniu and the Zeb family members Zfh1 and Zfh2 were all expressed in intestinal stem cells and downregulated in ECs and EEs. Thus, these EMT-linked transcription factors may work together to affect different aspects of midgut homeostasis and ISC differentiation. Indeed, Esg-positive ISCs and EBs are morphologically more similar to mesenchymal cells than they are epithelial, whereas Esg-negative EEs and ECs have the pronounced apical-basal polarity typical of epithelial cells. Esg+ cells often make striking lateral projections, suggestive of dynamic behavior, and they have the capacity to multilayer when their differentiation is blocked or they are forced to overproliferate. Furthermore, a number of epithelial-class genes are repressed in Esg+ progenitors and activated upon EC and/or EE differentiation. These include genes encoding the apico-lateral cortical Lgl-Dlg-Scrib-Crb complex, septate junction proteins (e.g., Ssk, Cora, Mesh) and polarity factors including Par3 and Par6. Strikingly, Scrib and Ssk both have Esg-binding sites in their promoters, and their expression is highly regulated by Esg. However, some gene targets that are central to EMT in mammalian cells show opposite trends in the fly's ISC lineage. For instance, Esg+ progenitors express significant levels of integrins, and E-cadherin-typically lost during EMT-is highly upregulated specifically in ISCs and EBs. Thus, the Esg-regulated differentiation of Drosophila ISCs only partially resembles a mesenchymal-to-epithelial transition (MET) (Korzelis, 2014).

    Esg's role in ISC maintenance nicely parallels the functions of other Snail family members in Drosophila and mammals. For instance, in Drosophila neuroblasts (neural stem cells), the Snail family member Worniu promotes self-renewal and represses neuronal differentiation. In mice, Snail family members have been associated with the regulation of the stem cell state in both normal and pathological conditions. For instance, mammary stem cells require the Snail family member Slug to retain their MaSC identity. Mouse Snai1 also represses the transition from the stem cell-like mitotically cycling trophoblast precursor cell to the endoreplicating trophoblast giant cell during rodent placental development. This process, which also requires a mitotic-to-endocycle switch upon differentiation, is strikingly similar to the role describe in this study for Esg in EC differentiation and its role during imaginal disc development (Korzelis, 2014).

    More interesting yet, mouse Snai1 is specifically expressed and required for stem cell maintenance in the crypts of the mouse intestine and expands the stem cell population when overexpressed. However, few studies highlight the target genes responsible for the function of Snail family members in stem cell maintenance. One example is from mouse muscle progenitors (myoblasts), where Snai1 and Snai2 repress expression from MyoD target promoters and this is required to maintain their progenitor state. The work presented in this study shows that Esg affects many aspects of the differentiation process and that it can form a transcriptional switch with one of the targets it represses (Pdm1) to balance self-renewal and differentiation in this stem cell lineage. Together, these studies suggest that the function of Snail family transcription factors as repressors of differentiation genes is ancient and widespread and may be an essential component in balancing self-renewal with differentiation in diverse animal stem cell lineages (Korzelis, 2014).

    Enteroendocrine cells support intestinal stem-cell-mediated homeostasis in Drosophila

    Intestinal stem cells in the adult Drosophila midgut are regulated by growth factors produced from the surrounding niche cells including enterocytes and visceral muscle. The role of the other major cell type, the secretory enteroendocrine cells, in regulating intestinal stem cells remains unclear. This study shows that newly eclosed scute loss-of-function mutant flies are completely devoid of enteroendocrine cells. These enteroendocrine cell-less flies have normal ingestion and fecundity but shorter lifespan. Moreover, in these newly eclosed mutant flies, the diet-stimulated midgut growth that depends on the insulin-like peptide 3 expression in the surrounding muscle is defective. The depletion of Tachykinin-producing enteroendocrine cells or knockdown of Tachykinin leads to a similar although less severe phenotype. These results establish that enteroendocrine cells serve as an important link between diet and visceral muscle expression of an insulin-like growth factor to stimulate intestinal stem cell proliferation and tissue growth (Amcheslavsky, 2014).

    Previous evidence shows that adult midgut mutant clones that have all the AS-C genes deleted are defective in EE formation while overexpression of scute (sc) or asense (ase) is sufficient to increase EE formation. Moreover, the Notch pathway with a downstream requirement of ase also regulates EE differentiation. To study the requirement of EEs in midgut homeostasis, attempts were made to delete all EEs by knocking down each of the AS-C transcripts using the ISC/EB driver esg-Gal4. The results show that sc RNAi was the only one that caused the loss of all EEs in the adult midgut. The esg-Gal4 driver is expressed in both larval and adult midguts, but the esg > sc RNAi larvae were normal while the newly eclosed adults had no EEs. Therefore, sc is likely required for all EE formation during metamorphosis when the adult midgut epithelium is reformed from precursors/stem cells (Amcheslavsky, 2014).

    The sc6/sc10-1 hemizygous mutant adults were also completely devoid of midgut EEs, while other hemizygous combinations including sc1, sc3B, and sc5 were normal in terms of EE number. Df(1)sc10-1 is a small deficiency that has both ac and sc uncovered. sc1 and sc3B each contain a gypsy insertion in far-upstream regions of sc, while sc5 and sc6 are 1.3 and 17.4 kb deletions, respectively, in the sc 3' regulatory region. The sc6/sc10-1 combination may affect sc expression during midgut metamorphosis and thus the formation of all adult EEs (Amcheslavsky, 2014).

    The atonal homolog 1 (Atoh1) is required for all secretory cell differentiation in mouse. However, esg-Gal4-driven atona; (ato) RNAi and the amorphic combination ato1/Df(3R)p13 showed normal EE formation. Nonetheless, older ato1/Df(3R)p13 flies exhibited a significantly lower increase of EE number, suggesting a role of ato in EE differentiation in adult flies (Amcheslavsky, 2014).

    In sc RNAi guts, the mRNA expression of allatostatin (Ast), allatostatin C (AstC), Tachykinin (Tk), diuretic hormone (DH31), and neuropeptide F (NPF) was almost abolished, consistent with the absence of all EEs. On the other hand, the mRNA expression of the same peptide genes in heads showed no significant change. Even though the EEs and regulatory peptides were absent from the midgut, the flies were viable and showed no apparent morphological defects. There was no significant difference in the number of eggs laid and the number of pupae formed from control and sc RNAi flies, suggesting that the flies probably have sufficient nutrient uptake to support the major physiological task of reproduction. However, when the longevity of these animals was examined, the EE-less flies after sc RNAi showed significantly shorter lifespan. In addition, when the number of EEs was increased in adult flies by esgGal4;tubGal80ts (esgts)-driven sc overexpression, an even shorter lifespan was observed. These results suggest that a balanced number of EEs is essential for the long-term health of the animal. Moreover, there may be important physiological changes in these EE-less flies that are yet to be uncovered, such as reduced intestinal growth described in detail below (Amcheslavsky, 2014).

    One of the phenotypic changes found for the sc RNAi/EE-less flies was that under normal feeding conditions, their midguts had a significantly narrower diameter than that of control midguts. When reared in poor nutrition of 1% sucrose, both wild-type (WT) and EE-less flies had thinner midguts. When reared in normal food, WT flies had substantially bigger midgut diameter, while EE-less flies had grown significantly less. The cross-section area of enterocytes in the EE-less midguts was smaller, suggesting that there is also a growth defect at the individual cell level (Amcheslavsky, 2014).

    A series of experiments showed that ingestion of food dye by the sc RNAi/EE-less flies was not lower than control flies. The measurement of food intake by optical density (OD) of gut dye contents also showed similar ingestion. The measurement of excretion by counting colored deposits and visual examination of dye clearing from guts showed that there was no significant change in food passage. The normal fecundity also suggested that the mutant flies likely had absorbed sufficient nutrient for reproduction. Nonetheless, another phenotype that was detected was a substantial reduction of intestinal digestive enzyme activities including trypsin, chymotrypsin, aminopeptidase, and acetate esterase. These enzyme activities exhibit strong reduction after starvation of WT flies. The EE-less flies therefore have a physiological response as if they experience starvation although they are provided with a normal diet (Amcheslavsky, 2014).

    A previous report has established that newly eclosed flies respond to nutrient availability by increasing ISC division that leads to a jump start of intestinal growth. When newly eclosed flies were fed on the poor diet of 1% sucrose, both WT and sc RNAi/EE-less guts had a very low number of p-H3-positive cells, which represent mitotic ISCs because ISCs are the only dividing cells in the adult midgut. When fed on normal diet, the WT guts had significantly higher p-H3 counts, but the sc RNAi/EE-less guts were consistently lower at all the time points. The sc6/sc10-1 hemizygous mutant combination exhibited a similarly lower mitotic activity on the normal diet (Amcheslavsky, 2014).

    When possible signaling defects were investigated in the EE-less flies,in addition to other gut peptide mRNAs, the level of Dilp3 mRNA was also found to be highly decreased in these guts while the head Dilp3 was normal. This is somewhat surprising, because Dilp3 is expressed not in the epithelium or EEs but in the surrounding muscle. Dilp3 promoter-Gal4-driven upstream activating sequence (UAS)-GFP expression (Dilp3 > GFP) was used to visualize the expression in muscle. Both control and sc RNAi under this driver showed normal muscle GFP expression, demonstrating that sc does not function within the smooth muscle to regulate Dilp3 expression. The esg-Gal4 and Dilp3-Gal4, and the control UAS-GFP samples showed the expected expression in both midgut precursors and surrounding muscles. When these combined Gal4 drivers were used to drive sc RNAi, the smooth muscle GFP signal was clearly reduced. These guts also exhibited no Prospero staining and overall fewer cells with small sizes as expected from esg > sc RNAi (Amcheslavsky, 2014).

    A previous report showed an increase of Dilp3 expression from the surrounding muscle in newly eclosed flies under a well-fed diet. This muscle Dilp3 expression precedes brain expression and is essential for the initial nutrient stimulated intestinal growth. The EE-less flies show similar growth and Dilp3 expression defects, suggesting that EE is a link between nutrient sensing and Dilp3 expression during this early growth phase (Amcheslavsky, 2014).

    WT and AS-C deletion (scB57) mutant clones in adult midguts did not exhibit a difference in their cell numbers. Moreover, esgts > sc RNAi in adult flies for 3 days but did not undergo a decrease of mitotic count or EE number. Together, these results suggest that sc is not required directly in ISC for proliferation, and they imply that the ISC division defects observed in the sc mutant/EE-less flies is likely due to the loss of EEs. To investigate this idea further, the esgts > system to was used to up- and downshift the expression of sc at various time points, and the correlation of sc expression, EE number, and ISC mitotic activity were measured. The overexpression of sc after shifting to 29°C for a few days correlated with increased EE number, expression of gut peptides, and increased ISC activity. Then, flies were downshifted back to room temperature to allow the Gal80ts repressor to function again. The sc mRNA expression was quickly reduced within 2 days and remained low for 4 days. Although there was no working antibody to check the Sc protein stability, the expression of a probable downstream gene phyllopod showed the same up- and downregulation, revealing that Sc function returned to normal after the temperature downshift. Meanwhile, the number of Pros+ cells and p-H3 count remained higher after the downshift. Therefore, the number of EEs, but not sc mRNA or function, correlates with ISC mitotic activity (Amcheslavsky, 2014).

    Another experiment that was independent of sc expression or expression in ISCs was performed. The antiapoptotic protein p35 was driven by the pros-Gal4 driver, which is expressed in a subset of EEs in the middle and posterior midgut. This resulted in a significant albeit smaller increase in EE number and a concomitant increase in mitotic activity, which was counted only in the middle and posterior midgut due to some EC expression of this driver in the anterior region. Therefore, the different approaches show consistent correlation between EE number and ISC division (Amcheslavsky, 2014).

    Dilp3 expression was significantly although modestly increased in flies that had increased EE number after sc overexpression, similar to that observed in fed versus fasted flies. Whether Dilp3 was functionally important in this EE-driven mitotic activity was tested. Flies were generated that contained a ubiquitous driver with temperature controlled expression, i.e., tub-Gal80ts/UAS-sc; tub-Gal4/UAS-Dilp3RNAi. These fly guts showed a significantly lower number of p-H3+ cells than that in the tub-Gal80ts/UAS-sc; tub-Gal4/+ control flies. These results demonstrate that the EE-regulated ISC division is partly dependent on Dilp3. The expression of an activated insulin receptor by esg-Gal4 could highly increase midgut proliferation, and this effect was dominant over the loss of EEs after scRNAi, which is consistent with an important function of insulin signaling in the midgut (Amcheslavsky, 2014).

    Normally hatched flies did not lower their EE number after esgts > sc RNAi, perhaps due to redundant function with other basic-helix-loop-helix proteins in adults. The expression of proapoptotic proteins by the prosts-Gal4 also could not reduce the EE number. Thus other drivers were screened and a Tk promoter Gal4 (Tk-Gal4) was identifed that had expression recapitulating the Tk staining pattern representing a subset of EEs. More importantly, when used to express the proapoptotic protein Reaper (Rpr), this driver caused a significant reduction in the EE number, Tk and Dilp3 mRNA, and mitotic count. The Tk-Gal4-driven expression of another proapoptotic protein, Hid, caused a less efficient killing of EEs and subsequently no reduction of p-H3 count. The knockdown of Tk itself by Tk-Gal4 also caused significant reduction of p-H3 count. A previous report revealed the expression by antibody staining of a Tk receptor (TkR86C) in visceral muscles, and the knockdown of TkR86C in smooth muscle by Dilp3-Gal4 or Mef2-Gal4 showed a modest but significant decrease in ISC proliferation. There was a concomitant reduction of Dilp3 mRNA in guts of all these experiments, while the head Dilp3 mRNA had no significant change in all these experiments. As a comparison, TkR99D or NPFR RNAi did not show the same consistent defect (Amcheslavsky, 2014).

    In conclusion, this study has shown that among the AS-C genes, sc is the one essential for the formation of all adult midgut EEs and is probably required during metamorphosis when the midgut is reformed. In newly eclosed flies, EEs serve as a link between diet-stimulated Dilp3 expression in the visceral muscle and ISC proliferation. Depletion of Tk-expressing EEs caused similar Dilp3 expression and ISC proliferation defects, although the defects appeared to be less severe than that in the sc RNAi/EE-less guts. The results together suggest that Tk-expressing EEs are part of the EE population required for this regulatory circuit. The approach reported in this study has established the Drosophila midgut as a model to dissect the function of EEs in intestinal homeostasis and whole-animal physiology (Amcheslavsky, 2014).

    Haemocytes control stem cell activity in the Drosophila intestine

    Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes were recruited to the intestine and secreted the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switched their response to DPP by inducing expression of Thickveins, a second type I receptor that had previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promoted infection resistance, but also contributed to the development of intestinal dysplasia in ageing flies. The study proposes that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

    Coordination of stem cell activity with inflammatory responses is critical for regeneration and homeostasis of barrier epithelia. The temporal sequence of cell interactions during injury-induced regeneration is only beginning to be understood. This study shows that intestinal stem cells (ISCs) are regulated by macrophage-like haemocytes during the early phase of regenerative responses of the Drosophila intestinal epithelium. On tissue damage, haemocytes are recruited to the intestine and secrete the BMP homologue DPP, inducing ISC proliferation by activating the type I receptor Saxophone and the Smad homologue SMOX. Activated ISCs then switch their response to DPP by inducing expression of Thickveins, a second type I receptor that has previously been shown to re-establish ISC quiescence by activating MAD. The interaction between haemocytes and ISCs promotes infection resistance, but also contributes to the development of intestinal dysplasia in ageing flies. It is proposed that similar interactions influence pathologies such as inflammatory bowel disease and colorectal cancer in humans (Ayyaz, 2015).

    The results extend the current model for the control of epithelial regeneration in the wake of acute infections in the Drosophila intestine. It is proposed that the control of ISC proliferation by haemocyte-derived DPP integrates with the previously described regulation of ISC proliferation by local signals from the epithelium and the visceral muscle, allowing precise temporal control of ISC proliferation in response to tissue damage, inflammation and infection (Ayyaz, 2015).

    The association of haemocytes with the intestine is extensive, and can be dynamically increased on infection or damage. In this respect, the current observations parallel the invasion of subepithelial layers of the vertebrate intestine by blood cells that induce proliferative responses of crypt stem cells during infection. A role for macrophages and myeloid cells in promoting tissue repair and regeneration has been described in adult salamanders and in mammals, where TGFβ ligands secreted by these immune cells can inhibit ISC proliferation, but can also contribute to tumour progression. The results provide a conceptual framework for immune cell/stem cell interactions in these contexts (Ayyaz, 2015).

    The observation that DPP/SAX/SMOX signalling is required for UPD-induced proliferation of ISCs suggests that SAX/SMOX signalling cooperates with JAK/STAT and EGFR signalling in the induction of ISC proliferation. Accordingly, while constitutive activation of EGFR/RAS or JAK/STAT signalling in ISCs is sufficient to promote ISC proliferation cell autonomously, this study found that this partially depends on Smox. Even in these gain-of-function conditions, ISC proliferation can thus be fully induced only in the presence of basal SMOX activity. As short-term overexpression of DPP in haemocytes does not induce ISC proliferation, it is further proposed that DPP/SAX/SMOX signalling can activate ISCs only when JAK/STAT and/or EGFR signalling are activated in parallel. However, long-term overexpression of DPP in haemocytes results in increased ISC proliferation, suggesting that chronic activation of immune cells disrupts normal signalling mechanisms and results in ISC activation even in the absence of tissue damage (Ayyaz, 2015).

    BMP TGFβ signalling pathways are critical for metazoan growth and development and have been well characterized in flies. Multiple ligands, receptors and transcription factors with highly context-dependent interactions and function have been described. This complexity is reflected by the sometimes conflicting studies exploring DPP/TKV/SAX signalling in the adult intestine. These studies consistently highlight two important aspects of BMP signalling in the adult Drosophila gut: ISCs can undergo opposite proliferative responses to BMP signals; and there are various sources of DPP that differentially influence ISC function in specific conditions. By characterizing the temporal regulation of BMP signalling activity in ISCs, the results resolve some of these conflicts: it is proposed that early in the regenerative response, haemocyte-derived DPP triggers ISC proliferation by activating SAX/SMOX signalling, and ISC quiescence is re-established by muscle-derived DPP as soon as TKV becomes expressed. Of note, some of the conflicting conclusions described in the literature may have originated from problems with the genetic tools used in some studies. This study have used two independent RNAi lines (BL25782 and BL33618) that effectively decrease dpp mRNA levels in haemocytes when expressed using HmlΔ::Gal4 (Ayyaz, 2015).

    The close association of haemocytes with the type IV collagen Viking suggests that the stimulation of ISC proliferation by haemocyte-derived DPP may also be controlled at the level of ligand availability, as suggested previously for DPP from other sources. The regulation of SAX/SMOX signalling by DPP observed in this study is surprising, but consistent with earlier reports showing that SAX can respond to DPP in certain contexts. Biochemical studies have suggested that heterotetrameric complexes between the type II receptor PUNT and the type I receptors SAX and TKV can bind DPP, and complexes with TKV/TKV homodimers preferentially bind DPP, and complexes with SAX/SAX homodimers preferentially bind GBB. In the absence of TKV, SAX has been proposed to sequester GBB, shaping the GBB activity gradient, but to fail to signal effectively. Expression of GBB in the midgut epithelium has recently been described, and ligand heterodimers between GBB and DPP are well established. Consistent with earlier reports, this study found that GBB knockdown in ECs significantly reduces ISC proliferation in response to infection. Complex interactions between haemocyte-derived DPP, epithelial GBB, and ISC-expressed SAX, PUNT and TKV thus probably shape the response of ISCs to damage, and will be an interesting area of further study (Ayyaz, 2015).

    Similar complexities exist in the regulation of transcription factors by SAX and TKV. Canonically, SMOX is regulated by Activin ligands (Activin, Dawdle, Myoglianin and maybe more), and the type I receptor Baboon. This study has tested the role of Activin and Dawdle in ISC regulation, and, in contrast to DPP, this study could not detect a requirement for these factors in the induction of ISC proliferation after Ecc15 infection. Furthermore, the data establish a requirement for haemocyte-derived DPP as well as for SAX expression in ISCs in the nuclear translocation of SMOX after a challenge. This study thus indicates that in this context, SAX responds to DPP and regulates SMOX. Regulation of SMOX by SAX has been described before, yet SAX is also known to promote MAD phosphorylation, but only in the presence of TKV. Consistent with such observations, this study has detected MAD phosphorylation in ISCs only in the late recovery phase on bacterial infection, when TKV is simultaneously induced in ISCs. During this recovery phase, ISCs maintain high SAX expression, but SMOX nuclear localization is not detected anymore, suggesting that SAX cannot activate SMOX in the presence of TKV, and might actually divert signals towards MAD instead. The data also suggest that Medea (the Drosophila SMAD4 homologue) is not required for SMOX activity. Although surprising, this observation is consistent with recent reports that SMAD proteins in mammals can translocate into the nucleus and activate target genes in a SMAD4-independent manner. The specific signalling readouts in ISCs when these cells are exposed to various BMP ligands and are expressing different combinations of receptors are thus likely to be complex (Ayyaz, 2015).

    The current findings demonstrate that the control of ISC proliferation by haemocyte-derived DPP is critical for tolerance against enteropathogens, but contributes to ageing-associated epithelial dysfunction, highlighting the importance of tightly controlled interactions between blood cells and stem cells in this tissue. Nevertheless, where haemocytes themselves are required for normal lifespan, loss of haemocyte-derived DPP does not impact lifespan. One interpretation of this finding is that beneficial (improved gut homeostasis) and deleterious (for example, reduced immune competence of the gut epithelium) consequences of reduced haemocyte-derived DPP cancel each other out over the lifespan of the animal. It will be interesting to test this hypothesis in future studies. Ageing is associated with systemic inflammation, and a role for immune cells in promoting inflammation in ageing vertebrates has been proposed. In humans, recruitment of immune cells to the gut is required for proper stem cell proliferation in response to luminal microbes, and prolonged inflammatory bowel disease further contributes to cancer development. It is thus anticipated that conserved macrophage/stem cell interactions influence the aetiology and progression of such diseases. The data confirm a role for haemocytes in age-related intestinal dysplasia in the fly intestine, and provide mechanistic insight into the causes for this deregulation. It can be anticipated that similar interactions between macrophages and intestinal stem cells may contribute to the development of IBDs, intestinal cancers, and general loss of homeostasis in the ageing human intestine (Ayyaz, 2015).

    Drosophila Pez acts in Hippo signaling to restrict intestinal stem cell proliferation

    The conserved Hippo signaling pathway acts in growth control and is fundamental to animal development and oncogenesis. Hippo signaling has also been implicated in adult midgut homeostasis in Drosophila. Regulated divisions of intestinal stem cells (ISCs), giving rise to an ISC and an enteroblast (EB) that differentiates into an enterocyte (EC) or an enteroendocrine (EE) cell, enable rapid tissue turnover in response to intestinal stress. The damage-related increase in ISC proliferation requires deactivation of the Hippo pathway and consequential activation of the transcriptional coactivator Yorkie (Yki) in both ECs and ISCs. This study identified Pez, an evolutionarily conserved FERM domain protein containing a protein tyrosine phosphatase (PTP) domain, as a novel binding partner of the upstream Hippo signaling component Kibra. Pez function (but not its PTP domain) is essential for Hippo pathway activity specifically in the fly midgut epithelium. Thus, Pez displays a tissue-specific requirement and functions as a negative upstream regulator of Yki in the regulation of ISC proliferation (Poernbacher, 2012).

    The WW domain protein Kibra has recently been shown to function as a tumor suppressor in the Hippo pathway. Because Kibra is an adaptor molecule, attempts were made to identify physical binding partners of Kibra to further explore upstream Hippo signaling. Affinity purification-mass spectrometry (AP-MS) analysis with Kibra as bait identified Pez as a novel interaction partner of Kibra in Drosophila cultured cells. The same result was recently obtained in a large-scale proteomic study of Drosophila cultured cells. The binding between Pez and Kibra was confirmed by reciprocal coimmunoprecipitation (co-IP) experiments with epitope-tagged proteins. Furthermore, a yeast two-hybrid (Y2H) experiment revealed that the Kibra-Pez interaction is robust and direct (Poernbacher, 2012).

    To address a possible function of Pez in the Hippo pathway, two loss-of-function alleles of Pez that were generated by different methods. Pez1 is an EMS-induced allele resulting in an early premature translational stop codon. Pez2 was generated by imprecise excision of the P element P{GawB}NP4748, removing most of the Pez coding sequence. Homozygotes for either Pez allele as well as heteroallelic Pez1/Pez2 flies are viable but smaller than controls. Combinations of the Pez alleles with the deficiency Df(2L)ED384 uncovering the Pez locus are also viable and cause a similar reduction in body size as the homozygous or heteroallelic combinations. One copy of a GFP-tagged Pez genomic rescue construct (gPez) restores normal body size. Therefore, both Pez1 and Pez2 are likely to represent strong or null alleles. For further experiments, heteroallelic Pez1/Pez2 flies were used as Pez mutant flies (Poernbacher, 2012).

    In addition to their reduced body size, Pez mutant flies exhibit a developmental delay of 2 days and decreased fertility, all hallmarks of starvation. Pez mutant larvae are small and have decreased triglyceride (TAG) stores and increased expression of the starvation marker genes lipase-3 and 4E-BP. Clones of Pez mutant cells in larval fat bodies did not affect lipid droplets, thus excluding a fat body-autonomous requirement for Pez in lipid metabolism. Surprisingly, overexpression of Drosophila Pez in the developing eye or wing decreased the size of the adult organs, indicating that Pez restricts growth rather than promoting it. It is proposed that the starvation-like phenotype of Pez mutants is due to indirect effects on metabolism arising from a failure in nutrient utilization. Clones of Pez mutant cells in wing imaginal discs did not show growth defects in comparison to their corresponding wild-type sister clones. However, Pez mutant flies exhibit hyperplasia and extensive multilayering of the adult midgut epithelium. One copy of gPez restores normal tissue architecture. The structure of the larval midgut epithelium, as well as that of the other larval and adult epithelia, is not disturbed in Pez mutants. Thus, Pez specifically functions to restrict growth of the adult midgut epithelium (Poernbacher, 2012).

    The Pez protein contains two conserved structural elements: an amino-terminal FERM domain (band 4.1-ezrin-radixin- moesin family of adhesion molecules) and a carboxyterminal protein tyrosine phosphatase (PTP) domain. A truncated version of the protein lacking the FERM domain (DFERM-Pez) or a phosphatase-dead protein (PezPD) still rescued the Pez mutant gut phenotype when overexpressed in ECs. However, overexpression of DFERM-Pez in the developing wing failed to decrease wing size, whereas overexpression of PezPD or of a truncated protein lacking the PTP domain (DPTP-Pez) caused a similar phenotype as overexpression of wild-type Pez, suggesting that the FERM domain is required for the growth-regulatory function of endogenous Pez but becomes dispensable when DFERM-Pez is overexpressed in ECs. In contrast, the potential phosphatase activity of Pez is clearly not needed for its function in growth control (Poernbacher, 2012).

    Two other FERM domain proteins, Merlin (Mer) and Expanded (Ex), act in upstream Hippo signaling to control organ size in Drosophila. Together with the WW domain protein Kibra, Ex and Mer constitute the KEM complex that assembles at the apical junction of epithelial cells and regulates the core Hippo pathway kinase cassette. Overexpression of Kibra, Ex, or Mer in ECs of Pez mutant flies significantly suppressed the Pez gut phenotypes. Thus, Pez is not an essential mediator of Hippo signaling downstream of the KEM complex. Mer and Ex did not detectably coimmunoprecipitate with Pez in Drosophila S2 cells. However, Kibra and Pez coimmunoprecipitated and colocalized in S2 cells. This was dependent on the first WW domain of Kibra, whereas the FERM and PTP domains of Pez as well as two potential ligands of WW domains, a PPPY motif and a PPSGY motif, in the central linker region of Pez were dispensable. A fragment encompassing a proline-rich stretch of Pez (amino acids 368-627; PezPro) was sufficient for the binding to Kibra, whereas the remaining linker region (amino acids 622-967; PezLink) did not bind Kibra. Importantly, knockdown of Kibra via Myo1A-Gal4 caused mild overgrowth of the adult midgut epithelium, and overexpressed Kibra recruited gPez-GFP from the cell cortex of ECs into cytoplasmic punctae. The subcellular localizations of overexpressed Kibra, Ex, or Mer were not affected when Pez was absent (Poernbacher, 2012).

    It is concluded that Pez and Kibra function together in a protein complex to regulate Hippo signaling in adult midgut ECs. The results establish that the Drosophila Pez protein acts as a component of upstream Hippo signaling, restricts transcriptional activity of Yki in epithelial cells of the adult midgut, and plays a crucial role in the control of ISC proliferation. Importantly, the involvement of Hippo signaling in intestinal regeneration is conserved in the mammalian system ] (Poernbacher, 2012).

    The two mammalian homologs of Drosophila Pez are the widely expressed, cytosolic nonreceptor tyrosine phosphatases PTPD1/PTPN21 and PTPD2/PTP36/PTPN14/Pez. All three proteins share a similar domain structure including the well-conserved terminal FERM and PTP domains. The central region shows extensive sequence divergence but it contains several shorter regions of conservation that may function as adaptors in signal transduction. PTPD1 is a component of a cortical scaffold complex nucleated by focal adhesion kinase (FAK) and thus regulates a proliferative signaling pathway through a scaffolding function. PTPD2 has been implicated in the regulation of cell adhesion, as an inducer of TGF-β signaling, and in lymphatic development of mammals and choanal development of humans. Interestingly, PTPD2 is a potential tumor suppressor, based on sporadic mutations in breast cancer cells and colorectal cancer cells. It is tempting to speculate that mammalian PTPD2 shares the function of its fly homolog as a component of Hippo signaling that restrains the oncogenic potential of gut regeneration (Poernbacher, 2012).

    Increased mitochondrial biogenesis preserves intestinal stem cell homeostasis and contributes to longevity in Indy mutant flies

    The Drosophila Indy (I'm not dead yet) gene encodes a plasma membrane transporter of Krebs cycle intermediates, with robust expression in tissues associated with metabolism. Reduced INDY alters metabolism and extends longevity in a manner similar to caloric restriction (CR); however, little is known about the tissue specific physiological effects of INDY reduction. This study focused on the effects of INDY reduction in the Drosophila midgut due to the importance of intestinal tissue homeostasis in healthy aging and longevity. The expression of Indy mRNA in the midgut changes in response to aging and nutrition. Genetic reduction of Indy expression increases midgut expression of the mitochondrial regulator spargel/dPGC-1, which is accompanied by increased mitochondrial biogenesis and reduced reactive oxygen species (ROS). These physiological changes in the Indy mutant midgut preserve intestinal stem cell (ISC) homeostasis and are associated with healthy aging. Genetic studies confirm that dPGC-1 mediates the regulatory effects of INDY, as illustrated by lack of longevity extension and ISC homeostasis in flies with mutations in both Indy and dPGC1. These data suggest INDY may be a physiological regulator that modulates intermediary metabolism in response to changes in nutrient availability and organismal needs by modulating dPGC-1 (Rogers, 2014).

    Caloric restriction (CR) extends lifespan in nearly all species and promotes organismal energy balance by affecting intermediary metabolism and mitochondrial biogenesis. Interventions that alter intermediary metabolism are though to extend longevity by preserving the balance between energy production and free radical production Indy (I'm Not Dead Yet) encodes a plasma membrane protein that transports Krebs' cycle intermediates across tissues associated with intermediary metabolism. Reduced Indy-mediated transport extends longevity in worms and flies by decreasing the uptake and utilization of nutrients and altering intermediate nutrient metabolism in a manner similar to CR. Furthermore, it was shown that caloric content of food directly affects Indy expression in fly heads and thoraces, suggesting a direct relationship between INDY and metabolism (Rogers, 2014 and references therein).

    dPGC-1/spargel is the Drosophila homolog of mammalian PGC-1, a transcriptional co-activator that promotes mitochondrial biogenesis by increasing the expression of genes encoding mitochondrial proteins. Upregulation of dPGC-1 is a hallmark of CR-mediated longevity and is thought to represent a response mechanism to compensate for energetic deficits caused by limited nutrient availability. Increases in dPGC-1 preserve mitochondrial functional efficiency without consequential changes in ROS. Previous analyses of Indy mutant flies revealed upregulation of mitochondrial biogenesis mediated by increased levels of dPGC-1 in heads and thoraces (Rogers, 2014 and references therein).

    Recently, dPGC-1 upregulation in stem and progenitor cells of the digestive tract was shown to preserve intestinal stem cell (ISC) proliferative homeostasis and extend lifespan. The Drosophila midgut is regenerated by multipotent ISCs, which replace damaged epithelial tissue in response to injury, infection or changes in redox environment. Low levels of reactive oxygen species (ROS) maintain stemness, self-renewal and multipotency in ISCs; whereas, age-associated ROS accumulation induces continuous activation marked by ISC hyper-proliferation and loss of intestinal integrity (Rogers, 2014 and references therein).

    This study describes a role for Indy as a physiological regulator that modulates expression in response to changes in nutrient availability. This is illustrated by altered Indy expression in flies following changes in caloric content and at later ages suggesting that INDY-mediated transport is adjusted in an effort to meet energetic demands. Further, role was characterized for dPGC-1 in mediating the downstream regulatory effects of INDY reduction, such as the observed changes in Indy mutant mitochondrial physiology, oxidative stress resistance and reduction of ROS levels. Longevity studies support a role for dPGC-1 as a downstream effector of Indy mutations as shown by overlapping longevity pathways and absence of lifespan extension without wild-type levels of dPGC-1. These findings show that Indy mutations affect intermediary metabolism to preserve energy balance in response to altered nutrient availability, which by affecting the redox environment of the midgut promotes healthy aging (Rogers, 2014).

    Reduction of Indy gene activity in fruit flies, and homologs in worms, extends lifespan by altering energy metabolism in a manner similar to caloric restriction (CR). Indy mutant flies on regular food share many characteristics with CR flies and do not have further longevity extension when aged on a CR diet. Furthermore, mINDY-/- mice on regular chow share 80% of the transcriptional changes observed in CR mice, supporting a conserved role for INDY in metabolic regulation that mimics CR and promotes healthy aging. This study shifted from systemic to the tissue specific effects of INDY reduction, focusing on the midgut due to the high levels of INDY protein expression in wild type flies and the importance of regulated intestinal homeostasis during aging. The evidence supports a role for INDY as a physiological regulator that senses changes in nutrient availability and alters mitochondrial physiology to sustain tissue-specific energetic requirements (Rogers, 2014).

    The age-associated increase in midgut Indy mRNA levels that can be replicated by manipulations that accelerate aging such as increasing the caloric content of food or exposing flies to paraquat. Conversely, it was also shown that CR decreases Indy mRNA in control midgut tissues, which is consistent with previous findings in fly muscle and mouse liver. Diet-induced variation in midgut Indy expression suggests that INDY regulates intermediary metabolism by modifying citrate transport to meet tissue or cell-specific bioenergetic needs. Specifically, as a plasma membrane transporter INDY can regulate cytoplasmic citrate, thereby affecting fat metabolism, respiration, and via conversion to malate, the TCA cycle. Reduced INDY-mediated transport activity in the midgut could prevent age-related ISC-hyperproliferation by decreasing the available energy needed to initiate proliferation, thereby preserving tissue function during aging. This is supported by findings that nutrient availability affects ISC proliferation in adult flies and that CR can affect stem cell quiescence and activation (Rogers, 2014).

    One of the hallmarks of CR-mediated longevity extension is increased mitochondrial biogenesis mediated by dPGC-1 (Spargel). Increased dPGC-1 levels and mitochondrial biogenesis have been described in the muscle of Indy mutant flies, the liver of mIndy-/- mice, and this study describes it in the midgut of Indy mutant flies. One possible mechanism for these effects can be attributed to the physiological effects of reduced INDY transport activity. Reduced INDY-mediated transport activity could lead to reduced mitochondrial substrates, an increase in the ADP/ATP ratio, activation of AMPK, and dPGC-1 synthesis. This is consistent with findings in CR flies and the livers of mINDY-/- mice. This study's analysis of mitochondrial physiology in the Indy mutant midgut shows upregulation of respiratory proteins, maintenance of mitochondrial potential and increased mitochondrial biogenesis, all of which are signs of enhanced mitochondrial health. The observed increase in dPGC-1 levels in Indy mutant midgut therefore appears to promote mitochondrial biogenesis and functional efficiency, representing a protective mechanism activated in response to reduced energy availability (Rogers, 2014).

    Genetic interventions that conserve mitochondrial energetic capacity have been shown to maintain a favorable redox state and regenerative tissue homeostasis. This is particularly beneficial in the fly midgut, which facilitates nutrient uptake, waste removal and response to bacterial infection. Indy mutant flies have striking increases in the steady-state expression of the GstE1 and GstD5 ROS detoxification genes. As a result, any increase in ROS levels, whether from mitochondrial demise or exposure to external ROS sources can be readily metabolized to prevent accumulation of oxidative damage. Such conditions not only promote oxidative stress resistance, but also preserve ISC homeostasis as demonstrated by consistent proliferation rates throughout Indy mutant lifespan and preserved intestinal architecture in aged Indy mutant midguts. Thus, enhanced ROS detoxification mechanisms induced by Indy reduction and subsequent elevation of dPGC-1 contributes to preservation of ISC functional efficiency, and may be a contributing factor to the long-lived phenotype of Indy mutant flies (Rogers, 2014).

    Several lines of evidence indicate that INDY and dPGC-1 are part of the same regulatory network in the midgut, in which dPGC-1 functions as a downstream effector of INDY. The similarity between dPGC-1 mRNA levels and survivorship of flies overexpressing dPGC-1 in esg-positive cells and Indy mutant flies suggests that Indy and dPGC-1 interact to extend lifespan. This is further supported by the lack of additional longevity extension when dPGC-1 is overexpressed in esg-positive cells of Indy mutant flies. Moreover, hypomorphic dPGC-1 flies in an Indy mutant background are similar to controls with respect to life span, declines in mitochondrial activity and ROS-detoxification. Together, these data suggest that dPGC-1 must be present to mediate the downstream physiological benefits and lifespan extension of Indy mutant flies (Rogers, 2014).

    There are some physiological differences between the effects of Indy mutation and dPGC-1 overexpression in esg-positive cells. While Indy mutant flies are less resistant to starvation and more resistant to paraquat, a recent report showed that overexpressing dPGC-1 in esg-positive cells has no effect on resistance to starvation or oxidative stress. Additionally, mice lacking skeletal muscle PGC-1α were found to lack mitochondrial changes associated with CR but still showed other CR-mediated metabolic changes. In the fly INDY is predominantly expressed in the midgut, fat body and oenocytes, though there is also low level expression in the malpighian tubules, salivary glands, antenae, heart and female follicle cell membranes. Thus, the effects of INDY on intermediary metabolism and longevity could be partially independent from dPGC-1 or related to changes in tissues other than the midgut (Rogers, 2014).

    This study suggests that INDY may function as a physiological regulator of mitochondrial function and related metabolic pathways, by modulating nutrient flux in response to nutrient availability and energetic demands. Given the localization of INDY in metabolic tissues, and importance of regulated tissue homeostasis during aging, these studies highlight INDY as a potential target to improved health and longevity. Reduced Indy expression causes similar physiological changes in flies, worms and mice indicating its regulatory role would be conserved. Further work should examine the interplay between Indy mutation and metabolic pathways, such as insulin signaling, which have been shown to promote stem cell maintenance and healthy aging in flies and mice. In doing so, the molecular mechanisms, which underlie Indy mutant longevity may provide insight for anti-aging therapies (Rogers, 2014).

    Origin and dynamic lineage characteristics of the developing Drosophila midgut stem cells
    Proliferating intestinal stem cells (ISCs) generate all cell types of the Drosophila midgut, including enterocytes, endocrine cells, and gland cells (e.g., copper cells), throughout the lifetime of the animal. Among the signaling mechanisms controlling the balance between ISC self-renewal and the production of different cell types, Notch (N) plays a pivotal role. This study investigates the emergence of ISCs during metamorphosis and the role of N in this process. Precursors of the Drosophila adult intestinal stem cells (pISCs) can be first detected within the pupal midgut during the first hours after onset of metamorphosis as motile mesenchymal cells. pISCs perform 2-3 rounds of parasynchronous divisions. The first mitosis yields only an increase in pISC number. During the following rounds of mitosis, dividing pISCs give rise to more pISCs, as well as the endocrine cells that populate the midgut of the eclosing fly. Enterocytes do not appear among the pISC progeny until around the time of eclosion. The "proendocrine" gene prospero (pros), expressed from mid-pupal stages onward in pISCs, is responsible to advance the endocrine fate in these cells; following removal of pros, pISCs continue to proliferate, but endocrine cells do not form. Conversely, the onset of N activity that occurs around the stage when pros comes on restricts pros expression among pISCs. Loss of N abrogates proliferation and switches on an endocrine fate among all pISCs. These results suggest that a switch depending on the activity of N and pros acts at the level of the pISC to decide between continued proliferation and endocrine differentiation (Takashima, 2016).

    Stem-cell-specific endocytic degradation defects lead to intestinal dysplasia in Drosophila

    UV radiation resistance-associated gene (UVRAG) is a tumor suppressor involved in autophagy, endocytosis and DNA damage repair, but how its loss contributes to colorectal cancer is poorly understood. This study shows that UVRAG deficiency in Drosophila intestinal stem cells leads to uncontrolled proliferation and impaired differentiation without preventing autophagy. As a result, affected animals suffer from gut dysfunction and short lifespan. Dysplasia upon loss of UVRAG is characterized by the accumulation of endocytosed ligands and sustained activation of STAT and JNK signaling, and attenuation of these pathways suppresses stem cell hyperproliferation. Importantly, the inhibition of early (dynamin-dependent) or late (Rab7-dependent) steps of endocytosis in intestinal stem cells also induces hyperproliferation and dysplasia. These data raise the possibility that endocytic, but not autophagic, defects contribute to UVRAG-deficient colorectal cancer development in humans (Nagy, 2016).

    UVRAG encodes a homolog of yeast Vps38 in metazoans. UVRAG/Vps38 and Atg14 are mutually exclusive subunits of two different Vps34 lipid kinase complexes, both of which contain Vps34, Vps15 and Atg6/Beclin 1. It is well established that Vps38 is required for endosome maturation and vacuolar and lysosomal protein sorting, whereas Atg14 is specific for autophagy in yeast. However, the function of UVRAG is much more controversial in mammalian cells. Although UVRAG was originally found to have dual roles in autophagy through promotion of autophagosome formation and fusion with lysosomes in various cultured cell lines based on, predominantly, overexpression experiments, recent reports have described that autophagosomes are normally generated and fused with lysosomes in the absence of UVRAG in cultured mammalian (HeLa) cells and in the Drosophila fat body (Nagy, 2016 and references therein).

    The discoveries of UVRAG mutations in colorectal cancer cells, and that its overexpression increases autophagy and suppresses the proliferation of certain cancer cell lines, altogether suggest that this gene functions as an autophagic tumor suppressor. Such a role for UVRAG is thought to be related to its binding to Beclin 1, a haploinsufficient tumor suppressor gene required for autophagy. UVRAG appears to play roles similar to yeast Vps38 in the Drosophila fat body, and developing eye and wing: its loss leads to the accumulation of multiple endocytic receptors and ligands in an endosomal compartment, impaired trafficking of Lamp1 and Cathepsin L to the lysosome, and defects in the biogenesis of lysosome-related pigment granules. However, whether this gene is also required for the maintenance of intestinal homeostasis in Drosophila was unclear because the loss of UVRAG did not lead to uncontrolled cell proliferation in the developing eye or wing according to these reports. The current results showing that Uvrag deficiency causes intestinal dysplasia suggest that this gene is also important for the proper functioning of the adult gut in Drosophila (Nagy, 2016). A surprising aspect of this work is that UVRAG appears to function independently of autophagy in the intestine. There are other lines of evidence that also support that UVRAG has a more important role in endocytic maturation than in autophagy. First, it has been shown that truncating mutations in UVRAG that are associated with microsatellite-unstable colon cancer cell lines do not disrupt autophagy. Second, UVRAG depletion in HeLa cells does not prevent the formation or fusion of autophagosomes with lysosomes, but it does interfere with Egfr degradation. Third, a very recent paper has shown that overexpression of the colorectal-cancer-associated truncated form of UVRAG promotes tumorigenesis independently of autophagy status, that is, both in control and Atg5-knockout cells. That paper, again, relied on the overexpression of full-length or truncated forms of UVRAG, rather than the analysis of cancer-related mutations of the endogenous locus. Fourth, the endocytic function of UVRAG has been found to be required for developmental axon pruning that is independent of autophagy in Drosophila (Nagy, 2016 and references therein).

    The results of this study indicate that UVRAG loss is accompanied with the sustained activation of JNK and STAT signaling in ISCs and EBs, and that these pathways are required for dysplasia in this setting. Sustained activation of these signaling routes is likely to be connected to the disruption of endocytic flux in the absence of UVRAG, because inhibiting endocytic uptake or degradation through dominant-negative dynamin expression or RNAi of Rab7, respectively, also leads to intestinal dysplasia. It is worth noting that the effects of inhibiting Shibire/dynamin function led to a much more severe hyperproliferation of ISCs and early death of animals. In line with this, the loss of early endocytic regulators, such as Rab5, in the developing eye causes overproliferation of cells and lethality during metamorphosis. Although eye development is not perturbed by the loss of the late endocytic regulators UVRAG or Rab7, these proteins are clearly important for controlling ISC proliferation and differentiation (Nagy, 2016).

    A recent paper shows that hundreds of RNAi lines cause the expansion of the esg-GFP compartment in 1-week-old animals, which might be due to an unspecific ISC stress response in some cases. However, several lines of evidence support that impaired UVRAG-dependent endocytic degradation is specifically required to prevent intestinal dysplasia. First of all, activation of JNK stress signaling in esg-GFP-positive cells induces short-term ISC proliferatio, and almost all stem cells are lost through apoptosis by the 2- to 3-week age, the time when the Uvrag-mutant phenotype becomes obvious. In fact, UVRAG loss resembles an early-onset age-associated dysplasia that is normally observed in old (30-60 days) flies and involves the simultaneous activation of both JNK and STAT signaling. Second, UVRAG RNAi in ISCs and EBs leads to paracrine activation of the cytokine Unpaired3 in enterocytes, one of the hallmarks of niche appropriation by Notch-negative tumors. However, autocrine expression of the Unpaired proteins and JNK activation is observed in Uvrag-knockdown cells, unlike in Notch-negative tumors, and EBs with active Notch signaling accumulate in the absence of UVRAG, so the two phenotypes are clearly different. Third, it is the loss of autophagy that could be expected to mimic a stress response and perhaps induce stem cell tumors, but this does not seem to be the case – ISCs with Atg5 or Atg14 RNAi proliferate less in 3-week-old animals and an overall decrease of the esg-GFP compartment is seen, as opposed to the Uvrag-deletion phenotype (Nagy, 2016).

    Taken together, this work indicates that endocytic maturation and degradation serves to prevent early-onset intestinal dysplasia in Drosophila, and its deregulation could be relevant for the development of colorectal cancer in humans (Nagy, 2016).

    Activation of the Tor/Myc signaling axis in intestinal stem and progenitor cells affects longevity, stress resistance and metabolism in Drosophila

    The TOR (target of rapamycin) signaling pathway and the transcriptional factor Myc play important roles in growBh control. Myc acts, in part, as a downstream target of TOR to regulate the activity and functioning of stem cells. Tbis study explored the role of TOR-Myc axis in stem and progenitor cells in the regulation of lifespan, stress resistance and metabolism in Drosophila. Goth overexpression of rheb and myc-rheb in midgut stem and progenitor cells decreased the lifespan and starvation resistance of flies. TOR activation caused higher survival under malnutrition conditions. Furthermore, gut-specific activation of JAK/STAT and insulin signaling pathways were demonstrated to control gut integrity. Both genetic manipulations had an impact on carbohydrate metabolism and transcriptional levels of metabolic genes. These findings indicate that activation of the TOR-Myc axis in midgut stem and progenitor cells influences a variety of traits in Drosophila (Strilbytska, 2016).

    Drosophila Sulf1 is required for the termination of intestinal stem cell division during regeneration

    Stem cell division is activated to trigger regeneration in response to tissue damage. The molecular mechanisms by which this stem cell mitotic activity is properly repressed at the end of regeneration are poorly understood. This study shows that a specific modification of heparan sulfate (HS) is critical in regulating Drosophila intestinal stem cell (ISC) division during normal midgut homeostasis and regeneration. Loss of the extracellular HS endosulfatase Sulf1 results in increased ISC division during normal homeostasis, which is caused by upregulation of mitogenic signaling including the JAK/STAT, EGFR, and Hedgehog pathways. Using a regeneration model, this study found that ISCs failed to properly halt division at the termination stage in Sulf1 mutants, showing that Sulf1 is required for terminating ISC division at the end of regeneration. It is proposed that post-transcriptional regulation of mitogen signaling by HS structural modifications provides a novel regulatory step for precise temporal control of stem cell activity during regeneration (Takemura, 2015).

    Diversity of fate outcomes in cell pairs under lateral inhibition

    Cell fate determination by lateral inhibition via Notch/Delta signalling has been extensively studied. Most formalised models consider Notch/Delta interactions in fields of cells, with parameters that typically lead to symmetry breaking of signalling states between neighbouring cells, commonly resulting in salt-and-pepper fate patterns. This study considers the case of signalling between isolated cell pairs. The bifurcation properties of a standard mathematical model of lateral inhibition was found to lead to stable symmetric signalling states. This model was applied to the adult intestinal stem cell (ISC) of Drosophila, whose fate is stochastic but dependent on the Notch/Delta pathway. A correlation was observed between signalling state in cell pairs and their contact area. This behaviour is intrepeted in terms of the properties of the model in the presence of population variability in contact areas, which affects the effective signalling threshold of individual cells. The results suggest that the dynamics of Notch/Delta signalling can contribute to explain stochasticity in stem cell fate decisions, and that the standard model for lateral inhibition can account for a wider range of developmental outcomes than previously considered (Guisoni, 2017).

    Tis11 mediated mRNA decay promotes the reacquisition of Drosophila intestinal stem cell quiescence

    Adult stem cell proliferation rates are precisely regulated to maintain long-term tissue homeostasis. Defects in the mechanisms controlling stem cell proliferation result in impaired regeneration and hyperproliferative diseases. Many stem cell populations increase proliferation in response to tissue damage and reacquire basal proliferation rates after tissue repair is completed. Although proliferative signals have been extensively studied, much less is known about the molecular mechanisms that restore stem cell quiescence. This study shows that Tis11, an Adenine-uridine Rich Element (ARE) binding protein that promotes mRNA degradation, is required to re-establish basal proliferation rates of adult Drosophila intestinal stem cells (ISC) after a regenerative episode. Tis11 limits ISC proliferation specifically after proliferation has been stimulated in response to heat stress or infection, and Tis11 expression and activity are increased in ISCs during tissue repair. Based on stem cell transcriptome analysis and RNA immunoprecipitation, it is proposed that Tis11 activation represents an integral part of a negative feedback mechanism that limits the expression of key components of several signaling pathways that control ISC function and proliferation. The results identify Tis11 mediated mRNA decay as an evolutionarily conserved mechanism of re-establishing basal proliferation rates of stem cells in regenerating tissues (McClelland, 2017).

    Drosophila dyskerin is required for somatic stem cell homeostasis

    Drosophila represents an excellent model to dissect the roles played by the evolutionary conserved family of eukaryotic dyskerins. These multifunctional proteins are involved in the formation of H/ACA snoRNP and telomerase complexes, both involved in essential cellular tasks. Since fly telomere integrity is guaranteed by a different mechanism, the specific role played by dyskerin in somatic stem cell maintenance was investigated. Focus was placed on Drosophila midgut, a hierarchically organized and well characterized model for stemness analysis. Surprisingly, the ubiquitous loss of the protein uniquely affects the formation of the larval stem cell niches, without altering other midgut cell types. The number of adult midgut precursor stem cells is dramatically reduced, and this effect is not caused by premature differentiation and is cell-autonomous. Moreover, only a few dispersed precursors found in the depleted midguts could maintain stem identity and the ability to divide asymmetrically, and show cell-growth defects or undergo apoptosis. Loss is mainly specifically dependent on defective amplification. These studies establish a strict link between dyskerin and somatic stem cell maintenance in a telomerase-lacking organism, indicating that loss of stemness can be regarded as a conserved, telomerase-independent effect of dyskerin dysfunction (Vicidomini, 2017).

    Intestinal stem cell pool regulation in Drosophila

    Intestinal epithelial renewal is mediated by intestinal stem cells (ISCs) that exist in a state of neutral drift, wherein individual ISC lineages are regularly lost and born but ISC numbers remain constant. To test whether an active mechanism maintains stem cell pools in the Drosophila midgut, partial ISC depletion was performed. In contrast to the mouse intestine, Drosophila ISCs failed to repopulate the gut after partial depletion. Even when the midgut was challenged to regenerate by infection, ISCs retained normal proportions of asymmetric division and ISC pools did not increase. The loss of differentiated midgut enterocytes (ECs), however, slows when ISC division is suppressed and accelerates when ISC division increases. This plasticity in rates of EC turnover appears to facilitate epithelial homeostasis even after stem cell pools are compromised. This study identifies unique behaviors of Drosophila midgut cells that maintain epithelial homeostasis (Jin, 2017).

    To achieve homeostasis in a stem cell pool, stem cell divisions typically give rise to one new stem cell and one cell that is destined to differentiate. This lineage asymmetry can be determined cell-intrinsically, for instance by the asymmetric partitioning of determinants during division, or by localized niche factors. In the latter case, lineage asymmetry may be observed only in populations, rather than by following each and every stem cell division. Studies in mice and flies have documented this sort of population asymmetry in ISC pools and have demonstrated the phenomenon of neutral drift, whereby individual stem cell lineages are born and extinguished at equivalent rates as a result of divisions that either duplicate stem cells or fail at self-renewal. In addition, in mice, dedifferentiation of progenitor cells within the crypt has been observed as a mechanism for restoring lost stem cells. However, the precise response of the gut after stem cell pools are compromised is not well understood in either Drosophila or mice. Understanding this response has considerable practical value, since many anti-cancer chemotherapies deplete intestinal and other stem cells, and thereby give rise to debilitating side effects such as gastrointestinal mucositis (Jin, 2017).

    In this study it was asked whether the fly intestinal stem cell pool is self-regulatory and capable of regeneration following the ablation of about 50% of the ISCs. Surprisingly, it was found that the fly's ISC population did not repopulate itself after ISC depletion. Instead, the remaining ISCs behaved essentially as in normal midguts: they divided at normal rates and responded normally to gut epithelial damage with increased division, but did not duplicate at higher frequencies or regenerate a normal-sized stem cell pool, even after long recovery periods. Nevertheless, midguts with about half the normal ISC number retained their normal size for many weeks, indicating that somehow homeostasis was maintained. In exploring this phenomenon it was found that the rate of stem cell division has a strong influence on the rate of loss of differentiated epithelial cells, both when ISC divisions were accelerated or retarded. Hence, it is suggested that homeostasis in ISC-depleted guts was made possible by a reduction in the rate of cell loss from the gut epithelium. One explanation for this may be that EC loss rates are substantially determined by ISC division rates, rather than directly by damage from digestive wear and tear and adverse interactions with the gut microbiota, as generally assumed. The demonstration that EC loss can be accelerated by promoting ISC proliferation is consistent with this view. It is speculated that, as between ECs and ISC tumors, competition between old and newborn ECs for attachment to the basement membrane may underlie these effects on EC lifespan. In support of this, midgut epithelial cell crowding induced by increased ISC proliferation due to stress was shown to be relieved by the loss of excess cells though apoptosis. The ability to alter the rate of epithelial replacement to match the capabilities of the stem cell pool represents an unexpected mechanism of homeostatic plasticity (Jin, 2017).

    Experiments were performed in which ISCs were completely ablated, and no recovery of the ISC pool was observed over the lifespan of flies. This is consistent with another recent report in which ISCs were completely ablated. In both of these cases the esg-Gal4 driver was used for depletion, allowing the conclusion that there are no esg- ISC precursors in the adult fly. Interestingly, in contrast to a recent report that found that flies lacking ISCs had almost normal lifespans, this study found that complete ISC ablation reduced fly survival. The data suggest that while flies can survive partial ISC loss for at least 4 weeks, complete ISC depletion results in a loss of midgut homeostasis and reduced survival. (Jin, 2017).

    The dynamics of ISC pool maintenance in the fly midgut have significant differences from those in the mammalian intestine. Symmetric ISC lineages are very often observed in the mouse intestine, while only 10% of ISC lineages are typically symmetric in the fly midgut. Strikingly, murine ISC pools readily recover after stem cell depletion whereas in the fly ISC depletion appears to be irreversible. Furthermore, in the murine intestine, selection of stem cells based on niche occupancy is important, and partially differentiated TA cells can revert into ISCs if they can access the niche. These phenomena are not observed in the fly midgut, which has a dispersed niche, no TA cells, and a fixed number of ISCs. The different behavior of ISCs in these two species could be due to differing requirements for stem cell capability. The mouse's lifespan is more than ten times longer than the fly's, so murine ISCs need to maintain gut homeostasis for much longer. Mammalian ISCs have to renew themselves many more times during the host's lifespan and also accumulate more genomic damage from DNA replication, which could alternatively drive cell death or transformation. Perhaps because of these pressures, the mammalian intestine evolved a more flexible system for stem cell pool control, which allows both better recovery from injury and the capability to select defective ISCs while maintaining a normal-sized stem cell pool (Jin, 2017).

    Control of intestinal stem cell function and proliferation by mitochondrial pyruvate metabolism

    Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. This study sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells (Schell, 2017).

    It was first observed almost 100 years ago that, unlike differentiated cells, cancer cells tend to avidly consume glucose, but not fully oxidize the pyruvate that is generated from glycolysis. This was originally proposed to be due to dysfunctional or absent mitochondria, but it has become increasingly clear that mitochondria remain functional and critical. Mitochondria are particularly important in proliferating cells because essential steps in the biosynthesis of amino acids, nucleotide and lipid occur therein. Most proliferating stem cell populations also exhibit a similar glycolytic metabolic program, which transitions to a program of mitochondrial carbohydrate oxidation during differentiation. The first distinct step in carbohydrate oxidation is import of pyruvate into the mitochondrial matrix, where it gains access to the pyruvate dehydrogenase complex (PDH) and enters the tricarboxylic acid (TCA) cycle as acetyl-CoA. The two proteins that assemble to form the mitochondrial pyruvate carrier (MPC) have been recently described. This complex is necessary and sufficient for mitochondrial pyruvate import in yeast, flies and mammals, and thereby serves as the junction between cytoplasmic glycolysis and mitochondrial oxidative phosphorylation. Decreased expression and activity of the MPC underlies the glycolytic program in colon cancer cells in vitro, and forced re-expression of the MPC subunits increased carbohydrate oxidation and impaired the ability of these cells to form colonies in vitro and tumours in vivo. This impairment of tumorigenicity was coincident with a loss of key markers and phenotypes associated with stem cells. This has prompted an examination of whether glycolytic non-transformed stem cells might also exhibit low MPC expression and mitochondrial pyruvate oxidation, which must increase during differentiation (Schell, 2017).

    The role of the MPC was studied in the ISCs of the fruit fly Drosophila, which share key aspects of their biology with mammalian ISCs. Both MPC1 and MPC2 are expressed in all four cell types of the intestine, with the lowest level of expression in the ISCs and the highest expression in the differentiated enteroendocrine cells. Confocal imaging of intestines dissected from dMPC1 mutants revealed that the epithelium exhibits multilayering unlike the normal single-cell layer seen in controls. This is a classic overgrowth phenotype that is associated with oncogene mutations in Drosophila. Accordingly, MARCM clonal analysis was used to determine whether a specific loss of the MPC in ISCs leads to an increase in their proliferation. On average, newly divided GFP-marked dMPC1 mutant clones are more than twofold larger than control clones, which were generated in parallel using a wild-type chromosome, indicating that the MPC is required in the ISC lineage to suppress proliferation. Because GFP-marked clones could include cells that differentiate into mature enterocytes or enteroendocrine cells, clonal analysis was conducted in the absence of Notch, thereby blocking ISC differentiation. Under these conditions, an approximately twofold increase was observed in the size of dMPC1 mutant ISC clones. To confirm and extend these results, MPC function was specifically disrupted in the ISCs by using the Dl-GAL4 driver in combination with UAS-GFP, which facilitates stem cell identification. Once again, approximately twofold more GFP-marked stem cells were observed relative to controls when either dMPC1 or dMPC2 expression was disrupted by RNA-mediated interference (RNAi) along with increased ISC proliferation as detected by staining for phosphorylated histone H3 (pHH3). Similar results were obtained when RNAi was targeted to the E1 or E2 subunits of PDH to specifically disrupt the next step in mitochondrial pyruvate oxidation. Importantly, an opposite phenotype was seen when Ldh was reduced by RNAi in the ISCs or progenitor cells. Ldh suppression is known to result in a significant increase in pyruvate levels, which can promote pyruvate oxidation. Taken together with the results with Pdh RNAi, these observations support the model that the MPC limits stem cell proliferation through promoting oxidative pyruvate metabolism in the mitochondria. It also appears to be sufficient as specific overexpression of MPC1 and MPC2 in ISCs or progenitors caused a reduction in stem cell proliferation, the opposite of the loss-of-function phenotype. This can be seen in either Pseudomonas-infected intestines, which undergo rapid stem cell proliferation, or under basal conditions in aged animals. Consistent with this, MPC overexpression under basal conditions had no effects on intestinal morphology, while the intestines from infected flies displayed a fully penetrant size reduction, which is probably due to the inability of ISCs to maintain tissue homeostasis. Taken together, these results demonstrate that mitochondrial pyruvate uptake and metabolism is both necessary and sufficient in a stem cell autonomous manner to regulate ISC proliferation and maintain intestinal homeostasis in Drosophila (Schell, 2017).

    Studies in Drosophila, intestinal organoids and mice provide strong evidence that the MPC is necessary and sufficient in a cell autonomous manner to suppress stem cell proliferation. Consistently, this study has demonstrated that ISCs maintain low expression of the subunits that comprise the MPC, which enforces a mode of carbohydrate metabolism wherein glucose is metabolized in the cytosol to pyruvate and other biosynthetic intermediates. This glycolytic metabolic program appears to be sufficient to drive robust and continuous stem cell proliferation. High mitochondrial content was observed in ISCs, which must be geared primarily toward biosynthetic functions and/or oxidation of other substrates such as fatty acids. Increased fatty acids, the metabolism of which is enhanced in MPC-deficient and MPC-inhibited organoids, have been shown to promote ISC expansion and proliferation via enhanced beta-catenin signalling and increasing tumour-initiating capacity. MPC expression increases following differentiation, consistent with the shift in demand from macromolecule biosynthesis to ATP production in support of post-mitotic differentiated cell function. A similar switch in MPC expression can be seen following differentiation of embryonic stem cells, haematopoietic stem cells and trophoblast stem cells. Conversely, MPC expression is reduced after reprogramming fibroblasts to induced pluripotent stem cells. This suggests that the effects of altering pyruvate flux that wad observed in this study might not be restricted to ISCs, but instead be representative of similar effects on multiple stem cell populations. Interestingly, Myc is known to drive a metabolic program that is similar to that observed following MPC loss, characterized by increased glycolysis and reliance on glutamine and fatty acid oxidation with reduced glucose oxidation. This suggests that Myc may play a role in repressing the MPC in stem cells, possibly acting downstream of Wnt/beta-catenin signalling. Consistent with this, Myc and its repressive co-factors localize to the Mpc1 promoter and Myc expression is strongly anti-correlated with Mpc1 expression (Schell, 2017).

    Taken together, these studies demonstrate that changes in the MPC and mitochondrial pyruvate metabolism are required to properly orchestrate the proliferation and homeostasis of intestinal stem cells. Importantly, this metabolic program, mediated at least partially by the MPC, appears to be instructive for cell fate, rather than a downstream consequence of cell fate. Future work will define the extent to which the results presented in this study relate to those showing that diet quality and quantity can modulate ISC behaviour. It is tempting to speculate that ISC metabolism is used as a signal for increased or decreased demand for intestinal epithelium. Perhaps of most importance will be to define the mechanisms whereby altered partitioning of pyruvate metabolism affects stem cell proliferation and fate. It is speculated that the robust changes that were observed in fatty acid oxidation and histone acetylation, which are probably downstream of altered metabolite utilization for acetyl-CoA production, play an important role. While the mechanisms are not as yet defined, these studies establish a paradigm wherein mitochondrial metabolism does not merely provide a permissive context for proliferation or differentiation, but rather plays a direct and instructive role in controlling stem cell fate (Schell, 2017).

    Oxidative stress induces stem cell proliferation via TRPA1/RyR-mediated Ca2+ signaling in the Drosophila midgut

    Precise regulation of stem cell activity is crucial for tissue homeostasis and necessary to prevent overproliferation. In the Drosophila adult gut, high levels of reactive oxygen species (ROS) has been detected with different types of tissue damage, and oxidative stress has been shown to be both necessary and sufficient to trigger intestinal stem cell (ISC) proliferation. However, the connection between oxidative stress and mitogenic signals remains obscure. In a screen for genes required for ISC proliferation in response to oxidative stress, this study identified two regulators of cytosolic Ca2+ levels, transient receptor potential A1 (TRPA1) and ryanodine receptor (RyR). Characterization of TRPA1 and RyR demonstrates that Ca2+ signaling is required for oxidative stress-induced activation of the Ras/MAPK pathway, which in turns drives ISC proliferation. These findings provide a link between redox regulation and Ca2+ signaling and reveal a novel mechanism by which ISCs detect stress signals (Xu, 2017).

    This study found that the two cation channels TRPA1 and RyR are critical for cytosolic Ca2+ signaling and ISC proliferation. Under homeostatic conditions, the basal activities of TRPA1 and RyR are required for maintaining cytosolic Ca2+ in ISCs to ensure their self-renewal activities and normal tissue turnover. Agonists, including but not limited to low levels of ROS, could be responsible for the basal activities of TRPA1 and RyR. Under tissue damage conditions, increased ROS stimulates the channel activities of TRPA1 to mediate increases in cytosolic Ca2+ in ISCs. As for RyR, besides its potential to directly sense ROS, it is known to act synergistically with TRPA1 in a positive feedback mechanism to release more Ca2+ from the ER into the cytosol upon sensing the initial Ca2+ influx through TRPA1 (Xu, 2017).

    Previously, Deng (2015) identified L-glutamate as a signal that can activate metabotropic glutamate receptor (mGluR) in ISCs, which in turn modulates the cytosolic Ca2+ oscillation pattern via phospholipase C (PLC) and inositol-1,4,5-trisphosphate (InsP3). Interestingly, L-glutamate and mGluR RNAi mainly affected the frequency of Ca2+ oscillation in ISCs, while their influence on cytosolic Ca2+ concentration was very weak. Strikingly, the number of mitotic cells induced by L-glutamate (i.e. an increase from a basal level of ~5 per midgut to ~10 per midgut) is far less than what has been observed in tissue damage conditions (depending on the severity of damage, the number varies from ~20 to more than 100 per midgut following damage). Consistent with this, in a screen for regulators of ISC proliferation in response to tissue damage, this study tested three RNAi lines targeting mGluR (BL25938, BL32872, and BL41668, which was used by Deng, 2015), and none blocked the damage response in ISCs, suggesting that L-glutamate and mGluR do not play a major role in damage repair of the gut epithelium (Xu, 2017).

    This study found that ROS can trigger Ca2+ increases through the redox- sensitive cation channels TRPA1 and RyR under damage conditions. In particular, it was demonstrated using voltage-clamp experiments that the TRPA1-D isoform, which is expressed in the midgut, is sensitive to the oxidant agent paraquat. In addition, the results of previous studies have demonstrated the direct response of RyR to oxidants via single channel recording and showed that RyR could amplify TRPA1-mediated Ca2+ signaling through the Ca2+-induced Ca2+ release (CICR) mechanism. Interestingly, expression of oxidant- insensitive TRPA1-C isoform in the ISCs also exhibits a tendency to induce ISC proliferation, indicating that ROS may not be the only stimuli for TRPA1 and RyR under physiological conditions. Possible other activators in the midgut may be irritant chemicals, noxious thermal/mechanical stimuli, or G-protein-coupled receptors (Xu, 2017).

    Altogether, the concentration of cytosolic Ca2+ in ISCs appears to be regulated by a number of mechanisms/inputs including mGluR and the ion channels TRPA1 and RyR. Although mGluR might make a moderate contribution to cytosolic Ca2+ in ISCs, TRPA1 and RyR have a much stronger influence on ISC Ca2+ levels. Thus, it appears that the extent to which different inputs affect cytosolic Ca2+ concentration correlates with the extent of ISC proliferation (Xu, 2017).

    Although, as a universal intracellular signal, cytosolic Ca2+ controls a plethora of cellular processes, we were able to demonstrate that cytosolic Ca2+ levels regulate Ras/MAPK activity in ISCs. Specifically, we found that trpA1 RNAi or RyR RNAi block Ras/MAPK activation in stem cells, and that forced cytosolic Ca2+ influx by SERCA RNAi induces Ras/MAPK activity. Moreover, Ras/MAPK activation is an early event following increases in cytosolic Ca2+, since increased dpErk signal was observed in stem cells expressing SERCA RNAi before they undergo massive expansion, and when Yki RNAi was co-expressed to block proliferation. It should be noted that a more variable pattern of pErk activation was observed with prolonged increases of cytosolic Ca2+, suggesting complicated regulations via negative feedback, cross-activation, and cell communication at late stages of Ca2+ signaling. This might explain why Deng failed to detect pErk activation after 4 days induction of Ca2+ signaling (Deng, 2015). Previously, Ras/MAPK activity was reported to increase in ISCs, regulating proliferation rather than differentiation, in regenerating midguts, which is consistent with the findings about TRPA1 and RyR (Xu, 2017).

    The Calcineurin A1/CREB-regulated transcription coactivator/CrebB pathway previously proposed to act downstream of mGluR-calcium signaling (Deng, 2015) is not likely to play a major role in high Ca2+-induced ISC proliferation, as multiple RNAi lines targeting CanA1 or CrebB were tested and none of them suppressed SERCA RNAi-induced ISC proliferation. In support of this model, it was also found that the active forms of CanA1/ CRTC/ CrebB cannot stimulate mitosis in ISCs when their cytosolic Ca2+ levels are restricted by trpA1 RNAi, whereas mitosis induced by the active forms of Ras or Raf is not suppressed by trpA1 RNAi (Xu, 2017).

    Prior to this study, it has been shown that paracrine ligands such as Vn from the visceral muscle, and autocrine ligands such as Spi and Pvf ligands from the stem cells, can stimulate ISC proliferation via RTK-Ras/MAPK signaling. It study found that multiple RTK ligands in the midgut are down-regulated by trpA1 RNAi expression in the ISCs, including spi and pvf1 that can be induced by SERCA RNAi. Further, it was demonstrated that high Ca2+ fails to induce ISC proliferation in the absence of EGFR. As spi is induced by EGFR-Ras/MAPK signaling in Drosophila cells, and DNA binding mapping (DamID) analyses indicate that spi might be a direct target of transcriptional factors downstream of EGFR-Ras/MAPK in the ISCs, the autocrine ligand Spi might therefore act as a positive feedback mechanism for EGFR-Ras/MAPK signaling in ISCs (Xu, 2017).

    In summary, this study identifies a mechanism by which ISCs sense microenvironment stress signals. The cation channels TRPA1 and RyR detect oxidative stress associated with tissue damage and mediate increases in cytosolic Ca2+ in ISCs to amplify and activate EGFR-Ras/MAPK signaling. In vertebrates, a number of cation channels, including TRPA1 and RyR, have been associated with tumor malignancy. The current findings, unraveling the relationship between redox-sensing, cytosolic Ca2+, and pro-mitosis Ras/MAPK activity in ISCs, could potentially help understand the roles of cation channels in stem cells and cancers, and inspire novel pharmacological interventions to improve stem cell activity for regeneration purposes and suppress tumorigenic growth of stem cells (Xu, 2017).

    Loss of the mucosal barrier alters the progenitor cell niche via JAK/STAT signaling

    The mucous barrier of the digestive tract is the first line of defense against pathogens and damage. Disruptions in this barrier are associated with diseases such as Crohn's disease, colitis and colon cancer, but mechanistic insights into these processes and diseases are limited. Loss of a conserved O-glycosyltransferase (PGANT4) in Drosophila has been shown to result in aberrant secretion of components of the peritrophic/mucous membrane in the larval digestive tract. This study shows that loss of pgant4 disrupts the mucosal barrier, resulting in epithelial expression of the IL-6-like cytokine Upd3, leading to activation of JAK/STAT signaling, differentiation of cells that form the progenitor cell niche and abnormal proliferation of progenitor cells. This niche disruption could be recapitulated by overexpressing upd3 and rescued by deleting upd3, highlighting a crucial role for this cytokine. Moreover, niche integrity and cell proliferation in pgant4-deficient animals could be rescued by overexpression of the conserved cargo receptor Tango1 and partially rescued by supplementation with exogenous mucins or treatment with antibiotics. These findings help elucidate the paracrine signaling events activated by a compromised mucosal barrier and provide a novel in vivo screening platform for mucin mimetics and other strategies to treat diseases of the oral mucosa and digestive tract (Zhang, L. 2017).

    The mucous barrier that lines the respiratory and digestive tracts is the first line of defense against pathogens and provides hydration and lubrication. This unique membrane separates the delicate epithelia from factors present in the external environment. In mammals, the mucous lining of the intestine allows nutrient penetration while conferring protection from both bacteria and the mechanical damage associated with digestion of solid food. The principal components of mucous membranes are mucins, a diverse family of proteins that are expressed in tissue-specific fashions. Whereas secreted mucins vary greatly in sequence and size, they all share highly O-glycosylated serine- and threonine-rich regions that confer unique structural and rheological properties, allowing the formation of hydrated gels. Deletion of specific components of the mucous membranes present in the lung (Muc5b) or the intestinal tract (Muc2) resulted in defects in mucociliary clearance or an increased incidence colorectal cancer, respectively. Changes in mucus production or the glycosylation status of mucins have also been associated with oral pathology in Sjogren's syndrome, the development of colitis and intestinal tumors in mice, and the progression of ulcerative colitis and colon cancer in humans. However, the detailed mechanisms by which loss/alteration of this protective layer results in epithelial pathology remain unknown (Zhang, L. 2017).

    Many components and factors that confer the unique properties of this protective lining, including the mucins and the enzymes that mediate their dense glycosylation, are conserved across species. Indeed, Drosophila melanogaster contains a similar protective lining known as the peritrophic membrane, consisting of a chitin scaffold that is bound by highly O-glycosylated mucins. During larval development, where animals ingest solid food and undergo massive growth in preparation for metamorphosis, specialized secretory cells (PR cells) of the anterior midgut produce this membrane, which is thought to protect epithelial cells of the digestive tract from mechanical and microbial damage. Previous work in Drosophila has shown that loss of one component of the peritrophic membrane resulted in a thinner and more permeable membrane, where adult flies were viable yet more susceptible to oral infections. However, the exact role of the entirety of this lining in the integrity and protection of cells of the digestive tract remains unknown (Zhang, L. 2017).

    Extensive research elucidating the development and function of the many cell types that comprise the Drosophila digestive tract has provided insights into mammalian digestive system formation and function. The Drosophila larval midgut is composed of specialized epithelial cells (enterocytes [ECs] and enteroendocrine cells) for digestion and nutrient absorption, as well as progenitor cells that will eventually form the adult midgut epithelium. These adult midgut progenitor cells (AMPs) reside in a protected niche, formed by peripheral cells (PCs) that wrap and shield them from external signaling. PCs are characterized by a unique crescent shape, with long processes that surround AMPs, restricting proliferation and differentiation until metamorphosis. The larval digestive tract therefore represents an ideal system to interrogate the role of the mucous layer in protection of both the epithelium and the progenitor cell niche at a stage when the mechanical and microbial stresses associated with the ingestion of solid food are abundant (Zhang, L. 2017).

    Previous work has shown that loss of a conserved UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase responsible for initiating O-linked glycosylation (PGANT4) resulted in aberrant secretion of components of the peritrophic membrane in the larval digestive tract (Tran, 2012). This study shows that pgant4 mutants are devoid of a peritrophic membrane, resulting in epithelial cell damage and expression of the IL-6-like inflammatory cytokine, unpaired 3 (Upd3). Upd3 expression resulted in increased JAK/STAT signaling in the progenitor cell niche, causing niche cell differentiation and aberrant progenitor cell proliferation. These effects were dependent on Upd3 and could be rescued by deleting upd3 or partially rescued by feeding animals antibiotics or exogenous mammalian intestinal mucins. Moreover, overexpression of the conserved extracellular matrix cargo receptor, Tango1 (transport and Golgi organization 1), in secretory cells of the digestive tract resulted in restoration of the peritrophic membrane and rescue of niche integrity. These results elucidate new mechanistic details regarding how a compromised mucous lining can influence epithelial integrity and the progenitor cell niche and provide an in vivo screening platform for compounds and strategies that could restore mucosal barrier function (Zhang, L. 2017).

    This study shows the peritrophic membrane is essential to protect the integrity of the epithelial cell layer and maintain an appropriate environment for the progenitor cell niche. Moreover, a dynamic and specific response to the loss of this membrane occurs via the production of the IL-6-like cytokine Upd3 from epithelial cells, which in turn signals to niche cells in a paracrine fashion, causing differentiation and morphological changes. This demonstrates the multipotent nature of PCs, which can respond to specific cytokines to alter their fate. Once the PC morphology and fate were altered, JAK/STAT signaling was activated in AMPs exposed to Upd3, causing aberrant cell proliferation/DNA replication. This represents the first example where loss of the protective mucous lining activates signaling from epithelial cells to alter the fate of niche cells and change the behavior of progenitor cells. These studies highlight the importance of this membrane in both epithelial and progenitor cell biology and elucidate the paracrine signaling cascade that is specifically activated when this barrier is compromised (Zhang, L. 2017).

    Interestingly, the mucinous peritrophic membrane could be restored by overexpression of the conserved cargo receptor Tango1. Tango1 is an essential protein that functions to package large extracellular matrix proteins, such as collagen and mucins, into secretory vesicles. Loss of the mammalian ortholog of Tango1 (Mia3) in a murine model resulted in lethality with global defects in collagen secretion and extracellular matrix composition. Alterations in Tango1/Mia3 expression have also been associated with colon and hepatocellular carcinomas in humans. Previous work in Drosophila demonstrated that PGANT4 glycosylates Tango1, protecting it from Dfur2-mediated proteolysis in the digestive tract. This study shows that Tango1 overexpression specifically in the secretory PR cells of the digestive tract can restore the mucinous membrane throughout the midgut to rescue epithelial viability and niche integrity, further demonstrating the crucial role of the peritrophic membrane in digestive system homeostasis and health. These results suggest the possibility of exogenous Tango1 expression as a potential strategy to restore secretion, mucous membranes, and/or extracellular matrix composition and confer epithelial protection (Zhang, L. 2017).

    The larval digestive system offers unique opportunities to investigate the role of the individual components of the mucinous membrane and restorative strategies in epithelial biology. Unlike the adult stage, the larval portion of the life cycle is devoted to continuous feeding and digestion to orchestrate the massive growth of cells and tissues in preparation for metamorphosis. As such, larvae consume many types of solid food and will readily ingest various compounds. Indeed, oral supplementation with an intestinal mucin (Muc2) partially rescued JAK/STAT signaling, suggesting that this could serve as a strategy for epithelial protection. Muc2 is a major component of the protective mucous membrane that lines the small intestine and colon of mammals. Muc2 is thought to confer lubrication for food passage as well as to form a barrier between microbes and epithelial cells of the digestive tract. The current results suggest that Muc2 supplementation could be providing similar properties in the Drosophila digestive tract. Interestingly, supplementation with the gastric mucin (Muc5AC) dramatically exacerbated JAK/STAT signaling, suggesting that the different structural, rheological, or binding properties of each mucin are mediating distinct cellular responses in this system. Current work is focused on deciphering the specific functional regions of various secreted mucins and testing their ability to confer epithelial protection using this in vivo system. Human diseases of the digestive tract are associated with disrupted mucinous linings, and disease severity is often correlated with the severity of barrier disruption. Interestingly, these diseases are also characterized by increased levels of the mammalian ortholog of Upd3 (IL-6), increased JAK/STAT activation, and increased cell proliferation, similar to what is seen in Drosophila, suggesting conserved mechanisms for responding to mucosal disruption/injury. It is widely known that immune cells are one source of IL-6 in mammals, but recent studies have demonstrated that mechanically damaged epithelial and endothelial cells also produce IL-6. This study demonstrated that epithelial expression of Upd3 is both necessary and sufficient for the changes in PC fate and AMP proliferation, as disruption of the niche could be recapitulated by overexpression of upd3 from ECs and rescued by deletion of upd3. How peritrophic membrane loss is signaling to up-regulate upd3 expression in ECs is currently unknown. However, previous studies in the adult Drosophila digestive system have shown up-regulation of upd, upd2, and upd3 in response to enteric infection or damage-inducing agents, such as bleomycin or dextran sulfate sodium, suggesting roles for both microbial insults and physical/mechanical damage to epithelial cells. Indeed, the results also suggest roles for microbial and mechanical damage in the absence of the peritrophic membrane, as both antibiotics and mucin supplementation were able to reduce upd3 expression and cell proliferation. This study demonstrates that the larval midgut can serve as a model system to study how cells/tissues sense and respond to damage as well as to decipher how upd3 is specifically activated in epithelial cells under various conditions (Zhang, L. 2017).

    As a mucous layer is present across most internal epithelial surfaces, understanding the mechanisms by which it confers protection and epithelial homeostasis will be informative in treating various diseases affecting the integrity of this layer. Mucosal healing has been proposed as a treatment option for inflammatory bowel disease and other diseases of the digestive tract that are characterized by destruction of the mucosa and epithelial surfaces. Likewise, mucins are a component in some oral treatments for dry mouth caused by head and neck irradiation or Sjogren's syndrome. Other therapeutics for various autoimmune and inflammatory diseases include JAK inhibitors (Jakinibs) and drugs directed against particular cytokines. This study has shown that genetic restoration of the peritrophic membrane can restore digestive system health and that antibiotic treatment or mucin supplementation can partially rescue damage-induced signaling cascades, suggesting that this Drosophila system may be a viable platform for testing compounds to remediate epithelial damage. Future studies will focus on testing newly emerging mucin mimetics (designed to confer epithelial protection and appropriate rheology/hydration), synthetic mucins (where the extent of glycosylation can be specifically modified), glycan-based hydrogels, and drugs that target conserved steps in the JAK/STAT signaling cascade. Lessons learned in Drosophila may inform future strategies for functional restoration of mucosal protection (Zhang, L. 2017).

    Nutritional control of stem cell division through S-Adenosylmethionine in Drosophila intestine

    The intestine has direct contact with nutritional information. The mechanisms by which particular dietary molecules affect intestinal homeostasis are not fully understood. In this study, S-adenosylmethionine (SAM) was identified as a universal methyl donor synthesized from dietary methionine, as a critical molecule that regulates stem cell division in Drosophila midgut. Depletion of either dietary methionine or SAM synthesis reduces division rate of intestinal stem cells. Genetic screening for putative SAM-dependent methyltransferases has identified protein synthesis as a regulator of the stem cells, partially through a unique diphthamide modification on eukaryotic elongation factor 2. In contrast, SAM in nutrient-absorptive enterocytes controls the interleukin-6-like protein Unpaired 3, which is required for rapid division of the stem cells after refeeding. This study sheds light upon a link between diet and intestinal homeostasis and highlights the key metabolite SAM as a mediator of cell-type-specific starvation response (Obata, 2018).

    Autophagy maintains stem cells and intestinal homeostasis in Drosophila

    Intestinal homeostasis is maintained by tightly controlled proliferation and differentiation of tissue-resident multipotent stem cells during aging and regeneration, which ensures organismal adaptation. This study shows that autophagy is required in Drosophila intestinal stem cells to sustain proliferation, and preserves the stem cell pool. Autophagy-deficient stem cells show elevated DNA damage and cell cycle arrest during aging, and are frequently eliminated via JNK-mediated apoptosis. Interestingly, loss of Chk2, a DNA damage-activated kinase that arrests the cell cycle and promotes DNA repair and apoptosis, leads to uncontrolled proliferation of intestinal stem cells regardless of their autophagy status. Chk2 accumulates in the nuclei of autophagy-deficient stem cells, raising the possibility that its activation may contribute to the effects of autophagy inhibition in intestinal stem cells. This study reveals the crucial role of autophagy in preserving proper stem cell function for the continuous renewal of the intestinal epithelium in Drosophila (Nagy, 2018).

    Ecdysone-regulated genomic networks in Drosophila: Midgut gene expression during metamorphosis

    During insect metamorphosis, each tissue displays a unique physiological and morphological response to the steroid hormone 20-hydroxyecdysone (ecdysone). Gene expression was assayed in five tissues during metamorphosis onset. Larval-specific tissues display major changes in genome-wide expression profiles, whereas tissues that survive into adulthood display few changes. In one larval tissue, the salivary gland, a computational approach was used to identify a regulatory motif and a cognate transcription factor involved in regulating a set of coexpressed genes. During the metamorphosis of another tissue, the midgut, genes encoding factors from the hedgehog, Notch, EGF, dpp, and wingless pathways are activated by the ecdysone regulatory network. Mutation of the ecdysone receptor abolishes their induction. Cell cycle genes are also activated during the initiation of midgut metamorphosis, and they are also dependent on ecdysone signaling. These results establish multiple new connections between the ecdysone regulatory network and other well-studied regulatory networks (Li, 2003).

    Developmental patterns of gene expression were studied from five different tissues and organs: central nervous system (CNS), wing imaginal disc (WD), larval epidermis and attached connective tissue (ED), midgut (MG), and salivary gland (SG), during late larval and early prepupal development when ecdysone triggers metamorphosis. At these stages of development, the five tissues display very different morphological and physiological responses to ecdysone. The wing imaginal disc responds to the hormone by initiating evagination, or unfolding, as it changes from a compact epithelial bilayer to an extended appendage. The salivary glands secrete glue proteins that are used to immobilize the puparium during metamorphosis. The cuticle attached to the larval epidermis undergoes a process of hardening and tanning to form the pupal case. The central nervous system (CNS) displays little morphological change during the late third instar ecdysone pulse, but the animal displays changes in behavior and in neurosecretory status. The two major types of cells in the larval midgut, larval epidermal cells and adult epidermal progenitor cells (midgut imaginal islands), respond in opposite ways to ecdysone. The larval epidermal cells initiate the process of programmed cell death, while the imaginal cells proliferate and form the adult midgut (Li, 2003).

    One tissue, the midgut, was selected to assay during its complete metamorphosis, which occurs from 18 hr before puparium formation (BPF) to 12 hr APF. During this 30 hr period, eleven time points were examined as the larval midgut is destroyed and replaced with the adult midgut. The two major cell types present in this organ are distinguishable by size. The larval epithelial cells are large, with decondensed polyploid nuclei, and undergo programmed cell death in response to ecdysone. Embedded among the larval cells are small diploid imaginal midgut cells, which proliferate in response to the hormone to form the adult epithelial cells. Additionally, the midgut contains relatively small numbers of muscle, tracheal, and endocrine cells (Li, 2003).

    In total, transcripts from a surprisingly large fraction of the genome, >30%, changed significantly during the metamorphosis of the midgut (18 hr BPF to 12 hr APF). Broad classes of temporally separable gene expression patterns are evident. These classes include sets of transcripts that rapidly decrease coincident with onset of programmed cell death in the larval cells, sets that are induced during early or late metamorphosis, and sets of transcripts expressed at highest levels during the middle period of the time course when the larval cells are in the final stages of cell death and the adult cells are rearranging to form new tissue (Li, 2003).

    Within these broad classes, specific sets of genes that have related functions and show parallel expression were identified, indicating that they make up gene batteries. Six such examples, included coregulated transcripts that encode proteins found in specific macromolecular complexes, biochemical pathways, organellar functions, and structural components of the cells that compose this tissue. Transcripts encoding proteasome components increase during the ecdysone pulse that triggers the onset of cell death in larval cells. Transcripts encoding glycolytic enzymes rapidly decrease during the initiation of metamorphosis, but gradually resume expression as the imaginal cells proliferate. Vacuolar ATPases shows a pattern similar to the glycolytic enzymes, whereas tubulin- and actin-encoding transcripts peak during the intense period of imaginal cell proliferation and migration as the adult midgut is formed. Transcripts encoding structural components of the peritrophic membrane of the mature larval gut gradually decrease during its replacement with adult tissue (Li, 2003).

    The expression patterns were examined of regulatory genes known to be involved in the ecdysone transcriptional hierarchy predicted to control the gene batteries that were identified. Also examined was the expression of genes with known roles in programmed cell death or cell cycle control. The expression of known ecdysone-responsive regulatory genes was consistent with previous observations in midgut. Although the larval midgut is composed of cell types that undergo divergent responses to ecdysone -- apoptosis and cell proliferation -- it was nonetheless possible to detect significant changes in transcript levels from genes encoding proteins involved in both processes. The apoptosis activator gene ark was expressed at 4 hr BPF. E93 and reaper, which encode proteins that serve as critical control points in the commitment to programmed cell death, were expressed at PF, as was the initiator caspase dronc. These midgut expression profiles were compared to those reported for salivary glands at and after 10 hr APF, when a prepupal pulse of ecdysone triggers apoptosis in that tissue; almost the entire genetic cascade was found to be similarly activated in salivary glands and midgut albeit at two distinct periods of development. However, one notable difference was observed at the top level of the cascade. In the salivary gland, E93 is activated by βFTZ-F1, whereas in the midgut the βFTZ-F1 gene is not induced until 6-8 hr after E93 is induced. The regulation of E93 therefore does not depend on βFTZ-F1 in the midgut, but must rely on another as yet unidentified factor(s). During midgut metamorphosis, developmental modulation of transcript levels were also observed for genes encoding DNA polymerases, cyclins, CDCs, and other cell cycle regulators, as well as genes encoding DNA repair proteins such as Hus1, Rad23, and PCNA/Mus209 (Li, 2003).

    Which of the genes that are differentially expressed at the onset of midgut metamorphosis require ecdysone signaling? Ecdysone-dependent transcriptional activity was removed using mutant Ecdysone Receptor (EcR) alleles, rescuing null EcR mutants to the third larval instar by using a heat shock-inducible EcR transgene. Gene expression was examined in mutant midguts that were isolated from mutant animals arrested at the end of the third larval stage (stage 2a mutants). 376 (76%) of the 495 genes that are significantly induced during the onset of midgut metamorphosis (18 hr BPF to 2 hr APF) required EcR function, whereas 296 (64%) of 460 transcripts that decline significantly in level during this time period require ecdysone signaling through EcR. Thus, a very large proportion of the genes that are developmentally regulated at the initiation of metamorphosis in this organ are under the control of the transcription factors that mediate the ecdysone signal. However, it does not appear that EcR function is a general requirement for transcription, because a significant fraction of differentially expressed genes are unaffected in EcR mutant tissue (Li, 2003).

    Of the several different classes of genes expressed during midgut metamorphosis, the regulation of all genes in the proteasome, tubulin/actin, and lysozyme clusters requires EcR to exhibit their normal changes in developmental expression. However, many genes in the v-ATPase cluster and nearly half the genes in the peritrophin cluster did not require EcR. The downregulation of hexokinase A, 6-phosphofructokinase, and pyruvate kinase genes in the glycolysis pathway were affected in the EcR mutants, while many others in this pathway were not. Hexokinase A, 6-phosphofructokinase, and pyruvate kinase are rate-controlling enzymes in the glycolytic pathway, indicating that their ecdysone dependence is functionally significant. The expression of the numerous known ecdysone receptor target genes such as E75, E74, broad, E23, and DHR3 required EcR as expected. The induction dynamics for the E74 and DHR3 transcription factor genes was as expected, as was their dependence on EcR. In contrast to E74 and DHR3, DHR78 has previously been described to reside upstream of EcR at the top of the ecdysone regulatory hierarchy -- the expression of EcR is dependent on the wild-type function of DHR78. However, DHR78 can also be induced by ecdysone in organ culture. The results demonstrate that DHR78 wild-type induction is indeed dependent on EcR function. Taken together, these data indicate a positive feedback loop between EcR and DHR78 during the onset of metamorphosis in the midgut (Li, 2003).

    Genes encoding factors involved in cell cycle and growth control, and in DNA repair, are also under the control of EcR. In spite of the role of ecdysone in stimulating cell proliferation during metamorphosis, no cell cycle genes have previously been linked to the ecdysone regulatory hierarchy. The induction of the cell cycle regulatory genes CyclinB, cdc2, and CyclinD were all observed to be dependent on EcR function. The rapid induction of cdc2 during the late third instar ecdysone pulse is similar to that observed for direct targets of EcR. The CyclinD gene is also induced at this time, but its maximal induction occurs several hours after that observed for cdc2. Cyclin D promotes cellular growth, whereas Cyclin B/Cdc2 controls G2/M transitions in proliferative cells. The dependence of these three genes on EcR function indicates that ecdysone may control cell proliferation, at least in part, through their regulation. Coordinate with the induction of CyclinB, cdc2, and CyclinD, the induction was observed of DNA polymerase-delta and DNA repair genes such as Rad23, and PCNA/mus209. The induction of these DNA repair and synthesis genes is also EcR dependent. The expression changes of these genes may be the result of the direct action of EcR, or due to the action of factors directly controlled by the ecdysone receptor complex. It is unlikely that the increase in expression of these genes is simply due to increased numbers of proliferative cells because the total number of divisions between 18 hr BPF and PF are few, and not all cell cycle or DNA repair genes showed an increase in expression at the initiation of metamorphosis. For example, the level of CyclinJ, which is known to be required during early embryonic division cycles, is actually reduced in expression from 18 hr BPF to PF. When the expression of cell death genes was examined in EcR mutant tissue, E93 induction was observed as well as induction of the Ark caspase activator and the dronc caspase gene required wild-type function of EcR (Li, 2003).

    Factors in several well-studied signaling pathways are induced during midgut metamorphosis. These include Wnt (dishevelled, armadillo, and zeste white 3), TGFβ/BMP (sara, daughters against dpp, and glass bottom boat), EGFR (torpedo/egfr, rhomboid/veinlet, vein, and keren/spitz2), and Notch pathway genes (delta, kuzbanian, suppressor of hairless, E(spl)malpha, and E(spl)mβ). All of these pathways are used during embryonic midgut development, and these data indicate they are reused during midgut metamorphosis. Genes in the Hedgehog signaling pathway (hedgehog, smoothened, and cubitus interruptus) changed significantly as well (Li, 2003).

    To determine whether any of the genes in these pathways are expressed as a consequence of ecdysone signaling, the EcR mutant expression data was examined for those genes that were induced during the late third instar ecdysone pulse. The induction of zeste white-3/shaggy, keren/spitz2, kuzbanian, and hedgehog are all dependent on the presence of functional EcR. The induction dynamics of the EGFR ligand gene keren/spitz2, the Notch proteolytic activation factor gene kuzbanian, and the shaggy/zeste white-3 kinase gene are similar to genes that are known direct targets of ecdysone signaling. The induction of hedgehog follows a secondary response pattern, as do genes from the E(spl) complex that are induced in response to Notch activation, although these induction kinetics are also consistent with these genes being partially activated directly by the ecdysone receptor and partially with other factors (i.e., they may be 'early-late' genes). These data show that the regulatory network controlled by ecdysone in midguts includes the activation of known components of the Wnt, EGFR, Hedgehog, and Notch pathways. Notably, ligand production for the EGF, Hedgehog, TGFβ/BMP, and Notch pathways is under control of ecdysone. The specific roles that each of these pathways plays during metamorphosis are currently unknown. These results nonetheless indicate new connections between ecdysone signaling and the activity of several other signaling pathways during the metamorphosis of this organ, either through direct targeting of the ecdysone receptor or through the actions of downstream factors (Li, 2003).

    Ecdysone-induced receptor tyrosine phosphatase PTP52F regulates Drosophila midgut histolysis by enhancement of autophagy and apoptosis

    The rapid removal of larval midgut is a critical developmental process directed by molting hormone ecdysone during Drosophila metamorphosis. To date, it remains unclear how the stepwise events can link the onset of ecdysone signaling to the destruction of larval midgut. This study investigated whether ecdysone-induced expression of receptor protein tyrosine phosphatase PTP52F regulates this process. The mutation of the Ptp52F gene caused significant delay in larval midgut degradation. Transitional endoplasmic reticulum ATPase (TER94), a regulator of ubiquitin proteasome system, was identified as a substrate and downstream effector of PTP52F in the ecdysone signaling. The inducible expression of PTP52F at the puparium formation stage resulted in dephosphorylation of TER94 on its Y800 residue, ensuring the rapid degradation of ubiquitylated proteins. One of the proteins targeted by dephosphorylated TER94 was found to be Drosophila inhibitor of apoptosis 1 (DIAP1), which was rapidly proteolyzed in cells with significant expression of PTP52F. Importantly, the reduced level of DIAP1 in response to inducible PTP52F was essential not only for the onset of apoptosis but also for the initiation of autophagy. This study demonstrates a novel function of PTP52F in regulating ecdysone-directed metamorphosis via enhancement of autophagic and apoptotic cell death in doomed Drosophila midguts (Santhanam, 2014).

    This study shows that ecdysone-induced expression of PTP52F and the subsequent tyrosine dephosphorylation of TER94 coordinate to construct upstream signaling determinants for a precise time-dependent degradation of larval midgut. The transient expression of Ptp52F gene at the PF stage is regulated by the functional EcR. Immediately after the level of endogenous PTP52F protein is detectable in larval midgut, TER94 becomes dephosphorylated on its Y800 residue. This modification may be critical to the rapid degradation of ubiquitylated proteins through a TER94-dependent regulation of ubiquitin proteasome system (UPS). Although the exact mechanism remains elusive, recent studies have suggested that only the tyrosine-dephosphorylated form and not the tyrosine-phosphorylated form of VCP interacts with cofactors for processing ubiquitylated substrates of UPS. Because VCP and TER94 share some evolutionarily conserved features, it is proposed that the same phosphorylation- and dephosphorylation-dependent mechanism may be adopted by TER94. Ubiquitylated DIAP1, a potential substrate of UPS, was found to be targeted by the Y800 dephosphorylated form of TER94. DIAP1 was rapidly degraded in cells in which levels of PTP52F were increased, as illustrated by in vivo observations in Drosophila midgut during metamorphosis. Consequently, the proteolysis of DIAP1 in response to inducible expression of PTP52F terminates the inhibitory effect on autophagy, allowing the initiation of autophagic cell death accompanied by apoptotic cell death for the destruction of the larval midgut tissues. Since the regulatory role of all Drosophila homologs of caspases have been ruled out in the process of larval midgut histolysis, it is likely that DIAP1 degradation-induced autophagic signaling may activate a yet-unknown pathway leading to the onset of apoptotic cell death in dying midgut. Additional experiments are needed to identify downstream effectors of PTP52F that modulate the cross talk between autophagy and apoptosis in the context of midgut maturation (Santhanam, 2014).

    Identification of TER94 as a substrate dephosphorylated by PTP52F in larval midgut is interesting and important. From the time of their original cloning and identification, Drosophila TER94 and its vertebrate ortholog VCP have been characterized as key mediators involved in ER-associated degradation (ERAD), a major quality control process in the protein secretary pathway. Additional investigations have demonstrated degradation of proteins with no obvious relationship to ERAD by a VCP-mediated process, suggesting that TER94 and VCP may perform general functions in the proteolysis of ubiquitylated proteins. However, it remains unknown how this process is regulated under physiological conditions. The current study presents evidence that TER94-dependent degradation of ubiquitylated proteins is enhanced by PTP52F-mediated dephosphorylation of the penultimate Y800 residue. It has been suggested that the penultimate tyrosine (Y805 in VCP and Y800 in TER94) must be in a dephosphorylated form in order to interact with substrate-processing cofactors, such as the peptides N-glycanase (PNGase) and Ufd3, during UPS-mediated proteolysis. In addition, tyrosine phosphorylation levels of VCP/TER94 determine how fast ubiquitylated proteins are degraded by the USP pathway. Clearly, the finding that PTP52F is responsible for dephosphorylation of the penultimate tyrosine residue is critical for uncovering the functional role of VCP/TER94 in the regulation of protein degradation under physiologically relevant conditions (Santhanam, 2014).

    This study has demonstrated that the timely degradation of DIAP1 in doomed larval midgut of developing flies is regulated by ecdysone-induced PTP52F. DIAP1 was identified to ubiquitylate proapoptotic proteins in living cells, thereby suppressing cell death signaling. Interestingly, DIAP1 can be ubiquitylated for degradation itself. The proteolytic process of ubiquitylated DIAP1 remained unclear until a recent report suggesting that TER94-mediated UPS pathway is involved in this process. This study has further shown that it is the dephosphorylated form of TER94 that is responsible for rapid DIAP1 degradation. In addition, although a previous study suggested that DIAP1 might suppress Atg1-mediated PCD, it was not known whether degradation of ubiquitylated DIAP1 could promote autophagy in vivo. This study has explored the underlying mechanism through which autophagic cell death is initiated by degradation of DIAP1. The data show that the constitutively tyrosine-phosphorylated form of TER94 acts as a gatekeeper ensuring the death signaling downstream of DIAP1 in'switch-off' mode. Developmental stage-dependent dephosphorylation of TER94 by inducible expression of PTP52F converts the autophagic death signaling into 'switch-on' mode through degradation of DIAP1. These findings thus explain, at least in part, how the massive destruction of larval midgut is precisely controlled by autophagic cell death. In conclusion, this study shows a novel function of PTP52F involved in the onset of autophagy and apoptosis essential for the removal of obsolete midgut tissues. Reversible tyrosine phosphorylation signaling controlled by PTP52F plays an indispensable role in the process of cell death-directed midgut maturation. Therefore, these findings open a new avenue for understanding the previously unexplored function of R-PTPs linked to regulation of autophagic and apoptotic cell death (Santhanam, 2014).

    Dpp regulates autophagy-dependent midgut removal and signals to block ecdysone production

    Animal development and homeostasis require the programmed removal of cells. Autophagy-dependent cell deletion is a unique form of cell death often involved in bulk degradation of tissues. In Drosophila the steroid hormone ecdysone controls developmental transitions and triggers the autophagy-dependent removal of the obsolete larval midgut. The production of ecdysone is exquisitely coordinated with signals from numerous organ systems to mediate the correct timing of such developmental programs. This study shows that blocking Dpp signaling induces premature autophagy, rapid cell death, and midgut degradation, whereas sustained Dpp signaling inhibits autophagy induction. Furthermore, Dpp signaling in the midgut prevents the expression of ecdysone responsive genes and impairs ecdysone production in the prothoracic gland. It is proposed that Dpp has dual roles: one within the midgut to prevent improper tissue degradation, and one in inter-organ communication to coordinate ecdysone biosynthesis and developmental timing (Denton, 2018).

    Apoptosis restores cellular density by eliminating a physiologically or genetically induced excess of enterocytes in the Drosophila midgut

    Using pathogens or high levels of opportunistic bacteria to damage the gut, studies in Drosophila have identified many signaling pathways involved in gut regeneration. Dying cells emit signaling molecules that accelerate intestinal stem cell proliferation and progenitor differentiation to replace the dying cells quickly. This process has been named 'regenerative cell death'. This study, mimicking environmental conditions, showed that the ingestion of low levels of opportunistic bacteria was sufficient to launch an accelerated cellular renewal program despite the brief passage of bacteria in the gut and the absence of cell death and this is is due to the moderate induction of the JNK pathway that stimulates stem cell proliferation. Consequently, the addition of new differentiated cells to the gut epithelium, without preceding cell loss, leads to enterocyte overcrowding. Finally, it was shown that a couple of days later, the correct density of enterocytes is promptly restored by means of a wave of apoptosis involving Hippo signaling and preferential removal of old enterocytes (Loudhaief, 2017).

    Suppression of insulin production and secretion by a Decretin hormone

    Decretins, hormones induced by fasting that suppress insulin production and secretion, have been postulated from classical human metabolic studies. From genetic screens, this study identified Drosophila Limostatin (Lst), a peptide hormone that suppresses insulin secretion. Lst is induced by nutrient restriction in gut-associated endocrine cells. limostatin deficiency leads to hyperinsulinemia, hypoglycemia, and excess adiposity. A conserved 15-residue polypeptide encoded by limostatin suppresses secretion by insulin-producing cells. Targeted knockdown of CG9918, a Drosophila ortholog of mammalian Neuromedin U receptors (NMURs), in insulin-producing cells phenocopied limostatin deficiency and attenuated insulin suppression by purified Lst, suggesting CG9918 encodes an Lst receptor. Human NMUR1 is expressed in islet β cells, and purified NMU suppressed insulin secretion from human islets. A human mutant NMU variant that co-segregates with familial early-onset obesity and hyperinsulinemia failed to suppress insulin secretion. The study proposes Lst as an index member of an ancient hormone class called decretins, which suppress insulin output. (Alfa, 2015).

    The coupling of hormonal responses to nutrient availability is fundamental for metabolic control. In mammals, regulated secretion of insulin from pancreatic b cells is a principal hormonal response orchestrating metabolic homeostasis. Circulating insulin levels constitute a dynamic metabolic switch, signaling the fed state and nutrient storage (anabolic pathways) when elevated, or starvation and nutrient mobilization (catabolic path ways) when decreased. Thus, insulin secretion must be precisely tuned to the nutritional state of the animal. Increased circulating glucose stimulates b cell depolarization and insulin secretion. In concert with glucose, gut-derived incretin hormones amplify glucose-stimulated insulin secretion (GSIS) in response to ingested carbohydrates, thereby tuning insulin output to the feeding state of the host (Alfa, 2015).

    While the incretin effect on insulin secretion during feeding is well-documented, counter-regulatory mechanisms that suppress insulin secretion during or after starvation are incompletely understood. Classical starvation experiments in humans and other mammals revealed that sustained fasting profoundly alters the dynamics of insulin production and secretion, resulting in impaired glucose tolerance, relative insulin deficits, and 'starvation diabetes'. Remarkably, starvation-induced suppression of GSIS was not reverted by normalizing circulating glucose levels, suggesting that the dampening effect of starvation on insulin secretion perdures and is uncoupled from blood glucose and macronutrient concentrations. Based on these observations, it has been postulated that hormonal signals induced by fasting may actively attenuate insulin secretion suggested that enteroendocrine 'decretin' hormones may constrain the release of insulin to prevent hypoglycemia. This concept is further supported by recent studies identifying a G protein that suppresses insulin secretion from pancreatic b cell. Thus, after nutrient restriction, decretin hormones could signal through G protein-coupled receptors (GPCRs) to attenuate GSIS from b cells (Alfa, 2015).

    The discovery of hormonal pathways regulating metabolism in mammals presents a formidable challenge. However, progress has revealed conserved mechanisms of metabolic regulation by insulin and glucagon-like peptides in Drosophila, providing a powerful genetic model to address unresolved questions relevant to mammalian metabolism. Similar to mammals, secretion of Drosophila insulin-like peptides (Ilps) from neuroendocrine cells in the brain regulates glucose homeostasis and nutrient stores in the fly. Ilp secretion from insulin-producing cells (IPCs) is responsive to circulating glucose and macronutrients and is suppressed upon nutrient withdrawal. Notably, recent studies have identified hormonal and GPCR-linked mechanisms regulating the secretion of Ilps from IPCs, suggesting further conservation of pathways regulating insulin secretion in the fly (Alfa, 2015).

    In mammals, the incretin hormones gastric inhibitory peptide (GIP) and glucagon-like peptide-1 (GLP-1) are secreted by enteroendocrine cells following a meal and enhance glucose-stimulated insulin production and secretion from pancreatic b cells. Thus, It was postulated that a decretin hormone would have the 'opposite' hallmarks of incretins. Specifically, a decretin (1) derives from an enteroendocrine source that is sensitive to nutrient availability, (2) is responsive to fasting or carbohydrate deficiency, and (3) suppresses insulin production and secretion from insulin-producing cells. However, like incretins, the action of decretins on insulin secretion would be manifest during feeding, when a stimulus for secretion is present (Alfa, 2015).

    This study identifed a secreted hormone, Limostatin (Lst), that suppresses insulin secretion following starvation in Drosophila. lst is regulated by starvation, and flies deficient for lst display phenotypes consistent with hyperinsulinemia. Lst production was shown to be localized to glucose-sensing, endocrine corpora cardiaca (CC) cells associated with the gut, and show that lst is suppressed by carbohydrate feeding. Using calcium imaging and in vitro insulin secretion assays, a 15-aa Lst peptide (Lst-15) was identified that is sufficient to suppress activity of IPCs and Ilp secretion. An orphan GPCR was identified in IPCs as a candidate Lst receptor. Moreover, Neuromedin U (NMU) is likely a functional mammalian ortholog of Lst that inhibits islet b cell insulin secretion. These results establish a decretin signaling pathway that suppresses insulin output in Drosophila (Alfa, 2015).

    Limostatin is a peptide hormone induced by carbohydrate restriction from endocrine cells associated with the gut that suppresses insulin production and release by insulin-producing cells. Thus, Drosophila Lst fulfills the functional criteria for a decretin and serves as an index member of this hormone class in metazoans. Results here also show that Lst signaling from corpora cardica cells may be mediated by the GPCR encoded by CG9918 in insulin-producing cells. In addition, the results reveal cellular and molecular features of a cell-cell signaling system in Drosophila with likely homologies to a mammalian entero-insular axis (Alfa, 2015).

    Reduction of nutrient-derived secretogogues, like glucose, is a primary mechanism for attenuating insulin output during starvation in humans and flies. Consistent with this, it was found that circulating Ilp2HF levels were reduced to a similar degree in lst mutant or control flies during prolonged fasting. Therefore, lst was dispensable for Ilp2 reduction during fasting. However, lst mutants upon refeeding or during subsequent ad libitum feeding had enhanced circulating Ilp2HF levels compared to controls, findings that demonstrate a requirement for Lst to restrict insulin output in fed flies. Thus, while induced by nutrient restriction, Lst decretin function was revealed by nutrient challenge. This linkage of feeding to decretin regulation of insulin output is reminiscent of incretin regulation and action (Campbell, 2013; Alfa, 2015 and references therein).

    Recent studies have demonstrated functional conservation in Drosophila of fundamental hormonal systems for metabolic regulation in mammals, including insulin, glucagon, and leptin (Rajan, 2012). This study used Drosophila to identify a hormonal regulator of insulin output, glucose, and lipid metabolism without an identified antecedent mammalian ortholog -- emphasizing the possibility for work on flies to presage endocrine hormone discovery in mammals. Gain of Lst function in these studies led to reduced insulin signaling, and hyperglycemia, consistent with prior work. By contrast, loss of Lst function led to excessive insulin production and secretion, hypoglycemia, and elevated triglycerides, phenotypes consistent with the recognized anabolic functions of insulin signaling in metazoans, and with the few prior metabolic studies of flies with insulin excess (Alfa, 2015).

    Prior studies show that somatostatin and galanin are mammalian gastrointestinal hormones that can suppress insulin secretion. Somatostatin-28 (SST-28) is a peptide derivative of the pro-somatostatin gene that is expressed widely, including in gastrointestinal cells and pancreatic islet cells. Islet somatostatin signaling is thought to be principally paracrine, rather than endocrine, and serum SST-28 concentrations increase post-prandially. Galanin is an orexigenic neuropeptide produced throughout the CNS and in peripheral neurons and has been reported to inhibit insulin secretion. Unlike enteroendocrine-derived hormones that act systemically, galanin is secreted from intrapancreatic autonomic nerve terminals and is thought to exert local effects. In addition, Galanin synthesis and secretion are increased by feeding and dietary fat. Thus, like incretins, output of SST- 28 and galanin are induced by feeding, but in contrast to incretins, these peptides suppress insulin secretion. Further studies are needed to assess the roles of these peptide regulators in the modulation of insulin secretion during fasting (Alfa, 2015).

    While sequence-based searches did not identify vertebrate orthologs of Lst, this study found that the postulated Lst receptor in IPCs, encoded by CG9918, is most similar to the GPCRs NMUR1 and NMUR2. In rodents, NMU signaling may be a central regulator of satiety and feeding behavior, and this role may be conserved in other organisms. In addition, NMU mutant mice have increased adiposity and hyperinsulinemia, but a direct role for NMU in regulating insulin secretion by insulin-producing cells was not identified. In rodents, the central effects of NMU on satiety are thought to be mediated by the receptor NMUR2; however, hyperphagia, hyperinsulinemia, and obesity were not reported in NMUR2 mutant mice. Together, these studies suggest that a subset of phenotypes observed in NMU mutant mice may instead reflect the activity of NMU on peripheral tissues like pancreatic islets, but this has not been previously shown. Notably, humans harboring the NMU R165W allele displayed obesity and elevated insulin C-peptide levels, without evident hyperphagia -- further suggesting that the central and peripheral effects of NMU reflect distinct pathways that may be uncoupled. This study has shown that NMU is produced abundantly in human foregut organs and suppresses insulin secretion from pancreatic b cells, supporting the view that NMU has important functions outside the CNS in regulating metabolism. Thus, like the incretin GLP-1, NMU may have dual central and peripheral signaling functions in the regulating metabolism. Demonstration that NMU is a mammalian decretin will require further studies on NMU regulation and robust methods to measure circulating NMU levels in fasting and re-feeding. In summary, these findings should invigorate searches for mammalian decretins with possible roles in both physiological and pathological settings (Alfa, 2015).

    A subset of enteroendocrine cells is activated by amino acids in the Drosophila midgut

    The intestine is involved in digestion and absorption, as well as the regulation of metabolism upon sensation of the internal intestinal environment. Enteroendocrine cells are thought to mediate these internal intestinal chemosensory functions. Using the CaLexA (calcium-dependent nuclear import of LexA) method, this study examined the enteroendocrine cell populations that are activated when flies are subjected to various dietary conditions such as starvation, sugar, high fat, protein, or pathogen exposure. A specific subpopulation of enteroendocrine cells in the posterior midgut that express Dh31 and tachykinin was found to be activated by the presence of proteins and amino acids (Park, 2016).

    To study the chemosensory functions of enteroendocrine cells in the Drosophila midgut, a method was needed to specifically label as many enteroendocrine cells as possible. In an independent study, Drosophila regulatory peptide genes were sought that are expressed in the enteroendocrine cells. The sum of expression of three regulatory peptide-GAL4 drivers (AstC-, Npf-, and Dh31-GAL4; for convenience, hereafter these three drivers together will be called EE-GAL4) were found to be expressed in about 80% of enteroendocrine cells in the midgut (Park, 2016).

    To visualize in vivo changes in cytoplasmic calcium ion concentration, the CaLexA (calcium-dependent nuclear import of LexA) system, which uses a calcium ion-sensitive synthetic transcription factor, was used. When calcium levels rise, modified nuclear factor of activated T cells (NFAT) is imported into the nucleus to transcriptionally activate GFP reporter expression. This CaLexA system has been successfully used to visualize neuronal activation and calcium ion changes in fat body tissue (Park, 2016).

    First, whether the CaLexA system could be used to monitor enteroendocrine cell activation, was tested. For this purpose, modified NFAT was expressed in enteroendocrine cells using EE-GAL4. GFP expression in the midgut was monitored after feeding adult flies with 200 mm sucrose for 5 days after eclosion. Sucrose was provided as a minimal nutrient source. Strong GFP expression was observed only in enteroendocrine cells in the middle midgut, while most enteroendocrine cells in the anterior and posterior midgut did not express GFP. Next, whether various dietary conditions could activate the enteroendocrine cells was tested, as well as whether stimuli or enteroendocrine cell specificity exists. Enteroendocrine cell activation in the middle midgut was observed for every tested condition including starvation, indicating that the middle midgut enteroendocrine cells are constitutively activated. The middle midgut is an acidic region, and acid secretion from the enteroendocrine cells likely constitutively occurs to maintain such an environment (Park, 2016).

    To quantitate enteroendocrine cell activation using the CaLexA system, the numbers of GFP-expressing cells was counted. GFP-expressing cells in the caudal half of the posterior midgut were counted, since enteroendocrine cell activation upon exposure to various dietary conditions was concentrated to this region. Only GFP-expressing cells costaining with Prospero were counted, excluding autofluorescent signals from food particles. These nonspecific signals can be observed as white dots that do not costain with Prospero, as seen for example in starvation or 50 mm NaCl conditions. When flies were provided with a single sugar diet composed of only sucrose, 14 ± 1.5 SEM GFP-expressing cells were observed in the caudal half of the posterior midgut. This can be considered a baseline for all of the conditions tested, with the exception of starvation, normal fly food, and high fat diet, since 200 mm sucrose was provided in all but these three conditions to induce a quantifiable level of feeding even in adverse conditions. A small number of posterior midgut enteroendocrine cells were activated when cornmeal-based fly food, commonly used in the lab, was provided. Significant activation was not observed when flies were exposed to conditions such as a diet of normal food with high fat composition, starvation, or a diet of 200 mm sucrose with the addition of 50 mm NaCl. In contrast, many enteroendocrine cells were activated in the posterior midgut upon oral infection with Erwinia carotovora carotovora 15 (Ecc15), which causes a gut immune respons. Enteroendocrine cell activation was observed at a similar level when flies were fed Pseudomonas aeruginosa, indicating that the enterondocrine cell response is not specific to a particular bacterial species. Enteroendocrine cell activation was also observed upon feeding on heat-inactivated Ecc15 or P. aeruginosa, at slightly decreased levels compared to untreated bacteria, but still higher than the 200 mm sucrose control flies. In contrast, enteroendocrine cells were not activated upon flies being fed uracil, which causes a gut immune response through acting as a DUOX-activating ligand in the Drosophila gut epithelia. These results indicated that enteroendocrine cell activation is not due to the detection of pathogenicity. Supporting this conclusion, ingestion of nonpathogenic E. coli or yeast also caused enteroendocrine cell activation. On the basis of these results, it is hypothesized that the enteroendocrine cells were being activated by the presence of protein. Enteroendocrine cell activation was observed in a dose-dependent manner when flies were fed various concentrations of casein peptone, providing evidence that protein cues were activating the enteroendocrine cells. Next, the twenty amino acids were all individually tested for enteroendocrine cell activation. The amino acids caused enteroendocrine cell activation, indicating that enteroendocrine cells are activated upon detection of most, if not all, amino acids. Aspartic acid and glutamic acid, which contain negatively charged side chains, caused a relatively low number of activated cells, but this is likely due to the lower concentration used due to solubility issues. The number of activated cells also increased in a dose-dependent manner, suggesting that these amino acids act as cues to activate enteroendocrine cells. For similar reasons, poorly water-soluble tyrosine also cannot be excluded as a potential activation cue for enteroendocrine cells (Park, 2016).

    Enteroendocrine cells in the posterior midgut are activated by protein and amino acids. GFP expression pattern induced by the CaLexA system in the caudal half of the posterior midgut upon exposure to the indicated dietary conditions (Park, 2016).

    Enteroendocrine cell activation by microorganisms, casein peptone, and amino acids was concentrated in enteroendocrine cells of the posterior midgut, in particular the caudal half. Enteroendocrine cells in this region can be largely divided into two populations, with one population expressing the regulatory peptides AstA and AstC, and the other expressing Dh31 and tachykinin. To examine which population of enteroendocrine cells in the caudal half of the posterior midgut are activated by microorganisms, casein peptone, and amino acids, the enteroendocrine cells were costained with AstA or Dh31 antisera. Although EE-GAL4 is expressed in both populations of enteroendocrine cells in the caudal half of the posterior midgut, most of the activated enteroendocrine cells belonged to the Dh31-expressing cell population. When Ecc15 was provided, an average of 68 cells label with Dh31 antiserum in the corresponding region, and 54 out of 61 cells that express GFP through the CaLexA system co-stain with Dh31 antiserum. Under the same conditions, an average of 96 cells are labeled by AstA antiserum, and only two of 64 cells expressing GFP through the CaLexA system co-stain with AstA antiserum . In conclusion, amino acids in proteins activate a specific subset of Dh31- and tachykinin-expressing enteroendocrine cells in the posterior midgut through an as yet unknown mechanism. Since the activation of these cells appears to be unrelated to pathogenicity of the protein source, it seems unlikely that these enteroendocrine cells are directly involved in eliciting an immune response to pathogens. The role of this enteroendocrine cell subgroup thus appears to mainly be detection of a potential nutrient source through its protein content. It is unclear whether activation of these cells by amino acids influences the production and/or secretion of Dh31 or other regulatory peptides. Weaker Dh31 antibody staining was not observed in GFP-expressing cells, which would be expected if Dh31 secretion was enhanced in the activated cells. It is formally possible that Dh31 secretion is enhanced in activated cells, and production is increased to compensate for the increased secretion, but the data are insufficient to provide support for either scenario. Tachykinin production is enhanced in the midgut upon nutrient deprivation, resulting in repression of lipogenesis in enterocytes. However, in this study, the enteroendocrine cells activated by proteins and amino acids were not activated upon starvation, and sucrose was coprovided as a minimal nutrient in all dietary conditions providing proteins and amino acids, with the sucrose-only basal level acting as a baseline for activation. This indicates that proteins and amino acids, and not other cues such as carbohydrate deprivation, act as specific cues to activate a particular subset of enteroendocrine cells. The molecular identity of potential protein or amino acid receptors in the enteroendocrine cells is as yet unknown. In mammals, several amino acids including L-glutamine have been shown to stimulate GLP1 secretion in vitro. CaSR, a primarily Gq-coupled calcium-sensing receptor, is expressed in the enteroendocrine cells, and CaSR activation has been associated with amino acid-stimulated gut hormone secretion. The umami taste receptor dimer T1R1/T1R3 and GPRC6A are also additional candidates for mediating GLP1 release in response to amino acids. This work provides an in vivo enteroendocrine system to investigate such possible amino acid receptors (Park, 2016).

    This study has shown that the CaLexA system can be used to monitor the activation of Drosophila midgut enteroendocrine cells upon exposure to specific stimuli. Activation of enteroendocrine cells upon exposure to sugar, fat, and bitter compounds was not observed using this method. It was found that proteins and most amino acids are capable of activating enteroendocrine cells in the posterior midgut. These activated cells are limited to a subpopulation of enteroendocrine cells that secrete specific regulatory peptides including Dh31 and tachykinin. This study provides an important step in studying the chemosensory functions of enteroendocrine cells (Park, 2016).

    A subset of neurons controls the permeability of the peritrophic matrix and midgut structure in Drosophila adults

    The metazoan gut performs multiple physiologic functions, including digestion and absorption of nutrients, and also serves as a physical and chemical barrier against ingested pathogens and abrasive particles. Maintenance of these functions and structures is partly controlled by the nervous system, yet the precise roles and mechanisms of the neural control of gut integrity remain to be clarified in Drosophila. This study screened for GAL4 enhancer-trap strains and labeled specific subsets of neurons. The strong inward rectifier potassium channel Kir2.1 was used to inhibit their neuronal activity. An NP3253 line was identified that is susceptible to oral infection by Gram-negative bacteria. The subset of neurons driven by the NP3253 line includes some of the enteric neurons innervating the anterior midgut, and these flies have a disorganized proventricular structure with high permeability of the peritrophic matrix and epithelial barrier. The findings of the present study indicate that neural control is crucial for maintaining the barrier function of the gut, and provide a route for genetic dissection of the complex brain-gut axis in the model organism Drosophila adults (Kenmoku, 2016).

    Metamorphosis of the Drosophila visceral musculature and its role in intestinal morphogenesis and stem cell formation

    This study has combined the use of specific markers with electron microscopy to follow the formation of the adult visceral musculature and its involvement in gut development during metamorphosis. Unlike the adult somatic musculature, which is derived from a pool of undifferentiated myoblasts, the visceral musculature of the adult is a direct descendant of the larval fibers, as shown by activating a lineage tracing construct in the larval muscle and obtaining labeled visceral fibers in the adult. However, visceral muscles undergo a phase of remodeling that coincides with the metamorphosis of the intestinal epithelium. During the first day following puparium formation, both circular and longitudinal syncytial fibers dedifferentiate, losing their myofibrils and extracellular matrix, and dissociating into mononuclear cells ("secondary myoblasts"). Towards the end of the second day, this process is reversed, and between 48 and 72h after puparium formation, a structurally fully differentiated adult muscle layer has formed. The musculature, the intestinal epithelium is completely renewed during metamorphosis. The adult midgut epithelium rapidly expands over the larval layer during the first few hours after puparium formation; in case of the hindgut, replacement takes longer, and proceeds by the gradual caudad extension of a proliferating growth zone, the hindgut proliferation zone (HPZ). The subsequent elongation of the hindgut and midgut, as well as the establishment of a population of intestinal stem cells active in the adult midgut and hindgut, requires the presence of the visceral muscle layer, based on the finding that ablation of this layer causes a severe disruption of both processes (Aghajanian, 2016).

    Regional cell-specific transcriptome mapping reveals regulatory complexity in the adult Drosophila midgut

    Deciphering contributions of specific cell types to organ function is experimentally challenging. The Drosophila midgut is a dynamic organ with five morphologically and functionally distinct regions (R1-R5), each composed of multipotent intestinal stem cells (ISCs), progenitor enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs), and visceral muscle (VM). To characterize cellular specialization and regional function in this organ, RNA-sequencing transcriptomes were generated of all five cell types isolated by FACS from each of the five regions, R1-R5. In doing so, transcriptional diversities were identified among cell types, and regional differences within each cell type were documented that define further specialization. Cell-specific and regional Gal4 drivers were validated; roles for transporter Smvt and transcription factors GATAe, Sna, and Ptx1 in global and regional ISC regulation were demonstrated, and the transcriptional response of midgut cells upon infection was studied. The resulting transcriptome database (http://flygutseq.buchonlab.com) will foster studies of regionalization, homeostasis, immunity, and cell-cell interactions (Dutta, 2015).

    Misregulation of an adaptive metabolic response contributes to the age-related disruption of lipid homeostasis in Drosophila

    Loss of metabolic homeostasis is a hallmark of aging and is commonly characterized by the deregulation of adaptive signaling interactions that coordinate energy metabolism with dietary changes. The mechanisms driving age-related changes in these adaptive responses remain unclear. This study characterized the deregulation of an adaptive metabolic response and the development of metabolic dysfunction in the aging intestine of Drosophila. Activation of the insulin-responsive transcription factor Foxo in intestinal enterocytes was found to be required to inhibit the expression of evolutionarily conserved lipases as part of a metabolic response to dietary changes. This adaptive mechanism becomes chronically activated in the aging intestine, mediated by changes in Jun-N-terminal kinase (JNK) signaling. Age-related chronic JNK/Foxo activation in enterocytes is deleterious, leading to sustained repression of intestinal lipase expression and the disruption of lipid homeostasis. Changes in the regulation of Foxo-mediated adaptive responses thus contribute to the age-associated breakdown of metabolic homeostasis (Karpac, 2013).

    This work identifies Foxo-mediated repression of intestinal lipases as a critical component of an adaptive response to dietary changes in Drosophila. Interestingly, misregulation of this metabolic response also contributes to the age-associated breakdown of lipid homeostasis, as elevated JNK signaling leads to chronic Foxo activation and subsequent disruption of lipid metabolism due to chronic repression of lipases. This age-related deregulation of an adaptive metabolic response is reminiscent of insulin resistance-like phenotypes in vertebrates, which can also be triggered by chronic activation of JNK, and thus highlights the antagonistic pleiotropy inherent in metabolic regulation. The adaptive nature of signaling interactions that drive pathology (such as JNK-mediated insulin resistance) has remained elusive in many instances, and this work provides a model for age-related changes in an adaptive regulatory process that ultimately lead to a pathological outcome. It is believed that this system can serve as a productive model to address a number of interesting questions with relevance to the loss of metabolic homeostasis in aging organisms (Karpac, 2013).

    In mammals, JNK has been shown to promote insulin resistance both cell-autonomously and systemically (through inflammation), subsequently affecting lipid homeostasis in various tissues. The current results further introduce a mechanism by which JNK can alter cellular and systemic lipid metabolism through the regulation of lipases, independent of changes in IIS. Thus, JNK-mediated Foxo activation in select tissues may be able to alter intracellular lipid metabolism, changing metabolic fuel substrates and disrupting metabolic homeostasis in other tissues without altering insulin responsiveness (Karpac, 2013).

    Whereas the current data show that Foxo activation leads to the transcriptional repression of intestinal lipases, especially LipA/Magro, it remains unclear if this control is direct or indirect. Foxo is classically described as an activator of transcription, but recent reports have shown that Foxo can transcriptionally repress genes through direct association with promoters. The promoter regions of LipA/Magro and CG6295 do not contain conserved Foxo transcription factor binding sites, suggesting that the regulation of these genes may be indirect, potentially through Foxo-regulated expression of secondary effectors. Thus, tissue-specific control of lipid homeostasis by IIS/Foxo might be achieved through the regulation of lipogenic or lipolytic transcription factors that can elicit global and direct changes in cellular lipid metabolism. Previous reports have shown that the nuclear receptor dHR96, a critical regulator of lipid and cholesterol homeostasis, promotes lipA/magro expression. However, dhr96 expression is upregulated in aging intestines, suggesting that the age-related repression of intestinal lipases is not merely due to decreases in dHR96 levels. dhr96 transcript levels are strongly induced in genetic conditions where Foxo is activated and intestinal lipases are repressed, again suggesting that Foxo does not mediate its effects on lipase transcription by antagonizing dhr96 expression. Furthermore, age-related changes that are independent of JNK/Foxo activation may also contribute to the repression of intestinal lipase expression and disruption of lipid metabolism, such as an age-associated decline in feeding/food intake (Karpac, 2013).

    The reasons for the increase in JNK and Foxo activity in aging enterocytes remain to be explored. Buchon (2009) has also shown that age-related activation of JNK in the intestinal epithelium is dependent on the presence of commensal bacteria, as maintaining animals axenically reduces activation of JNK in the first 30 days of life. Thus, bacteria-induced inflammation and subsequent JNK activation appears to be a likely cause, in part, for age-related increases in Foxo activity. In a separate study, however, this laboratory found that Foxo activation still occurs in intestines of old (40-day-old), axenically reared flies, suggesting that age-related activation of Foxo may also occur through JNK-independent processes. Supporting this idea, the results show that inhibiting JNK function in enterocytes can attenuate, although not completely inhibit, this Foxo activation. Additional factors, such as sirtuins or histone deacetylases, recently shown to deacetylate and activate Foxo in response to endocrine signals, may also lead to age-related increases in intestinal Foxo activity (Karpac, 2013).

    Interactions between JNK and IIS/Foxo are mediated by various mechanisms. In mammals, JNK phosphorylates the insulin receptor substrate (IRS), inhibiting insulin signaling transduction. Whereas JNK has clearly been shown to antagonize IIS (activate Foxo) in C. elegans and Drosophila, that exact mechanism by which Foxo activation is achieved may be divergent in mammals. For example, no IRS homolog has been identified in worms, and the JNK phosphorylation site in mammalian IRS is not conserved in flies. The current data show that JNK-mediated Foxo activation in the aging fly intestine is not achieved through IIS antagonism upstream of Akt, suggesting either a direct interaction between Foxo and JNK or changes in other regulators of Foxo. Recent studies have shown that JNK-mediated phosphorylation of 14-3-3 proteins results in the release of their binding partners, including Foxo. The conservation of 14-3-3 proteins between vertebrates and invertebrates makes 14-3-3 an interesting candidate in promoting Foxo function via JNK in the aging fly intestine. This chronic intestinal Foxo activation and subsequent metabolic changes, provide a physiological system in Drosophila to genetically dissect the crosstalk between IIS/Foxo and JNK signaling. Detailed analysis of these signaling interactions promises to provide important insight into the pleiotropic effects of IIS/Foxo function and the pathogenesis of age-related metabolic diseases (Karpac, 2013).

    The data further reveal the pleiotropic consequences of Foxo activation in regard to healthspan and longevity in Drosophila. Overexpressing Foxo in the fat body or muscle of flies leads to lifespan extension. The data presented here show that chronic Foxo activation in intestinal enterocytes disrupts lipid metabolism by deregulating intestinal lipases and thus highlight how cell- and tissue-specific consequences of Foxo function play an important role in determining either the beneficial (i.e., lifespan extension) or pathological (i.e., disruption of lipid metabolism) outcome of Foxo activation (Karpac, 2013).

    Recent work in C. elegans has begun to explore the relationship between lipid metabolism and longevity, revealing that increases in intestinal lipase expression can extend lifespan. The beneficial effects of elevated lipase expression appear to be mediated by increases in specific types of fatty acids, which can activate autophagy and lead to lifespan extension. The current study identifies Foxo-mediated repression of intestinal lipases as a critical component of an adaptive response to dietary changes in Drosophila. Interestingly, misregulation of this metabolic response also contributes to the age-associated breakdown of lipid homeostasis, as elevated JNK signaling leads to chronic Foxo activation and subsequent disruption of lipid metabolism due to chronic repression of lipases. This age-related deregulation of an adaptive metabolic response is reminiscent of insulin resistance-like phenotypes in vertebrates, which can also be triggered by chronic activation of JNK, and thus highlights the antagonistic pleiotropy inherent in metabolic regulation. The adaptive nature of signaling interactions that drive pathology (such as JNK-mediated insulin resistance) has remained elusive in many instances, and the current work provides a model for age-related changes in an adaptive regulatory process that ultimately lead to a pathological outcome. This system can serve as a productive model to address a number of interesting questions with relevance to the loss of metabolic homeostasis in aging organisms (Karpac, 2013).

    The results further introduce a mechanism by which JNK can alter cellular and systemic lipid metabolism through the regulation of lipases, independent of changes in IIS. Thus, JNK-mediated Foxo activation in select tissues may be able to alter intracellular lipid metabolism, changing metabolic fuel substrates and disrupting metabolic homeostasis in other tissues without altering insulin responsiveness (Karpac, 2013).

    Whereas the data show that Foxo activation leads to the transcriptional repression of intestinal lipases, especially LipA/Magro, it remains unclear if this control is direct or indirect. Foxo is classically described as an activator of transcription, but recent reports have shown that Foxo can transcriptionally repress genes through direct association with promoters. The promoter regions of LipA/Magro and CG6295 do not contain conserved Foxo transcription factor binding sites, suggesting that the regulation of these genes may be indirect, potentially through Foxo-regulated expression of secondary effectors. Thus, tissue-specific control of lipid homeostasis by IIS/Foxo might be achieved through the regulation of lipogenic or lipolytic transcription factors that can elicit global and direct changes in cellular lipid metabolism. Previous reports have shown that the nuclear receptor dHR96, a critical regulator of lipid and cholesterol homeostasis, promotes lipA/magro expression. However, dhr96 expression is upregulated in aging intestines, suggesting that the age-related repression of intestinal lipases is not merely due to decreases in dHR96 levels. dhr96 transcript levels are strongly induced in genetic conditions where Foxo is activated and intestinal lipases are repressed, again suggesting that Foxo does not mediate its effects on lipase transcription by antagonizing dhr96 expression. Furthermore, age-related changes that are independent of JNK/Foxo activation may also contribute to the repression of intestinal lipase expression and disruption of lipid metabolism, such as an age-associated decline in feeding/food intake (Karpac, 2013).

    The reasons for the increase in JNK and Foxo activity in aging enterocytes remain to be explored. Age-related activation of JNK in the intestinal epithelium is dependent on the presence of commensal bacteria, as maintaining animals axenically reduces activation of JNK in the first 30 days of life. Thus, bacteria-induced inflammation and subsequent JNK activation appears to be a likely cause, in part, for age-related increases in Foxo activity. In a separate study, however, it was found that Foxo activation still occurs in intestines of old (40-day-old), axenically reared flies, suggesting that age-related activation of Foxo may also occur through JNK-independent processes. Supporting this idea, the current results show that inhibiting JNK function in enterocytes can attenuate, although not completely inhibit, this Foxo activation. Additional factors, such as sirtuins or histone deacetylases, recently shown to deacetylate and activate Foxo in response to endocrine signals, may also lead to age-related increases in intestinal Foxo activity (Karpac, 2013).

    Interactions between JNK and IIS/Foxo are mediated by various mechanisms. In mammals, JNK phosphorylates the insulin receptor substrate (IRS), inhibiting insulin signaling transduction. JNK has also been shown to directly phosphorylate and activate Foxo in mammalian cell culture, that exact mechanism by which Foxo activation is achieved may be divergent in mammals. For example, no IRS homolog has been identified in worms, and the JNK phosphorylation site in mammalian IRS is not conserved in flies. The data show that JNK-mediated Foxo activation in the aging fly intestine is not achieved through IIS antagonism upstream of Akt, suggesting either a direct interaction between Foxo and JNK or changes in other regulators of Foxo. Recent studies have shown that JNK-mediated phosphorylation of 14-3-3 proteins results in the release of their binding partners, including Foxo. This chronic intestinal Foxo activation and subsequent metabolic changes, provide a physiological system in Drosophila to genetically dissect the crosstalk between IIS/Foxo and JNK signaling. Detailed analysis of these signaling interactions promises to provide important insight into the pleiotropic effects of IIS/Foxo function and the pathogenesis of age-related metabolic diseases (Karpac, 2013).

    The data further reveal the pleiotropic consequences of Foxo activation in regard to healthspan and longevity in Drosophila. Overexpressing Foxo in the fat body or muscle of flies leads to lifespan extension. Overexpression of selected cytoprotective Foxo target genes in stem cells, on the other hand, is sufficient to prevent age-associated dysplasia and extend lifespan. The data presented here show that chronic Foxo activation in intestinal enterocytes disrupts lipid metabolism by deregulating intestinal lipases and thus highlight how cell- and tissue-specific consequences of Foxo function play an important role in determining either the beneficial (i.e., lifespan extension) or pathological (i.e., disruption of lipid metabolism) outcome of Foxo activation (Karpac, 2013).

    Recent work in C. elegans has begun to explore the relationship between lipid metabolism and longevity, revealing that increases in intestinal lipase expression can extend lifespan. The beneficial effects of elevated lipase expression appear to be mediated by increases in specific types of fatty acids, which can activate autophagy and lead to lifespan extension. Interventions that promote lipid homeostasis with age, such as JNK/Foxo inhibition in intestinal enterocytes, might thus affect healthspan and/or longevity through means other than primarily maintaining energy homeostasis (Karpac, 2013).

    High sugar diet disrupts gut homeostasis though JNK and STAT pathways in Drosophila

    The incidence of diseases associated with a high sugar diet has increased in the past years, and numerous studies have focused on the effect of high sugar intake on obesity and metabolic syndrome. However, how a high sugar diet influences gut homeostasis is still poorly understood. This study used Drosophila melanogaster as a model organism and supplemented a culture medium with 1 M sucrose to create a high sugar condition. The results indicate that a high sugar diet promoted differentiation of intestinal stem cells through upregulation of the JNK pathway and downregulation of the JAK/STAT pathway. Moreover, the number of commensal bacteria decreased in the high sugar group. These data suggests that the high caloric diet disrupts gut homeostasis and highlights Drosophila as an ideal model system to explore gastrointestinal disease (Zhang, X. 2017).

    The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila

    Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. This study investigated stem-cell death in the digestive system of adult Drosophila melanogaster. It was found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spares differentiated cells. The dying stem cells are engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduce CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers (Singh, 2016)

    To investigate the molecular mechanism behind the resistance of CSCs to therapeutics, the death of stem cells with different degrees of quiescence was studied in the adult Drosophila digestive system, including intestinal stem cells (ISCs). Expression of the proapoptotic genes rpr and p53 effectively ablated differentiated cells but had little effect on stem cells (Singh, 2016).

    In mammals, treatment-resistant leukaemic stem cells (LSCs) can be eliminated by a two-step protocol involving initial activation by interferon-α (IFNα) or colony-stimulating factor (G-CSF), followed by targeted chemotherapy. In Drosophila, activation of the hopscotch (also known as JAK)-Stat92E signalling pathway induces hyperplastic stem cells, which are overproliferating, but retain their apico-basal polarity and differentiation ability. A slightly different two-step protocol was conducted in Drosophila stem cells by overexpressing the JAK-Stat92E pathway ligand unpaired (upd) and rpr together. The induction of upd + rpr using the temperature-sensitive (ts) mutant esg-Gal4 (esgts > upd + rpr effectively ablated all of the ISCs and RNSCs through apoptosis within four days. Consistent with this result, expressing a gain-of-function Raf mutant (Rafgof) also accelerated apoptotic cell death of hyperplastic ISCs (Singh, 2016).

    Expressing a constitutively active form of Ras oncogene at 85D (also known as RasV12) in RNSCs and the knockdown of Notch activity in ISCs can transform these cell types into CSC-like neoplastic stem cells, which were not only overproliferating, but also lost their apico-basal polarity and differentiation abilit. It ws found that expressing rpr in RasV12-transformed RNSCs or in ISCs expressing a dominant-negative form of Notch (NDN) caused the ablation of only a proportion of the transformed RNSCs and few transformed ISCs and it did not affect differentiated cells; substantial populations of the neoplastic stem cells remained even seven days after rpr induction (Singh, 2016).

    These results suggest that the activation of proliferation can accelerate the apoptotic cell death of hyperplastic stem cells, but that a proportion of actively proliferating neoplastic RNSCs and ISCs are resistant to apoptotic cell death. Neoplastic tumours in Drosophila are more similar to high-grade malignant human tumours than are the hyperplastic Drosophila tumours (Singh, 2016).

    Vesicle-mediated COPI and COPII are essential components of the trafficking machinery for vesicle transportation between the endoplasmic reticulum and the Golgi. In addition, the COPI complex regulates the transport of lipolysis enzymes to the surface of lipid droplets for lipid droplet usage. In a previous screen, it was found that knockdown of COPI components (including Arf79F, the Drosophila homologue of ADP-ribosylation factor 1 (Arf1)) rather than COPII components resulted in stem-cell death, suggesting that lipid-droplet usage (lipolysis) rather than the general trafficking machinery between the endoplasmic reticulum and Golgi is important for stem-cell survival (Singh, 2016)

    To further investigate the roles of these genes in stem cells, a recombined double Gal4 line of esg-Gal4 and wg-Gal4 was used to express genes in ISCs, RNSCs, and HISCs (esgts wgts > X). Knockdown of these genes using RNA interference (RNAi) in stem cells ablated most of the stem cells in 1 week. However, expressing Arf79FRNAi in enterocytes or in differentiated stellate cells in Malpighian tubules did not cause similar marked ablation. These results suggest that Arf79F knockdown selectively kills stem cells and not differentiated cells (Singh, 2016).

    It was also found that expressing Arf79FRNAi in RasV12-transformed RNSCs ablated almost all of the transformed stem cells. Similarly, expressing Arf79FRNAi in NDN-transformed ISCs ablated all of the cells within one week, but restored differentiated cells to close to their normal levels within one week (Singh, 2016).

    δ-COP- and γ-COP-mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique, and it was found that the COPI complex cell-autonomously regulated stem cell survival. In summary, knockdown of the COPI-Arf79F complex effectively ablated normal and transformed stem cells but not differentiated enterocytes or stellate cells (Singh, 2016)

    In the RNAi screen acyl-CoA synthetase long-chain (ACSL), an enzyme in the Drosophila lipolysis-β-oxidation pathway, and bubblegum (bgm), a very long-chain fatty acid-CoA ligase, were also identified. RNAi-mediated knockdown of Acsl and bgm effectively killed ISCs and RNSCs, but killed HISCs less effectively. Expressing AcslRNAi in RasV12-transformed RNSCs also ablated almost all of the transformed RNSCs in one week (Singh, 2016).

    Brummer (bmm) is a triglyceride lipase, the Drosophila homologue of mammalian ATGL, the first enzyme in the lipolysis pathway. Scully (scu) is the Drosophila orthologue of hydroxy-acyl-CoA dehydrogenase, an enzyme in the β-oxidation pathway. Hepatocyte nuclear factor 4 (Hnf4) regulates the expression of several genes involved in lipid mobilization and β-oxidation. To determine whether the lipolysis-β-oxidation pathway is required for COPI-Arf79F-mediated stem cell survival, upstream activating sequence (UAS)-regulated constructs (UAS-bmm, UAS-Hnf4, and UAS-scu) were also expressed in stem cells that were depleted of Arf79F, β-COP, or ζ-COP. Overexpressing either scu or Hnf4 significantly attenuated the stem cell death caused by knockdown of the COPI-Arf79F complex. Expressing UAS-Hnf4 MARCM clones also rescued the stem cell death phenotype induced by γ-COP knockdown. However, bmm overexpression did not rescue the stem-cell death induced by Arf79F knockdown. Since there are several other triglyceride lipases in Drosophila in addition to bmm, another lipase may redundantly regulate the lipolysis pathway (Singh, 2016)

    To further investigate the function of lipolysis in stem cells, the expression of a lipolysis reporter (GAL4-dHFN4; UAS-nlacZ which consisted of hsp70-GAL4-dHNF4 combined with a UAS-nlacZ reporter gene was investigated. The flies were either cultured continuously at 29°C or heat-shocked for 30 min at 37°C, 12 h before dissection. Without heat shock, the reporter was expressed only in ISCs and RNSCs of mature adult flies, but not in enteroendocrine cells, enterocytes, quiescent HISCs or quiescent ISCs of freshly emerged young adult flies (less than 3 days old. Expressing δ-COPRNAi almost completely eliminated the reporter expression, suggesting that the reporter was specifically regulated by the COPI complex. After heat shock or when a constitutively active form of JAK (hopTum-l) was expressed, the reporter was strongly expressed in ISCs, RNSCs and HISCs, but not in enteroendocrine cells or enterocytes. These data suggest that COPI-complex-regulated lipolysis was active in stem cells, but not in differentiated cells, and that the absence of the reporter expression in quiescent HISCs at 29°C was probably owing to weak hsp70 promoter activity rather than to low lipolysis in these cells (Singh, 2006).

    Lipid storage was futher investigated, and it was found that the size and number of lipid droplets were markedly increased in stem cells after knockdown of Arf79F (Singh, 2016).

    Arf1 inhibitors (brefeldin A, golgicide A, secin H3, LM11 and LG8) and fatty-acid-oxidation (FAO) inhibitors (triacsin C, mildronate, etomoxir and enoximone) were used, and it was found that these inhibitors markedly reduced stem-cell tumours in Drosophila through the lipolysis pathway but had a negligible effect on normal stem cells (Singh, 2016)

    These data together suggest that the COPI-Arf1 complex regulates stem-cell survival through the lipolysis-β-oxidation pathway, and that knockdown of these genes blocks lipolysis but promotes lipid storage. Further, the transformed stem cells are more sensitive to Arf1 inhibitors and may be selectively eliminated by controlling the concentration of Arf1 inhibitors (Singh, 2016)

    These data suggest that neither caspase-mediated apoptosis nor autophagy-regulated cell death regulates the stem-cell death induced by the knockdown of components of the COPI-Arf79F complex. Therefore whether necrosis regulates the stem-cell death induced by knockdown of the COPI-Arf79F complex was investigated. Necrosis is characterized by early plasma membrane rupture, reactive oxygen species (ROS) accumulation and intracellular acidification. Propidium iodide detects necrotic cells with compromised membrane integrity, the oxidant-sensitive dye dihydroethidium (DHE) indicates cellular ROS levels and LysoTracker staining detects intracellular acidification. The membrane rupture phenotype was detected only in esg and the propidium iodide signal was observed only in ISCs from flies that had RNAi-induced knockdown of expression of COPI-Arf79F components, and not in cells from wild-type flies. In the esgts wgts > AcslRNAi flies, all of the ISCs and RNSCs were ablated after four days at 29°C, but a fraction of the HISCs remained, and these were also propidium iodide positive, indicating that the HISCs were dying slowly. This slowness may have been due to either a lower GAL4 (wg-Gal4) activity in these cells compared to ISCs and RNSCs (esg-Gal4) or quiescence of the HISCs. Furthermore, strong propidium iodide signals were detected in transformed ISCs from esgts > NDN + Arf79FRNAi but not esgts flies, indicating that the transformed stem cells were dying through necrosis (Singh, 2016)

    Similarly, DHE signals were detected only in ISCs from esgts > Arf79FRNAi flies, indicating that the dying ISCs had accumulated ROS and were intracellularly acidified. Overexpressing catalase (a ROS-chelating enzyme) rescued the stem-cell death specifically induced by the γ-COP mutant clone, and the ROS inhibitor NAC blocked the Arf1 inhibitor-induced death of RasV12-induced RNSC tumours. These data together suggest that knockdown of the COPI-Arf1 complex induced the death of stem cells or of transformed stem cells (RasV12-RNSCs, NDN-ISCs) through ROS-induced necrosis. Although ISCs, RNSCs, and HISCs exhibit different degrees of quiescence, they all rely on lipolysis for survival, suggesting that this is a general property of stem cells (Singh, 2016)

    Cases were noticed where the GFP-positive material of the dying ISCs was present within neighbouring enterocytes, suggesting that these enterocytes had engulfed dying ISCs (Singh, 2016)

    The JNK pathway, autophagy and engulfment genes are involved in the engulfment of dying cells. Therefore, whether these genes are required for COPI-Arf79F-regulated ISC death was investigated. The following was found: (1) ISC death activated JNK signalling and autophagy in neighbouring enterocytes; (2) knockdown of these genes in enterocytes but not in ISCs rescued ISC death to different degrees; (3) the drpr-mbc-Rac1-JNK pathway in enterocytes is not only necessary but also sufficient for ISC death; and (4) inhibitors of JNK and Rac1 could block Arf1-inhibitor-induced cell death of the RasV12-induced RNSC tumours. These data together suggest that the drpr-mbc-Rac1-JNK pathway in neighbouring differentiated cells controls the engulfment of dying or transformed stem cells (Singh, 2016)

    The finding that the COPI-Arf79F-lipolysis-β-oxidation pathway regulated transformed stem-cell survival in the fly led to an investigation of whether the pathway has a similar role in CSCs. WTwo Arf1 inhibitors (brefeldin A and golgicide A) and two FAO inhibitors (triascin C and etomoxir) were tested on human cancer cell lines, and it was found that the growth, tumoursphere formation and expression of tumour-initiating cell markers of the four cancer cell lines were significantly suppressed by these inhibitors, suggesting that these inhibitors suppress CSCs. In mouse xenografts of BSY-1 human breast cancer cells, a novel low-cytotoxicity Arf1-ArfGEF inhibitor called AMF-26 was reported to induce complete regression in vivo in five days. Together, this report and the current results suggest that inhibiting Arf1 activity or blocking the lipolysis pathway can kill CSCs and block tumour growth (Singh, 2016)

    Stem cells or CSCs are usually localized to a hypoxic storage niche, surrounded by a dense extracellular matrix, which may make them less accessible to sugar and amino acid nutrition from the body's circulatory system. Most normal cells rely on sugar and amino acids for their energy supply, with lipolysis playing only a minor role in their survival. The current results suggest that stem cells and CSCs are metabolically unique; they rely mainly on lipid reserves for their energy supply, and blocking COPI-Arf1-mediated lipolysis can starve them to death. It was further found that transformed stem cells were more sensitive than normal stem cells to Arf1 inhibitors. Thus, selectively blocking lipolysis may kill CSCs without severe side effects. Therefore, targeting the COPI-Arf1 complex or the lipolysis pathway may prove to be a well-tolerated, novel approach for eliminating CSCs (Singh, 2016)

    Reduced gut acidity induces an obese-like phenotype in Drosophila melanogaster and in mice

    In order to identify genes involved in stress and metabolic regulation, this study carried out a Drosophila P-element-mediated mutagenesis screen for starvation resistance. A mutant, m2, was isolated that showed a 23% increase in survival time under starvation conditions. The P-element insertion was mapped to the region upstream of the vha16-1 gene, which encodes the c subunit of the vacuolar-type H+-ATPase. It was found that vha16-1 is highly expressed in the fly midgut, and that m2 mutant flies are hypomorphic for vha16-1 and also exhibit reduced midgut acidity. This deficit is likely to induce altered metabolism and contribute to accelerated aging, since vha16-1 mutant flies are short-lived and display increases in body weight and lipid accumulation. Similar phenotypes were also induced by pharmacological treatment, through feeding normal flies and mice with a carbonic anhydrase inhibitor (acetazolamide) or proton pump inhibitor (PPI, lansoprazole) to suppress gut acid production. This study may thus provide a useful model for investigating chronic acid suppression in patients (Lin, 2015).

    Hs3st-A and Hs3st-B regulate intestinal homeostasis in Drosophila adult midgut

    Intrinsic and extrinsic signals as well as the extracellular matrix (ECM) tightly regulate stem cells for tissue homeostasis and regenerative capacity. Little is known about the regulation of tissue homeostasis by the ECM. Heparan sulfate proteoglycans (HSPGs), important components of the ECM, are involved in a variety of biological events. Two heparin sulfate 3-O sulfotransferase (Hs3st) genes, Hs3st-A and Hs3st-B, encode the modification enzymes in heparan sulfate (HS) biosynthesis. This study demonstrates that Hs3st-A and Hs3st-B are required for adult midgut homeostasis. Depletion of Hs3st-A in enterocytes (ECs) results in increased intestinal stem cell (ISC) proliferation and tissue homeostasis loss. Moreover, increased ISC proliferation is also observed in Hs3st-B null mutant alone, or in combination with Hs3st-A RNAi. Hs3st-A depletion-induced ISC proliferation is effectively suppressed by simultaneous inhibition of the EGFR signaling pathway, suggesting that tissue homeostasis loss in Hs3st-A-deficient intestines is due to increased EGFR signaling. Furthermore, this study found that Hs3st-A-depleted ECs are unhealthy and prone to death, while ectopic expression of the antiapoptotic p35 is able to greatly suppress tissue homeostasis loss in these intestines. Together, these data suggest that Drosophila Hs3st-A and Hs3st-B are involved in the regulation of ISC proliferation and midgut homeostasis maintenance (Guo, 2014).

    More Drosophila enteroendocrine peptides: Orcokinin B and the CCHamides 1 and 2

    Antisera to orcokinin B, CCHamide 1, and CCHamide 2 recognize enteroendocrine cells in the midgut of the Drosophila and its larvae. Although the antisera to CCHamide 1 and 2 are mutually cross-reactive, polyclonal mouse antisera raised to the C-terminals of their respective precursors allowed the identification of the two different peptides. In both larva and adult, CCHamide 2 immunoreactive endocrine cells are large and abundant in the anterior midgut and are also present in the anterior part of the posterior midgut. The CCHamide 2 immunoreactive endocrine cells in the posterior midgut are also immunoreactive with antiserum to allatostatin C. CCHamide 1 immunoreactivity is localized in endocrine cells in different regions of the midgut; those in the caudal part of the posterior midgut are identical with the allatostatin A cells. In the larva, CCHamide 1 enteroendocrine cells are also present in the endocrine junction and in the anterior part of the posterior midgut. Like in other insect species, the Drosophila orcokinin gene produces two different transcripts, A and B. Antiserum to the predicted biologically active peptide from the B-transcript recognizes enteroendocrine cells in both larva and adult. These are the same cells as those expressing beta-galactosidase in transgenic flies in which the promoter of the orcokinin gene drives expression of this enzyme. In the larva, a variable number of orcokinin-expressing enteroendocrine cells are found at the end of the middle midgut, while in the adult, those cells are most abundant in the middle midgut, while smaller numbers are present in the anterior midgut. In both larva and adult, these cells also express allatostatin C. A specific polyclonal antiserum was also made to the NPF precursor in order to determine more precisely the expression of this peptide in the midgut. Using this antiserum, expression in the midgut was found to be the same as described previously using transgenic flies, while in the adult, midgut expression appears to be concentrated in the middle midgut, thus suggesting that in the anterior midgut only minor quantities of NPF are produced (Veenstra, 2014).

    Robust intestinal homeostasis relies on cellular plasticity in enteroblasts mediated by miR-8-Escargot switch

    This study used tracing methods that allow simultaneously capturing the dynamics of intestinal stem and committed progenitor cells (called enteroblasts) and intestinal cell turnover with spatiotemporal resolution. Intestinal stem cells (ISCs) divide 'ahead' of demand during Drosophila midgut homeostasis. Their newborn enteroblasts, on the other hand, take on a highly polarized shape, acquire invasive properties and motility. They extend long membrane protrusions that make cell-cell contact with mature cells, while exercising a capacity to delay their final differentiation until a local demand materializes. This cellular plasticity is mechanistically linked to the epithelial-mesenchymal transition (EMT) programme mediated by escargot, a snail family gene. Activation of the conserved microRNA miR-8/miR-200 in 'pausing' enteroblasts in response to a local cell loss promotes timely terminal differentiation via a reverse EMT by antagonizing escargot. These findings unveil that robust intestinal renewal relies on hitherto unrecognized plasticity in enteroblasts and reveal their active role in sensing and/or responding to local demand (Antonello, 2015).

    The robustness of intestinal cell renewal relies on cellular plasticity in committed progenitor cells and a rather loose regulation of ISCs proliferation. One key finding is that stem cells divide continually and generate a 'stock' of committed progenitor cells that do not terminally differentiate right away but postpone their final differentiation for long time intervals in the absence of a local epithelial cell loss. Accordingly, one noticeable change in newborn progenitor cells after their (enterocyte) fate commitment is their transformation from rounded cells to spindle-shaped cells that appear to actively monitor their surroundings by extending long membrane actin-rich protrusions that make cell-cell contact with mature epithelial cells and their mother ISCs. Timely terminal differentiation with epithelial cell loss is orchestrated by activation of a conserved pro-epithelial microRNA, in turn, directly repressing the repressors of differentiation. A microRNA-induced repression of the repressors of differentiation provides a faster mechanism than one involving a transcriptional regulator since synthesizing a miRNA likely requires less time than synthesizing a protein. Importantly, mutual antagonism between the microRNA (MiR-8/miR-200) and its targets (Escargot/Snail2 and Zfh1/ZEB) may serve to slow down the mesenchymal-to-epithelial process inside individual mesenchymal/progenitor cells until they are successfully integrated in the epithelium. Consistently, abrupt transition as in mir-8 overexpressing midguts results in erroneous tissue repair (Antonello, 2015).

    Supply and demand in business production involves frequently two alternative solutions called 'make-to-stock' and 'make-to-order'. In 'make-to-stock' or MTS, production is continuous so that response to customers can be supplied immediately. However, as production is not based on actual demand, the MTS solution is not robust against fluctuations in demand and errors in forecasting can result in shortages (if there is insufficient residual stock) or overproduction. In 'make-to-order', or MTO, production only starts upon receiving a customer's order, thereby precisely matching production to demand. However, the MTO generates a delay in the response and can be less efficient and competitive than the MTS paradigm. The dynamics of stem cells and committed progenitor cells in the midgut suggests a hybrid solution between MTS and MTO -- reminiscent to the business solution known as delayed differentiation. Thus, in basal homeostasis, production of new cells to replace cell loss occurs in two stages: (1) a 'make-to-stock' stage where committed progenitor cells are continually generated and 'stocked' in an 'undifferentiated' state; and (2) a 'make-to-order' stage where terminal differentiation takes place only in response to a local demand. In mice and humans, the rapid turnover that occurs in the small intestinal epithelium is thought to be the result of continual shedding of superficial cells balanced by the continual stem cell production. The mechanism described in this study may be more general than expected and could account for how murine cells after fate commitment like the secretory-committed cells defer for long periods their terminal differentiation (Buczacki et al, 2013; Antonello, 2015).

    Escargot/Snail2 sustains the undifferentiated state and self-renewing divisions of midgut intestinal stem cells. However, the committed progenitor cells also express escargot and apparently at higher levels than the stem cells. It is hypothesized that below a certain threshold level, Escargot maintains stemness and a partial EMT that may facilitate regular cell division and a topologically confined position at the base of the intestinal epithelium. Conversely, when Escargot surpasses a certain threshold level, it promotes a full EMT that confers invasive properties and motility for the successful response and integration of the newly differentiated cells in the preexisting epithelium. Intriguingly, the enteroendocrine cells appear to escape from this block in terminal differentiation and differentiate at the normal rate in the absence of escargot. There is as yet no explanation for the behaviour of these progenitor cells (Antonello, 2015).

    Mechanistically, the different levels of escargot could be achieved via Notch signalling pathway, which is prominently activated in enterocyte-committed progenitors. Notch signalling activates directly zfh1 gene and Zfh1, a homolog of the mammalian stemness and EMT-determinant Zeb1,2, and binds to the escargot promoter region, and this study shows that Zfh1 acts genetically upstream of escargot. Thus, progenitor cells receiving Notch signalling might enhance escargot transcriptional levels via Notch-induced zfh1 transcription. Such regulatory mechanism would explain, for example, that loss of Notch results in stem-like/round cells (Antonello, 2015).

    In mammalian cell culture, the EMT process has been linked to the acquisition of stem-like nature via an interplay between the ZEB1,2 and Snail transcription factors and the microRNAs of the miR-200 family. Moreover, EMT determinants often regulate each other to promote EMT. Thus, the interactions between Escargot/Snail2, zfh1/Zeb and miR-8/miR-200 that were identified in this study exemplify the conservation of the regulatory mechanisms involved in EMT/MET and stemness in an in vivo context and a normal physiology of an adult organism. However, this study shows that escargot-zfh1 promotes stemness and full EMT/invasive properties in distinct cell populations and likely at different concentration levels, highlighting the utility of Drosophila midgut as a model to dissect out mechanisms linking physiological EMT to cellular plasticity and stemness as well as provide novel insights linking polyploidy and EMT towards stemness (Antonello, 2015).

    Although midgut mesenchymal/progenitor cells have motility, most of them maintain their own local area as clearly defined by Flybow clonal analysis. This situation is similar to the leading edge mesenchymal cells during collective cell migration. Midgut enteroblasts retain contact via E-cadherin with their mother ISC, a process that might be regulated by escargot as in tracheal cells. Cell-cell contact is crucial to sustain Notch signalling in committed progenitor cells and likely to help to stabilize polarity of enteroblasts and their membrane protrusions that contact mature cells. Through these protrusions, mesenchymal/enteroblasts might actively monitor their surroundings. When a protrusion detects changes in tension and mechanical forces generated during the elimination of a dying cells, a positive input might be created that triggers the activation of expression of the microRNA mir-8 in the particular progenitor cell which, in turn, promotes the epithelial state and integration of the newly differentiated cell in the epithelium. Adhesion via E-cadherin could facilitate communication between an epithelial cells and a mesenchymal/progenitor cell in its vicinity so that a single, newly differentiated cell fills the gap left by the cleared cell (Antonello, 2015).

    Dynamic pseudopodia in migrating cells have been proposed as a mechanism for temporal and spatial sensing during cell migration. Direction sensing is also consistent with time-lapse data showing individual progenitor cells re-adjusting position in the homeostatic midguts. Transduction of mechanical cues via YAP and TAZ (called Yorkie in flies) is functionally involved in differentiation of mesenchymal stem cells. Hence, Drosophila Hippo/Yorkie-YAP in mature enterocytes is a primary candidate pathway for a potential transduction of mechanical cues activating mir-8 in response to cell death (Antonello, 2015).

    In summary, the miR-8-escargot-zfh1 axis and the EMT/MET programme provides a conceptual shift of the current stem cell-centred view of tissue renewal and offers a starting point for investigating how mature cells speak with neighbouring committed progenitor cells to ensure that epithelial cell loss and cell addition are kept in balance (Antonello, 2015).

    miR-263a regulates ENaC to maintain osmotic and intestinal stem cell homeostasis in Drosophila

    Proper regulation of osmotic balance and response to tissue damage is crucial in maintaining intestinal stem cell (ISC) homeostasis. The Drosophila genome encodes an exceptionally large number of DEG/ENaC subunits termed Pickpocket (Ppk) 1-31. This study found that Drosophila miR-263a downregulates the expression of epithelial sodium channel (ENaC) subunits in enterocytes (ECs) to maintain osmotic and ISC homeostasis. In the absence of miR-263a, the intraluminal surface of the intestine displays dehydration-like phenotypes, Na+ levels are increased in ECs, stress pathways are activated in ECs, and ISCs overproliferate. Furthermore, miR-263a mutants have increased bacterial load and expression of antimicrobial peptides. Strikingly, these phenotypes are reminiscent of the pathophysiology of cystic fibrosis (CF) in which loss-of-function mutations in the chloride channel CF transmembrane conductance regulator can elevate the activity of ENaC, suggesting that Drosophila could be used as a model for CF. Evidence is provided that overexpression of miR-183, the human ortholog of miR-263a, can also directly target the expressions of all three subunits of human ENaC (Kim, 2016).

    The Drosophila intestinal system is an attractive model for studying signaling events that control stem cell homeostasis given its anatomical and functional similarities to human epithelial systems, including the intestine. The adult midgut is continuously damaged during feeding as well as by chemicals and pathogens they encounter in the food, and thus needs to be constantly renewed. The renewal process requires tight regulation of the activities of multiple conserved signaling pathways in response to various types of intestinal epithelial injuries. These responses promote both intestinal stem cell (ISC) proliferation and enteroblast (EB) differentiation, expediting the rapid generation of new midgut epithelial cells to replace damaged (Kim, 2016).

    MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. In the past few years, miRNAs have been shown to play an important role in stem cell homeostasis by regulating differentiation and self-renewal. This study found that a well-conserved miRNA, miR-263a, is necessary for maintaining ISC homeostasis. Deletion of miR-263a in the adult midgut enterocytes (ECs) activates a stress response that, in turn, activates signaling pathways required for ISC proliferation, resulting in midgut hyperplasia. Well-conserved subunits of the epithelial sodium channel (ENaC) were found to be biologically important targets of miR-263a,and regulation of these subunits by miR-263a was found to be critical for maintaining proper osmotic homeostasis in the midgut epithelium. Remarkably, many of the phenotypes of miR-263a mutants are reminiscent of the pathophysiology of cystic fibrosis (CF), an autosomal recessive disorder caused by mutations in the gene encoding the chloride channel CF transmembrane conductance regulator (CFTR). In CF patients, loss-of-function mutations in the CFTR can elevate the activity of ENaC through a mechanism that is not fully understood. ENaC is present at the apical plasma membrane in many epithelial tissues throughout the body to regulate sodium reabsorption, and control total body salt and water homeostasis. The most common symptoms of CF are potential lethal blockages of distal small intestines, airway mucus obstruction, and chronic airway inflammation, which are consistent with the model that upregulation in ENaC activity increases sodium and water reabsorption, ultimately leading to dehydration of the intraluminal surface and reduction in mucus transport. Interestingly, this study provides evidence that overexpression of miR-183, the human ortholog of miR-263a, can also directly target all three subunits of human ENaC to regulate its activity. Altogether, these findings describe the role of a miRNA in regulating ENaC levels and suggest that the Drosophila intestine could be used as a model for CF (Kim, 2016).

    In CF, two different models have been proposed regarding the role of hydration and salt concentration in normal airway defense. The hydration model proposes that increased absorption of fluid by the epithelium leads to dehydrated mucus and impaired mucociliary clearance that contributes to the establishment of an environment promoting colonization of the lungs by bacteria. In contrast, the salt model proposes that the salt content of airway fluid in CF is too high and thus prevents salt-sensitive defensin molecules in the airway surface liquid from killing bacteria, leading to increased susceptibility to lung infections. In Drosophila, the phenotypes associated with perturbation of ENaC are consistent with the hydration model, as misregulation of ENaC subunits in miR-263a mutants result in increased sodium reabsorption across the midgut epithelium. Furthermore, a dehydration-like phenotype of the PM, which is analogous to mucous secretions in the vertebrate digestive tract, was observed. Consistent with the PM providing protection against abrasive food particles and pathogens, miR-263a mutants appear more susceptible to bacterial infections as they succumb to P. aeruginosa infection more rapidly than the controls. In addition, increased bacterial load and antimicrobial peptide levels, and disruption of the intestinal pH were observed in miR-263a mutants. Interestingly, ECs in miR-263a mutants appear swollen, which is likely due to increased water reabsorption through osmosis. Finally, an activation was observed of stressed pathways characteristic of damaged ECs, which correlates with increased proliferation of ISCs (Kim, 2016).

    Consistent with previous reports that cell swelling can activate the JNK pathway, JNK signaling is activated in miR-263a mutants that have large ECs. In addition, the JAK/STAT and EGFR pathways that regulate ISC proliferation are hyperactivated. Similarly, in CF airway and small intestine epithelia, cells in the airway epithelium and submucosal glands are more proliferative than cells in non-CF airways. In addition, in all CF mouse models in which CFTR has been deleted, goblet cell hyperplasia was observed in the small intestine (Kim, 2016).

    Although the existence of Drosophila CFTR is yet to be determined, given its phenotypic similarities to the pathophysiology of CF, miR-263a mutants may provide a cost-effective and high-throughput animal model for identifying potential therapeutics that can specifically target ENaC in vivo, as the Drosophila gut is amenable to large-scale small-molecule screens. In addition, miR-183 might itself be a potential therapeutic agent for regulating ENaC activity in CF, based on the data that overexpression of miR-183 can directly target the expression of all three ENaC subunits in CFBE41o cells. Thus, possibly a combinational therapy for CF using the CFTR potentiator, Ivacaftor (also known as Kalydeco, which improves the transport of chloride through the mutated CFTR, together with overexpression of miR-183, could be imagined (Kim, 2016).

    Control of lipid metabolism by Tachykinin in Drosophila

    The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. This study demonstrates that Tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila and that TKs repress lipogenesis in enterocytes (ECs) associated with TKR99D receptor and protein kinase A (PKA) signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions (Song, 2014).

    Previous studies in mammals have indicated that a few gut secretory hormones, like GLP1 and GLP2, are involved in intestinal lipid metabolism. However, due to gene and functional redundancy, mammalian genetic models for gut hormones and/or their receptors with severe metabolic defects are not available. This study has establish that Drosophila TKs produced from EEs coordinate midgut lipid metabolic processes. The studies clarify the roles of TK hormones in intestinal lipogenesis and establish Drosophila as a genetic model to study the regulation of lipid metabolism by gut hormones (Song, 2014).

    Six mature TKs, TK1-TK6, are processed and secreted from TK EEs in both the brain and midgut (Reiher, 2011). Using a specific Gal4 driver line, gene expression in TK EEs was specifically manipulated, leading to the demonstration that loss of gut TKs results in an increase in midgut lipid production. Further, this study showed that TKs regulate intestinal lipid metabolism associated with TKR99D, but not TKR86C, which is consistent with the expression of these receptors. Consistent with previous reports that TK/TKR99D signaling regulates cAMP level and PKA activation, loss of gut TKs is associated with a reduction in PKA activity in ECs, and overexpression of a PKA catalytic subunit was able to reverse the increased intestinal lipid production associated with loss of TKR99D. In addition, the transcription factor SREBP that triggers lipogenesis was controlled by TK/TKR99D/PKA signaling. Taken together, these results suggest that TKs produced from EEs regulate midgut lipid metabolism via TKR99D/PKA signaling and regulation of, at least, SREBP-induced lipogenesis in ECs (Song, 2014).

    Interestingly, this study reveals that TKs derived from either the brain or gut exhibit distinct functions: TKs derived from gut control intestinal lipid metabolism, whereas TKs derived from brain control behavior. This is reminiscent of the distinct functions of mammalian secreted regulatory peptides, where different spatial expressions or deliveries of peptides like Ghrelin can result in distinct physiological functions. In addition, some prohormones encode multiple mature peptides that can have multiple functions. For example, processing of proglucagon in the pancreas α cells preferentially gives rise to glucagon, which antagonizes the effect of insulin. In intestine L cells, however, proglucagon is mostly processed into GLP1 to promote insulin release. These studies of TKs exemplify how secreted regulatory peptides derived from different tissues can be associated with fundamentally diverse physiological functions. Clearly, additional studies examining the function of secreted peptides in a cell-type- and tissue-specific manner are needed to fully appreciate and unravel their complex roles both in flies and mammals (Song, 2014).

    There is a growing body of studies emphasizing that intestinal lipid metabolism is key to the control of systemic lipid homeostasis. For example, chemicals such as orlistat, designed to inhibit dietary lipid digestion/absorption in the intestine, efficiently reduce obesity. In addition, mammalian inositol-requiring enzyme 1β deficiency-induced abnormal chylomicron assembly in the small intestine results in hyperlipidemia. Similarly, in Drosophila, dysfunction of intestinal lipid digestion/absorption caused by Magro/LipA deficiency eventually decreases whole-body lipid storage and starvation resistance in Drosophila. Further, intestinal lipid transport, controlled by lipoproteins, is essential for systemic lipid distribution and energy supply in other tissues. Consistent with these observations, this study demonstrates that increased midgut lipid synthesis associated with gut TK deficiency is sufficient to elevate systemic lipid storage. Although TK ligands and TK receptors show high homologies between mammals and fruit flies, whether mammalian TK signaling plays a similar role in intestinal lipid metabolism is largely unknown. Future studies will reveal whether mammalian TK signaling affects intestinal lipid metabolism as in Drosophila. If this is the case, it may provide a therapeutic opportunity for the treatment of intestinal lipid metabolic disorder and obesity (Song, 2014).

    Production and secretion of gut hormones are precisely regulated under various physiological conditions. Similar to previous observations that starvation induces gut TK secretion in other insects, this study found that nutrient deprivation promotes TK production in EEs. Interestingly, feeding of amino-acid-enriched yeast, but not coconut oil or sucrose, potently suppressed gut TK levels, indicating that amino acids may act directly on TK production in EEs. It has been reported that dietary nutrients regulate gut hormone production through certain receptors located on the cell membrane of EEs in mammals. Future studies will be necessary to elucidate the detailed mechanism by which nutrients regulate TK production from EEs (Song, 2014).

    Transforming growth factor beta/Activin signaling functions as a sugar-sensing feedback loop to regulate digestive enzyme expression

    Organisms need to assess their nutritional state and adapt their digestive capacity to the demands for various nutrients. Modulation of digestive enzyme production represents a rational step to regulate nutriment uptake. However, the role of digestion in nutrient homeostasis has been largely neglected. This study analyzed the mechanism underlying glucose repression of digestive enzymes in the adult Drosophila midgut. Glucose represses the expression of many carbohydrases and lipases. The data reveal that the consumption of nutritious sugars stimulates the secretion of the transforming growth factor β (TGF-β) ligand, Dawdle, from the fat body. Dawdle then acts via circulation to activate TGF-β/Activin signaling in the midgut, culminating in the repression of digestive enzymes that are highly expressed during starvation. Thus, this study not only identifies a mechanism that couples sugar sensing with digestive enzyme expression but points to an important role of TGF-β/Activin signaling in sugar metabolism (Chng, 2004).

    Digestive enzymes expression is subjected to complex regulation. However, apart from the regulation of magro (lipase) by the nutrient-sensitive DHR96 and dFOXO (Karpac, 2013). It is noteworthy that an arbitrary threshold for RNA-seq analysis has rejected several genes whose repression was more subtle. For this, it has been have independently verified Amy-p, Amy-d, CG9466, CG9468, and CG6283 to be repressed by glucose through qRT-PCR. Thus, the actual repertoire of carbohydrases and lipases affected by glucose could be potentially larger (Karpac, 2013).

    To date, little is known about the contribution of digestion on sugar homeostasis. It seems likely that glucose repression of carbohydrases and lipases is aimed at reducing the amount of sugars and lipids that are available for absorption. Consistent with this view, glucose transmembrane transporters were also found among genes that were downregulated by dietary glucose. A high-sugar diet in Drosophila is associated with dire consequences such as hyperglycemia, insulin resistance, and increased fat accumulation. Thus, reducing both carbohydrases and lipases expression may restrict the nutritional load available for absorption into the circulation when carbohydrate stores in the organism are sufficient and fats are accumulating. In accordance with this, early postprandial glucose level was elevated in the hemolymph when TGF-β/Activin pathway function was compromised in the midgut, a condition associated with elevated digestive enzymes expression. However, when the levels of TAG, glycogen, glucose, and trehalose were monitored after 2 weeks on a high-sugar diet, no significant differences were observed between flies whereby Smad2 or Babo were knocked down in the midgut and control. Sugar homeostasis is a tightly regulated process involving multiple tissues. One possibility would be that the postprandial increase in glucose was counteracted by early acting satiety response when hemolymph glucose level passed a certain threshold, thus limiting the net amount of glucose entering the circulation. Clearly, the role of glucose repression in sugar homeostasis and metabolism warrants additional research. An understanding of how the repertoire of digestive enzymes respond to other nutriments in the diet will provide insights into how an organism may rebalance its diet after ingestion and improve understanding of nutrients homeostasis (Karpac, 2013).

    In this study, it was also shown that digestive enzyme repression is induced only by nutritious carbohydrates in the diet. Arabinose, a sweet-tasting sugar with no nutritional value, and L-glucose, another nonutilizable sugar did not suppress amylase and maltase expression. Hence, postprandial activation of gustatory receptors in the gut are considered to be an unlikely mechanism for glucose repression of digestive enzymes. Instead, all these are suggestive of an underlying sugar-sensing mechanism to ensure that carbohydrate digestive capacity toward utilizable carbohydrate sources are not comprised until nutritional sufficiency is attained (Karpac, 2013).

    In Drosophila, sugar homeostasis is often associated with the AKH and insulin signaling, whereas insulin signaling is also modulated by proteins and amino acids in the diet. Recently, it has been shown that Daw expression is modulated by insulin signaling, and Daw was identified as a target of dFOXO (Bai, 2013), raising the possibility that glucose repression may be similarly affected by insulin signaling. Surprisingly, disrupting both AKH and insulin signaling did not compromise glucose repression. Instead, this study identified a key role for TGF-β/Activin signaling in this process. Whereas Daw expression may be modulated by insulin signaling, the results clearly showed that glucose repression is mediated through an insulin-independent mechanism. More recently, Ghosh (2014) has demonstrated that Daw is required for insulin secretion, suggesting that the TGF-β/Activin pathway may function upstream of the insulin signaling. It is also noteworthy that, whereas compromising insulin signaling is known to raise circulating sugar levels, this did not affect the ability of flies to repress digestive enzymes in response to dietary glucose. One possible explanation is that Daw expression in response to glucose is dependent on the nutritional state perceived cell autonomously by the fat body cells. Thus, if nutrient sensing in these cells is not compromised, Daw induction and glucose repression can be achieved. Future research should clarify the mechanism underlying Daw induction by nutritious sugar and define the possible interactions between TGF-β/Activin and other sugar-sensing mechanisms (Karpac, 2013).

    The TGF-β/Activin pathway in Drosophila has been previously studied in the context of larval brain development, neuronal remodeling, wing disc development, and, more recently, aging and pH homeostasis. This study addresses the physiological function of the TGF-β/Activin pathway in the adult midgut. When the TGF-β/Activin signaling was disrupted in the adult midgut, glucose repression was abolished. Conversely, increasing TGF-β/Activin signaling in the midgut, through the overexpression of the constitutive active form of Babo or Smad2, was sufficient to repress both amylase and maltase expression. Furthermore, glucose repression is mediated by the TGF-β ligand Daw, produced and secreted from the fat body, a metabolic tissue functionally analogous to the mammalian liver and adipose tissue. Thus, this study uncovers a physiological role for the TGF-β/Activin pathway in adapting carbohydrate and lipase digestion in response to the nutritional state of the organism. Because many features of digestion and absorption are conserved between flies and mammals, it will be of interest to investigate the role of TGF-β/Activin pathway in mammalian digestion (Karpac, 2013).

    Recent studies have attributed a role for Daw in aging and pH homeostasis, two processes tightly linked to metabolism. Thus, it is likely that Daw induced from the fat body in response to carbohydrate in the diet will induce a more global response instead of a local response, affecting only digestive enzyme expression. As such, Daw may act as a central mediator for glucose homeostasis by regulating sugar level in the circulation. When there are sufficient carbohydrates in the diet, Daw expression restricts the expression of carbohydrase and glucose transporters. Concurrently, at the postabsorption level, Daw in the circulation may act directly or indirectly (via insulin signaling) to maintain circulating sugar level. A broader role for Daw in sugar homeostasis is reinforced by the findings that Daw mutant larvae were more sensitive to a high-sugar diet. Similarly, this study found overexpression of Daw, but not Myo, Mav, or Actβ, renders flies sensitive to sugar starvation. Along this line, in C. elegans, the TGF-β signaling is reported to be elevated and required in neurons for satiety. There were also several observations that hyperglycemia is linked to increased TGF-β activity in mammals. Hence, the role of TGF-β/Activin signaling in sugar homeostasis requires further investigation in Drosophila and other organisms (Karpac, 2013).

    In conclusion, this study revealed a remarkable resilience in the regulation of carbohydrate and lipid-acting enzymes expression to ensure that digestive capacity in the midgut is not compromised before certain metabolic criteria in the fat body is attained. The study also unraveled a role of the TGF-β/Activin-signaling pathway in the adult Drosophila midgut, which has not been appreciated. It reinforced the notion that the gut is not a passive tube for nutriment flow. Rather, it dynamically modulates digestive enzyme expression in response to the organism’s nutritional state through endocrine signals derived from other metabolic tissues (Karpac, 2013).

    Drosophila Pez acts in Hippo signaling to restrict intestinal stem cell proliferation

    The conserved Hippo signaling pathway acts in growth control and is fundamental to animal development and oncogenesis. Hippo signaling has also been implicated in adult midgut homeostasis in Drosophila. Regulated divisions of intestinal stem cells (ISCs), giving rise to an ISC and an enteroblast (EB) that differentiates into an enterocyte (EC) or an enteroendocrine (EE) cell, enable rapid tissue turnover in response to intestinal stress. The damage-related increase in ISC proliferation requires deactivation of the Hippo pathway and consequential activation of the transcriptional coactivator Yorkie (Yki) in both ECs and ISCs. This study identified Pez, an evolutionarily conserved FERM domain protein containing a protein tyrosine phosphatase (PTP) domain, as a novel binding partner of the upstream Hippo signaling component Kibra. Pez function (but not its PTP domain) is essential for Hippo pathway activity specifically in the fly midgut epithelium. Thus, Pez displays a tissue-specific requirement and functions as a negative upstream regulator of Yki in the regulation of ISC proliferation (Poernbacher, 2012).

    The WW domain protein Kibra has recently been shown to function as a tumor suppressor in the Hippo pathway. Because Kibra is an adaptor molecule, attempts were made to identify physical binding partners of Kibra to further explore upstream Hippo signaling. Affinity purification-mass spectrometry (AP-MS) analysis with Kibra as bait identified Pez as a novel interaction partner of Kibra in Drosophila cultured cells. The same result was recently obtained in a large-scale proteomic study of Drosophila cultured cells. The binding between Pez and Kibra was confirmed by reciprocal coimmunoprecipitation (co-IP) experiments with epitope-tagged proteins. Furthermore, a yeast two-hybrid (Y2H) experiment revealed that the Kibra-Pez interaction is robust and direct (Poernbacher, 2012).

    To address a possible function of Pez in the Hippo pathway, two loss-of-function alleles of Pez that were generated by different methods. Pez1 is an EMS-induced allele resulting in an early premature translational stop codon. Pez2 was generated by imprecise excision of the P element P{GawB}NP4748, removing most of the Pez coding sequence. Homozygotes for either Pez allele as well as heteroallelic Pez1/Pez2 flies are viable but smaller than controls. Combinations of the Pez alleles with the deficiency Df(2L)ED384 uncovering the Pez locus are also viable and cause a similar reduction in body size as the homozygous or heteroallelic combinations. One copy of a GFP-tagged Pez genomic rescue construct (gPez) restores normal body size. Therefore, both Pez1 and Pez2 are likely to represent strong or null alleles. For further experiments, heteroallelic Pez1/Pez2 flies were used as Pez mutant flies (Poernbacher, 2012).

    In addition to their reduced body size, Pez mutant flies exhibit a developmental delay of 2 days and decreased fertility, all hallmarks of starvation. Pez mutant larvae are small and have decreased triglyceride (TAG) stores and increased expression of the starvation marker genes lipase-3 and 4E-BP. Clones of Pez mutant cells in larval fat bodies did not affect lipid droplets, thus excluding a fat body-autonomous requirement for Pez in lipid metabolism. Surprisingly, overexpression of Drosophila Pez in the developing eye or wing decreased the size of the adult organs, indicating that Pez restricts growth rather than promoting it. It is proposed that the starvation-like phenotype of Pez mutants is due to indirect effects on metabolism arising from a failure in nutrient utilization. Clones of Pez mutant cells in wing imaginal discs did not show growth defects in comparison to their corresponding wild-type sister clones. However, Pez mutant flies exhibit hyperplasia and extensive multilayering of the adult midgut epithelium. One copy of gPez restores normal tissue architecture. The structure of the larval midgut epithelium, as well as that of the other larval and adult epithelia, is not disturbed in Pez mutants. Thus, Pez specifically functions to restrict growth of the adult midgut epithelium (Poernbacher, 2012).

    The Pez protein contains two conserved structural elements: an amino-terminal FERM domain (band 4.1-ezrin-radixin- moesin family of adhesion molecules) and a carboxyterminal protein tyrosine phosphatase (PTP) domain. A truncated version of the protein lacking the FERM domain (DFERM-Pez) or a phosphatase-dead protein (PezPD) still rescued the Pez mutant gut phenotype when overexpressed in ECs. However, overexpression of DFERM-Pez in the developing wing failed to decrease wing size, whereas overexpression of PezPD or of a truncated protein lacking the PTP domain (DPTP-Pez) caused a similar phenotype as overexpression of wild-type Pez, suggesting that the FERM domain is required for the growth-regulatory function of endogenous Pez but becomes dispensable when DFERM-Pez is overexpressed in ECs. In contrast, the potential phosphatase activity of Pez is clearly not needed for its function in growth control (Poernbacher, 2012).

    Two other FERM domain proteins, Merlin (Mer) and Expanded (Ex), act in upstream Hippo signaling to control organ size in Drosophila. Together with the WW domain protein Kibra, Ex and Mer constitute the KEM complex that assembles at the apical junction of epithelial cells and regulates the core Hippo pathway kinase cassette. Overexpression of Kibra, Ex, or Mer in ECs of Pez mutant flies significantly suppressed the Pez gut phenotypes. Thus, Pez is not an essential mediator of Hippo signaling downstream of the KEM complex. Mer and Ex did not detectably coimmunoprecipitate with Pez in Drosophila S2 cells. However, Kibra and Pez coimmunoprecipitated and colocalized in S2 cells. This was dependent on the first WW domain of Kibra, whereas the FERM and PTP domains of Pez as well as two potential ligands of WW domains, a PPPY motif and a PPSGY motif, in the central linker region of Pez were dispensable. A fragment encompassing a proline-rich stretch of Pez (amino acids 368-627; PezPro) was sufficient for the binding to Kibra, whereas the remaining linker region (amino acids 622-967; PezLink) did not bind Kibra. Importantly, knockdown of Kibra via Myo1A-Gal4 caused mild overgrowth of the adult midgut epithelium, and overexpressed Kibra recruited gPez-GFP from the cell cortex of ECs into cytoplasmic punctae. The subcellular localizations of overexpressed Kibra, Ex, or Mer were not affected when Pez was absent (Poernbacher, 2012).

    It is concluded that Pez and Kibra function together in a protein complex to regulate Hippo signaling in adult midgut ECs. The results establish that the Drosophila Pez protein acts as a component of upstream Hippo signaling, restricts transcriptional activity of Yki in epithelial cells of the adult midgut, and plays a crucial role in the control of ISC proliferation. Importantly, the involvement of Hippo signaling in intestinal regeneration is conserved in the mammalian system ] (Poernbacher, 2012).

    The two mammalian homologs of Drosophila Pez are the widely expressed, cytosolic nonreceptor tyrosine phosphatases PTPD1/PTPN21 and PTPD2/PTP36/PTPN14/Pez. All three proteins share a similar domain structure including the well-conserved terminal FERM and PTP domains. The central region shows extensive sequence divergence but it contains several shorter regions of conservation that may function as adaptors in signal transduction. PTPD1 is a component of a cortical scaffold complex nucleated by focal adhesion kinase (FAK) and thus regulates a proliferative signaling pathway through a scaffolding function. PTPD2 has been implicated in the regulation of cell adhesion, as an inducer of TGF-β signaling, and in lymphatic development of mammals and choanal development of humans. Interestingly, PTPD2 is a potential tumor suppressor, based on sporadic mutations in breast cancer cells and colorectal cancer cells. It is tempting to speculate that mammalian PTPD2 shares the function of its fly homolog as a component of Hippo signaling that restrains the oncogenic potential of gut regeneration (Poernbacher, 2012).

    Suppressor of Deltex mediates Pez degradation and modulates Drosophila midgut homeostasis

    Pez functions as an upstream negative regulator of Yorkie (Yki) to regulate intestinal stem cell (ISC) proliferation and is essential for the activity of the Hippo pathway specifically in the Drosophila midgut epithelium. This study reports that Suppressor of Deltex (Su(dx)) acts as a negative regulator of Pez. Su(dx) was shown to target Pez for degradation both in vitro and in vivo. Overexpression of Su(dx) induced proliferation in the fly midgut epithelium, which could be rescued by overexpressed Pez. The study also demonstrated that the interaction between Su(dx) and Pez, bridged by WW domains and PY/PPxY motifs, is required for Su(dx)-mediated Pez degradation. Furthermore, Kibra, a binding partner of Pez, was shown to stabilize Pez via WW-PY/PPxY interaction. Moreover, PTPN14, a Pez mammalian homolog, is degraded by overexpressed Su(dx) or Su(dx) homologue WWP1 in mammalian cells. These results reveal a previously unrecognized mechanism of Pez degradation in maintaining the homeostasis of Drosophila midgut (Wang, 2015).

    The protein tyrosine phosphatase Pez is the Drosophila homologue of non-receptor type protein tyrosine phosphatase 14 (PTPN14), a regulator of the TGF-β pathway (Smith, 1995; Wyatt, 2007; Wyatt, 2008). PTPN14 overexpression activates TGF-β signalling and causes epithelial-mesenchymal transition (EMT) (Wyatt, 2007). Its overexpression is also correlated with lymphatic function, choanal development, angiogenesis and hereditary haemorrhagic telangiectasia (Au, 2010; Benzinou, 2012). Recent studies have revealed that PTPN14 negatively regulates the oncogenic function of Yes-associated protein (YAP) through retaining YAP in the cytoplasm and sustaining the phosphorylation state of YAP (Huang, 2013, Liu, 2013; Wang, 2012; Michaloglou, 2013). YAP is the transcription co-activator downstream of Hippo signalling to mediate the expression of various genes to promote growth, and its upregulation was found in a variety of human tumours and cancers (Wang, 2015).

    Drosophila midgut, where the intestinal stem cells (ISCs) are under tight control to maintain homeostasis, has been developed as an excellent model to study adult stem cells in recent years. The Hippo signalling pathway has been shown to play an essential role in the regulation of ISC proliferation. Pez has been identified as a negative upstream regulator of Yorkie (Yki), the Drosophila homologue of YAP, and is required for the activity of the Hippo pathway in the regulation of ISC proliferation (Poernbacher, 2012). However, how the stability and function of Pez are regulated remains unclear (Wang, 2015).

    Suppressor of Deltex (Su(dx)) is a member of the NEDD4 (neural precursor cell-expressed developmentally downregulated gene 4) family E3 ubiquitin ligase (Cornell, 1999). There are three typical NEDD4 family members in Drosophila, dSmurf, Su(dx) and NEDD4. Each of them contains an N-terminal phospholipid binding C2 domain, four WW domains and a C-terminal HECT-type ligase domain. Su(dx) was first reported as a negative regulator of the Notch signaling pathway (Fostier, 1998). It downregulates the expression of Notch target genes through promoting Notch endosomal sorting (Hori, 2004; Wilkin, 2004; Wang, 2015 and references therein).

    This study shows that Su(dx) targets Pez for degradation both in vitro and in vivo. Su(dx) overexpression induces cell proliferation in Drosophila midgut by downregulating Pez protein levels. It was also demonstrated that Su(dx) directly interacts with Pez via its WW domains and Pez's PY/PPPY motifs. This interaction subsequently promotes Pez ubiquitylation. Furthermore, Kibra, a WW domain containing Pez binding partner, was found to stabilize Pez on interaction. Moreover, evidence is provided that overexpression of Su(dx) or its homologue WWP1 is able to degrade PTPN14 in mammalian culture cells, indicating a possibility that a conserved mechanism of Pez degradation may play an essential role in maintaining tissue homeostasis (Wang, 2015).

    This study reports the identification of Su(dx) as an E3 ligase of Pez. Su(dx) was first identified as an E3 ligase regulating the Notch signalling pathway. But the direct substrate of Su(dx) was unclear. The present study identified Pez as a Su(dx) substrate. Furthermore, whether Pez regulates the Notch signalling pathway was examined. Pez knockdown induced notched wings and a decrease of Cut in wing discs that is very similar to what have caused by Su(dx) overexpression, indicating a dysfunction of the Notch pathway. However, another typical marker of the Notch pathway, wingless, was not affected by the absence of Pez. It is possible that Pez is not a canonical regulator of the Notch pathway and Su(dx) might have other substrates under this circumstance (Wang, 2015).

    According to the observations, Su(dx) overexpression in ECs only induced midgut epithelial proliferation to some extent, and it did not fully mimic the loss of pez induced phenotypes. It is speculated that the difference was largely due to the incomplete degradation efficiency of Pez by Su(dx) overexpression in ECs (Wang, 2015).

    PTPN14 has been reported as an inhibitor of YAP1 in mammalian cells. It can suppress the activity of YAP1 through retaining YAP1 in the cytoplasm and sustaining the phosphorylation state of YAP1 (Wang, 2012). However, in the current experiments, Pez overexpression slightly upregulates Yki phosphorylation level without obvious Yki localization change in S2 cells. It is speculated that the mechanism of YAP regulation by PTPN14 may not be conserved in Drosophila (Wang, 2015).

    Furthermore, this work presents evidence that Kibra, a WW domain containing Pez partner, stabilizes Pez, providing an interesting model that WW–PY/PPxY interaction play a role in the regulation of protein stabilization. In addition, it was found that other WW-containing proteins, such as Sav, were unable to stabilize Pez. On the basis of these observations, the regulation of Pez stabilization by Su(dx) and Kibra is speculated to be a specific event (Wang, 2015).

    It was also found that PTPN14, the human homologue of Pez, can be degraded by overexpressed Su(dx) and its human homologue WWP1. However, in the following experiments, it was found that WWC1, the human homologue of Kibra, did not stabilize PTPN14. These data suggest that, although the similar regulation of Pez/PTPN14 by degradation exists in Drosophila and mammalian cells, the detailed mechanism may vary (Wang, 2015).

    It has been reported that PTPN14 sporadic mutations were found in breast cancer cells and colorectal cancer cells, indicating a potential tumour suppressor function of PTPN14. Moreover, amplification and overexpression of WWP1 has been found in breast and prostate cancers. Therefore, the current study may provide new insights into cancer development. Further characterization of the relationship of Su(dx)-Pez in mice and examination of their correlation in clinical cancers may provide potential targeting therapy for cancer treatments (Wang, 2015).

    A tetraspanin regulates septate junction formation in Drosophila midgut

    Septate junctions (SJs) are membrane specializations that restrict the free diffusion of solutes via the paracellular pathway in invertebrate epithelia. In arthropods, two morphologically different types of SJs are observed: pleated SJs (pSJs) and smooth SJs (sSJs), which are present in ectodermally- and endodermally-derived epithelia, respectively. Recent identification of sSJ-specific proteins, Mesh and Snakeskin (Ssk), in Drosophila indicates that the molecular compositions of sSJs and pSJs differ. A deficiency screen based on immunolocalization of Mesh, identified a tetraspanin family protein, Tetraspanin 2A (Tsp2A), as a novel protein involved in sSJ formation in Drosophila. Tsp2A specifically localizes at sSJs in the midgut and Malpighian tubules. Compromised (Tsp2A) expression caused by RNAi or the CRISPR/Cas9 system is associated with defects in the ultrastructure of sSJs, changes localization of other sSJ proteins, and impairs barrier function of the midgut. In most Tsp2A-mutant cells, Mesh fails to localize to sSJs and is distributed through the cytoplasm. Tsp2A forms a complex with Mesh and Ssk and these proteins are mutually interdependent for their localization. These observations suggest that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).

    Epithelia separate distinct fluid compartments within the bodies of metazoans. For this epithelial function, specialized intercellular junctions, designated as occluding junctions, regulate the free diffusion of solutes through the paracellular pathway. In vertebrates, tight junctions act as occluding junctions, whereas, in invertebrates, septate junctions (SJs) are the functional counterparts of tight junctions. SJs form circumferential belts around the apicolateral regions of epithelial cells. In transmission electron microscopy, SJs are observed between the parallel plasma membranes of adjacent cells, with ladder-like septa spanning the intermembrane space. SJs are subdivided into several morphological types that differ among different animal phyla, and several phyla possess multiple types of SJs that vary among different types of epithelia (Izumi, 2016).

    In arthropods, two types of SJs exist: pleated SJs (pSJs) and smooth SJs (sSJs). pSJs are found in ectodermally-derived epithelia and surface glia surrounding the nerve cord, while sSJs are found mainly in endodermally-derived epithelia, such as the midgut and the gastric caeca. The outer epithelial layer of the proventriculus (OELP) and the Malpighian tubules also possess sSJs, although these epithelia are ectodermal derivatives. The criteria distinguishing these two types of SJs are the arrangement of the septa. In oblique sections of lanthanum-treated preparations, the septa of pSJs are visualized as regular undulating rows but those in sSJs are observed as regularly spaced parallel lines. In freeze-fracture images, the rows of intramembrane particles in pSJs are separated from one another, whereas those in sSJs are fused into ridges. To date, more than 20 pSJ-related proteins, including pSJ components and regulatory proteins involved in pSJ assembly, have been identified and characterized in Drosophila. In contrast, few genetic and molecular analyses have been carried out on sSJs. Recently, two sSJ-specific membrane proteins, Ssk and Mesh, have been identified and characterized (Izumi, 2014; Izumi, 2012; Yanagihashi, 2012). Ssk consists of 162 amino acids and has four membrane-spanning domains, two short extracellular loops, cytoplasmic N- and C-terminal domains, and a cytoplasmic loop (Yanagihashi, 2012). Mesh has a single-pass transmembrane domain and a large extracellular region containing a NIDO domain, an Ig-like E set domain, an AMOP domain, a vWD domain, and a sushi domain (Izumi, 2012). Mesh transcripts are predicted to be translated into three isoforms of which the longest isoform consists of 1,454 amino acids. In Western blot studies, Mesh is detected as a main ~90 kDa band and a minor ~200 kDa band (Izumi, 2012). Compromised expression of ssk or mesh causes defects in the ultrastructure of sSJs and in the barrier function of the midgut against a 10-kDa fluorescent tracer (Izumi, 2012; Yanagihashi, 2012). Ssk and Mesh physically interact with each other and are mutually dependent for their sSJ localization (Izumi, 2012). Thus, Mesh and Ssk play crucial roles in the formation and barrier function of sSJs (Izumi, 2016).

    Tetraspanins are a family of integral membrane proteins in metazoans with four transmembrane domains, N- and C-terminal short intracellular domains, two extracellular loops and one short intracellular turn. Among several protein families with four transmembrane domains, tetraspanins are characterized especially by the structure of the second extracellular loop. It contains a highly conserved cysteine-cysteine-glycine (CCG) motif and 2 to 4 other cysteine residues. These cysteines form 2 or 3 disulfide bonds within the loop. Tetraspanins are believed to play a role in membrane compartmentalization and are involved in many biological processes, including cell migration, cell fusion and lymphocyte activation, as well as viral and parasitic infections. Several tetraspanins regulate cell-cell adhesion but none are known to be involved in the formation of epithelial occluding junctions. In the Drosophila genome, there are 37 tetraspanin family members, and some have been characterized by genetic analyses. Lbm, CG10106 and CG12143 participate in synapse formation. Sun associates with light-dependent retinal degeneration. TspanC8 subfamily members, including Tsp3A, Tsp86D and Tsp26D, are involved in the Notch-dependent developmental processes via the regulation of a transmembrane metalloprotease, ADAM10 (Dornier, 2012). However, the functions of most other Drosophila tetraspanins remain obscure (Izumi, 2016).

    This study identified a tetraspanin family protein, Tsp2A, as a novel molecular component of sSJs in Drosophila. Tsp2A is required for sSJ formation and for the barrier function of Drosophila midgut. Tsp2A and two other sSJ-specific membrane proteins Mesh and Ssk show mutually dependent localizations at sSJs and form a complex with each other. Therefore, it is concluded that Tsp2A cooperates with Mesh and Ssk to organize sSJs (Izumi, 2016).

    Of the sSJ-specific components, Mesh is a membrane-spanning protein and has an ability to induce cell-cell adhesion, implying that it is a cell adhesion molecule and may be one of the components of the electron-dense ladder-like structures in sSJs (Izumi, 2012). In contrast, both Ssk and Tsp2A are unlikely to act as cell adhesion molecules in sSJs because each of the two extracellular loops of Ssk (25 and 22 amino acids, respectively) appear to be too short to bridge the 15-20-nm intercellular space of sSJs. Furthermore, overexpression of EGFP-Tsp2A in Drosophila S2 cells did not induce cell aggregation, which is a criterion for cell adhesion activity (Izumi, 2016).

    Several observations in Tsp2A-mutants may provide clues for understanding the role of Tsp2A in sSJ formation. In most Tsp2A-mutant midgut epithelial cells, Mesh fails to localize to the apicolateral membranes but was distributed in the cytoplasm, possibly to specific intracellular membrane compartments. To further examine where Mesh was localized in Tsp2A-mutant cells, the midgut was doublestained with the anti-Mesh antibody and the antibodies against typical markers of various intracellular membrane compartments, including the Golgi apparatus (anti-GM130), early endosomes (anti-Rab5), recycling endosomes (anti-Rab11) and lysosomes (anti-LAMP1). However, it was not possible to detect any overlap between staining by these markers and that of Mesh. The staining pattern in Tsp2A-mutant midgut epithelial cells produced with the anti-KDEL antibody, which labels endoplasmic reticulum, was similar, although not identical with that produced by the anti-Mesh antibody (Izumi, 2016).

    Interestingly, some tetraspanins are known to control the intracellular trafficking of their partners. For instance, a mammalian tetraspanin, CD81 is necessary for normal trafficking or for surface membrane stability of a phosphoglycoprotein, CD19, in lymphoid B cells. The TspanC8 subgroup proteins, which all possess eight cysteine residues in their large extracellular domain, regulate the exit of a metalloproteinase, ADAM10, from the ER and differentially control its targeting to either late endosomes or to the plasma membrane. Consequently, TspanC8 proteins regulate Notch signaling via the activation of ADAM10 in mammals, Drosophila and Caenorhabditis elegans. If Mesh is retained in the trafficking pathway from endoplasmic reticulum to plasma membrane in Tsp2A-mutant cells, Tsp2A may have an ability to promote the intracellular trafficking of Mesh in the secretory pathway. To clarify the role of Tsp2A in sSJ formation, it will be necessary to determine the intracellular membrane compartment where Mesh was localized in Tsp2A-mutant cells (Izumi, 2016).

    Tsp2A, Mesh and Ssk are mutually dependent for their localization at sSJs. Consistent with this intimate relationship, the co-immunoprecipitation experiment revealed that Tsp2A physically interacts with Mesh and Ssk in vivo. However, the amount of Ssk observed in the co-immunoprecipitation with EGFP-Tsp2A was barely enriched relative to that in the extracts of embryos expressing EGFP-Tsp2A. This was particularly striking in comparison to the degree of enrichment of Mesh in the co-immunoprecipitation with EGFP-Tsp2A. To interpret these results, the detailed manner of the interaction between Tsp2A, Mesh and Ssk proteins needs to be further clarified. Many tetraspanin family proteins are known to interact with one another and with other integral membrane proteins to form a dynamic network of proteins in cellular membranes. Tetraspanins are also believed to have a role in membrane compartmentalization. Given such functional properties of tetraspanins, Tsp2A may determine the localization of sSJs at the apicolateral membrane region by membrane domain formation (Izumi, 2016).

    In the Tsp2A-mutant midgut epithelial cells, Lgl was distributed throughout the basolateral membrane region, whereas it was localized in the apicolateral membrane region in the wild-type. In view of the role of Lgl in the formation of the apical-basal polarity of ectodermally-derived epithelial cells, it is of interest to consider whether this abnormal localization of Lgl in the Tsp2A-mutant affects epithelial polarity. However, in the Tsp2A-mutant midgut epithelial cells, Dlg still showed polarized concentration into the apicolateral membrane region and the Lgl never leaked into the apical membrane domain. These observations suggest that the lack of Tsp2A does not affect the gross apical-basal polarity of the midgut epithelial cells (Izumi, 2016).

    Some tetraspanins have been reported to be involved in the regulation of cell-cell adhesion. A mammalian tetraspanin, CD151, regulates epithelial cell-cell adhesion through PKC- and Cdc42-dependent actin reorganization, or through complex formation with α3γ1 integrin. A mammalian tetraspanin, CD9, is concentrated in the axoglial paranodal region in the brain and in the peripheral nervous system, and CD9 knockout mice display defects in the formation of paranodal septate junctions and in the localization of paranodal proteins. Paranodal septate junctions have electron-dense ladder-like structures and their molecular organization is similar to that of pSJs but tetraspanins involved in pSJ formation have not been reported in Drosophila (Izumi, 2016).

    Interactions between several tetraspanins and claudins, the key integral membrane proteins involved in the organization and function of tight junctions, are also known. Claudin-11 forms a complex with OAP-1/Tspan-3 and chemical crosslinking reveals a direct association between claudin-1 and CD9. Furthermore, the interaction between claudin-1 and CD81 is shown to be required for hepatitis C virus infectivity. To date, no tight junction defect has been reported in CD9 knockout mice, CD81 knockout mice, or CD9/CD81 double knockout mice. Further investigation is necessary to clarify whether the interactions between tetraspanins and tight junction proteins are involved in the formation and function of tight junctions (Izumi, 2016).

    A systematic analysis of Drosophila regulatory peptide expression in enteroendocrine cells

    The digestive system is gaining interest as a major regulator of various functions including immune defense, nutrient accumulation, and regulation of feeding behavior. Intestinal stem cells constantly divide and differentiate into enterocytes that secrete digestive enzymes and absorb nutrients, or enteroendocrine cells that secrete regulatory peptides. This study systemically examined the expression of 45 regulatory peptide genes in the Drosophila midgut, and verified that at least 10 genes are expressed in the midgut enteroendocrine cells through RT-PCR, in situ hybridization, antisera, and 25 regulatory peptide-GAL transgenes. The Drosophila midgut is highly compartmentalized, and individual peptides in enteroendocrine cells were observed to express in specific regions of the midgut. It was also confirmed that some peptides expressed in the same region of the midgut are expressed in mutually exclusive enteroendocrine cells. These results indicate that the midgut enteroendocrine cells are functionally differentiated into different subgroups. Through this study, a basis has been established to study regulatory peptide functions in enteroendocrine cells as well as the complex organization of enteroendocrine cells in the Drosophila midgut (Chen, 2016b).

    A role for the Drosophila zinc transporter Zip88E in protecting against dietary zinc toxicity

    Zinc absorption in animals is thought to be regulated in a local, cell autonomous manner with intestinal cells responding to dietary zinc content. The Drosophila zinc transporter Zip88E shows strong sequence similarity to Zips 42C.1, 42C.2 and 89B as well as mammalian Zips 1, 2 and 3, suggesting that it may act in concert with the apically-localised Drosophila zinc uptake transporters to facilitate dietary zinc absorption by importing ions into the midgut enterocytes. However, the functional characterisation of Zip88E presented in this study indicates that Zip88E may instead play a role in detecting and responding to zinc toxicity. Larvae homozygous for a null Zip88E allele are viable yet display heightened sensitivity to elevated levels of dietary zinc. This decreased zinc tolerance is accompanied by an overall decrease in Metallothionein B transcription throughout the larval midgut. A Zip88E reporter gene is expressed only in the salivary glands, a handful of enteroendocrine cells at the boundary between the anterior and middle midgut regions, and in two parallel strips of sensory cell projections connecting to the larval ventral ganglion. Zip88E expression solely in this restricted subset of cells is sufficient to rescue the Zip88E mutant phenotype. Together, these data suggest that Zip88E may be functioning in a small subset of cells to detect excessive zinc levels and induce a systemic response to reduce dietary zinc absorption and hence protect against toxicity (Richards, 2017).

    Pleiotropic and novel phenotypes in the Drosophila gut caused by mutation of drop-dead
    <{>Normal gut function is vital for animal survival. In Drosophila, mutation of the gene drop-dead (drd) results in defective gut function, as measured by enlargement of the crop and reduced food movement through the gut, and drd mutation also causes the unrelated phenotypes of neurodegeneration, early adult lethality and female sterility. This work shows that adult drd mutant flies lack the peritrophic matrix (PM), an extracellular barrier that lines the lumen of the midgut and is found in many insects including flies, mosquitos and termites. The use of a drd-gal4 construct to drive a GFP reporter in late pupae and adults revealed drd expression in the anterior cardia, which is the site of PM synthesis in Drosophila. Moreover, the ability of drd knockdown or rescue with several gal4 drivers to recapitulate or rescue the gut phenotypes (lack of a PM, reduced defecation, and reduced adult survival 10-40 days post-eclosion) was correlated to the level of expression of each driver in the anterior cardia. Surprisingly, however, knocking down drd expression only in adult flies, which has previously been shown not to affect survival, eliminated the PM without reducing defecation rate. These results demonstrate that drd mutant flies have a novel phenotype, the absence of a PM, which is functionally separable from the previously described gut dysfunction observed in these flies. As the first mutant Drosophila strain reported to lack a PM, drd mutants will be a useful tool for studying the synthesis of this structure (Conway, 2018).

    The cis-regulatory dynamics of embryonic development at single-cell resolution

    This study investigate the dynamics of chromatin regulatory landscapes during embryogenesis at single-cell resolution. Using single-cell combinatorial indexing assay for transposase accessible chromatin with sequencing (sci-ATAC-seq), chromatin accessibility was profiled in over 20,000 single nuclei from fixed Drosophila embryos spanning three embryonic stages: stage 5 blastoderm nuclei; 6-8 h after egg laying, to capture a midpoint in embryonic development when major lineages in the mesoderm and ectoderm are specified; and 10-12 h after egg laying, when cells are undergoing terminal differentiation. The results show that there is spatial heterogeneity in the accessibility of the regulatory genome before gastrulation, a feature that aligns with future cell fate, and that nuclei can be temporally ordered along developmental trajectories. During mid-embryogenesis, tissue granularity emerges such that individual cell types can be inferred by their chromatin accessibility while maintaining a signature of their germ layer of origin. Analysis of the data reveals overlapping usage of regulatory elements between cells of the endoderm and non-myogenic mesoderm, suggesting a common developmental program that is reminiscent of the mesendoderm lineage in other species. 30,075 distal regulatory elements were identified that exhibit tissue-specific accessibility. The germ-layer specificity of a subset of these predicted enhancers was validated in transgenic embryos, achieving an accuracy of 90%. Overall, these results demonstrate the power of shotgun single-cell profiling of embryos to resolve dynamic changes in the chromatin landscape during development, and to uncover the cis-regulatory programs of metazoan germ layers and cell types (Cusanovich, 2018).

    Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster

    At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K(+)] in the hemolymph (hyperkalemia). Cold-acclimation, the physiological response to a prior low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. This study tested whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to seven days at warm (25 ° C) or cold (10 ° C) acclimation conditions. The cold acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K(+) balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K(+) secretion rates in the cold, as well as reduced rectal K(+) reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, the activities were measured of Na(+)-K(+)-ATPase and V-type H(+)-ATPase at the Malpighian tubules, midgut, and hindgut. Na(+)/K(+)-ATPase and V-type H(+)-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold acclimated flies, and were not predictive of the observed alterations in K(+) transport. These results suggest that modification of Malpighian tubule and gut ion and water transport likely prevents cold-induced hyperkalemia in cold-acclimated flies and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na(+)/K(+)- and V-type H(+)-ATPases (Yerushalmi, 2018).

    Age-associated loss of lamin-B leads to systemic inflammation and gut hyperplasia

    Aging of immune organs, termed as immunosenescence, is suspected to promote systemic inflammation and age-associated disease. The cause of immunosenescence and how it promotes disease, however, has remained unclear. This study reports that the Drosophila fat body, a major immune organ, undergoes immunosenescence and mounts strong systemic inflammation that leads to deregulation of immune deficiency (IMD) signaling in the midgut of old animals. Inflamed old fat bodies secrete circulating peptidoglycan recognition proteins that repress IMD activity in the midgut, thereby promoting gut hyperplasia. Further, fat body immunosenecence is caused by age-associated lamin-B reduction specifically in fat body cells, which then contributes to heterochromatin loss and derepression of genes involved in immune responses. As lamin-associated heterochromatin domains are enriched for genes involved in immune response in both Drosophila and mammalian cells, these findings may provide insights into the cause and consequence of immunosenescence during mammalian aging (Chen, 2014).

    By analyzing gene expression changes upon aging in fat bodies and midguts, it was shown that an increase of immune response in the fat body is accompanied by a striking reduction in the midgut. Specifically, it was demonstrate that the age-associated increase in Immune deficiency (IMD) signaling in fat bodies leads to reduction of IMD activity in the midgut, which in turn contributes to midgut hyperplasia. This fat body to midgut effect requires peptidoglycan recognition proteins (PGRPs) secreted from fat body cells and is mediated by both bacteria dependent and independent pathways. Therefore, fat body aging contributes to systemic inflammation, which contributes to the disruption of gut homeostasis. Importantly, it was shown that the age-associated lamin-B loss in fat body cells causes the derepression of a large number of immune responsive genes, thereby resulting in fat body-based systemic inflammation (Chen, 2014).

    B-type lamins have long been suggested to have a role in maintaining heterochromatin and gene repression. Consistently, this study's global analyses of fat body depleted of lamin-B revealed a loss of heterochromatin and derepression of a large number of immune responsive genes. This is further supported by ChIP-qPCR analyses of H3K9me3 on specific IMD regulators. Recent studies in different cell types show that tethering genes to nuclear lamins do not always lead to their repression. Deleting B-type lamins or all lamins in mouse ES cells or trophectdoderm cells does not result in derepression of all genes in LADs. In light of these studies, it is suggested that the transcriptional repression function of lamin-B could be gene and cell type dependent. Interestingly, GO analyses revealed a significant enrichment of immune responsive genes in Lamin-associated domains (LADs) in four different mammalian cell types and Drosophila Kc cells. Since the large-scale pattern of LADs is conserved in different cell types in mammals, it is possible that the immune-responsive genes are also enriched in LADs in the fly fat body cells. Supporting this notion, the IKKγ, key, which is one of the two derepressed IMD regulators and was found to exhibit H3K9me3 reduction and gene activation, is localized to LADs in Kc cells. It is speculated that lamin-B might play an evolutionarily conserved role in repressing a subset of inflammatory genes in certain tissues, such as the immune organs, in the absence of infection or injury. Consistently, senescence-associated lamin-B1 loss in mammalian fibroblasts is correlated with senescence-associated secretory phenotype senescence-associated secretory phenotype (SASP). Although the in vivo relevance of fibroblast SASP in chronic inflammation and aging-associated diseases in mammals remains to be established, the findings in Drosophila provide insights and impetus to investigate the role of lamins in immunosenescence and systemic inflammation in mammals (Chen, 2014).

    Lamin-B gradually decreases in fat body cells of aging flies, whereas lamin-C amount remains the same. Since it has been recently shown that the assembly of an even and dense nuclear lamina is dependent on the total lamin concentration, the age-associated appearance of lamin-B and lamin-C gaps around the nuclear periphery of fat body cells is likely caused by the drop of the lamin-B level. How aging triggers lamin-B loss is unknown, but it appears to be posttranscriptional, because lamin-B transcripts in fat bodies remain unchanged upon aging. Interestingly, among the tissues examined, no changes of lamin-B and lamin-C proteins were found in cells in the heart tube, oenocytes, or gut epithelia in old flies. Therefore, the age-associated lamin-B loss does not occur in all cell types in vivo. A systematic survey to establish the cell/tissue types that undergo age-associated reduction of lamins in both flies and mammals should provide clues to the cause of loss. Deciphering how advanced age leads to lamin loss should open the door to further investigate the cellular mechanism that contributes to chronic systemic inflammation and how it in turn promotes age-associated diseases in humans (Chen, 2014).

    Old Drosophila gut is known to exhibit increased microbial load, which would cause increased stress response and activation of tissue repair, thereby leading to midgut hyperplasia. Systemic inflammation caused by lamin-B loss in fat body leads to repression of local midgut IMD signaling. The upregulation of targets of IMD in the aged whole gut has been recently reported, while a downregulation of target genes was observed in the current analyses of the midgut. However, the previous study found a similar upregulation of the genes when performing RNA-seq of the whole gut (Chen, 2014).

    These studies reveal an involvement of bacteria in the repression of midgut IMD signaling by the PGRPs secreted from the fat body. How PGRPs from the fat body repress midgut IMD is still unknown. One possibility is that the body cavity bacteria contribute to the maintenance of midgut IMD activity, and the increased circulating PGRPs limit these bacteria. The circulating PGRPs may also reduce midgut IMD activity indirectly by affecting other tissues. The evidence suggests that lamin-B loss could also contribute to midgut hyperplasia independent of the IMD pathway. While it will be important to further address these possibilities, the findings have revealed a fat body mediated inflammatory pathway that can lead to reduced migut IMD, increased gut microbial accumulation, and midgut hyperplasia upon aging (Chen, 2014).

    Interestingly, microbiota changes also occur in aging human intestine and have been linked to altered intestinal inflammatory states and diseases. Although, much effort has been devoted to understand how local changes in aging mammalian intestines affect gut microbial community, the cause remains unclear. The findings in Drosophila reveal the importance of understanding the impact of immunosenescence and systemic inflammation on gut microbial homeostasis. Indeed, if increased circulating inflammatory cytokines perturb the ability of local intestine epithelium and the gut-associated lymphoid tissue to maintain a balanced microbial community, the unfavorable microbiota in the old intestine would cause chronic stress response and tissue repair, thereby leading to uncontrolled cell growth as observed in age-associated cancers (Chen, 2014).

    Genetic, molecular and physiological basis of variation in Drosophila gut immunocompetence

    Gut immunocompetence involves immune, stress and regenerative processes. To investigate the determinants underlying inter-individual variation in gut immunocompetence, enteric infection was performed of 140 Drosophila lines with the entomopathogenic bacterium Pseudomonas entomophila, and extensive variation was observed in survival. Using genome-wide association analysis, several novel immune modulators were identified. Transcriptional profiling further shows that the intestinal molecular state differs between resistant and susceptible lines, already before infection, with one transcriptional module involving genes linked to reactive oxygen species (ROS) metabolism contributing to this difference. This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines. This study revealed how relatively minor, but systematic variation can mediate overt physiological differences that determine enteric infection susceptibility (Bou Sleiman, 2015).

    Distinct shifts in microbiota composition during Drosophila aging impair intestinal function and drive mortality

    Alterations in the composition of the intestinal microbiota have been correlated with aging and measures of frailty in the elderly. However, the relationships between microbial dynamics, age-related changes in intestinal physiology, and organismal health remain poorly understood. This study shows that dysbiosis of the intestinal microbiota, characterized by an expansion of the Gammaproteobacteria, is tightly linked to age-onset intestinal barrier dysfunction in Drosophila. Indeed, alterations in the microbiota precede and predict the onset of intestinal barrier dysfunction in aged flies. Changes in microbial composition occurring prior to intestinal barrier dysfunction contribute to changes in excretory function and immune gene activation in the aging intestine. In addition, a distinct shift in microbiota composition was shown to follow intestinal barrier dysfunction, leading to systemic immune activation and organismal death. These results indicate that alterations in microbiota dynamics could contribute to and also predict varying rates of health decline during aging in mammals (Clark, 2015).

    Preventing age-related decline of gut compartmentalization limits microbiota dysbiosis and extends lifespan

    Compartmentalization of the gastrointestinal (GI) tract of metazoans is critical for health. GI compartments contain specific microbiota, and microbiota dysbiosis is associated with intestinal dysfunction. Dysbiosis develops in aging intestines, yet how this relates to changes in GI compartmentalization remains unclear. The Drosophila GI tract is an accessible model to address this question. This study shows that the stomach-like copper cell region (CCR) in the middle midgut controls distribution and composition of the microbiota. It was found that chronic activation of JAK/Stat signaling in the aging gut induces a metaplasia of the gastric epithelium, CCR decline, and subsequent commensal dysbiosis and epithelial dysplasia along the GI tract. Accordingly, inhibition of JAK/Stat signaling in the CCR specifically prevents age-related metaplasia, commensal dysbiosis and functional decline in old guts, and extends lifespan. These results establish a mechanism by which age-related chronic inflammation causes the decline of intestinal compartmentalization and microbiota dysbiosis, limiting lifespan (Li, 2016).

    Gut microbiota in Drosophila melanogaster interacts with Wolbachia but does not contribute to Wolbachia-mediated antiviral protection

    Animals experience near constant infection with microorganisms. A significant proportion of these microbiota reside in the alimentary tract. There is a growing appreciation for the roles gut microbiota play in host biology. The gut microbiota of insects, for example, have been shown to help the host overcome pathogen infection either through direct competition or indirectly by stimulating host immunity. These defenses may also be supplemented by coinfecting maternally inherited microbes such as Wolbachia. The presence of Wolbachia in a host can delay and/or reduce death caused by RNA viruses. Whether the gut microbiota of the host interacts with Wolbachia, or vice versa, the precise role of Wolbachia in antiviral protection is not known. This study used 16S rDNA sequencing to characterize changes in gut microbiota composition in Drosophila melanogaster associated with Wolbachia infection and antibiotic treatment. Subsequently, it was tested whether changes in gut composition via antibiotic treatment alter Wolbachia-mediated antiviral properties. It was found that both antibiotics and Wolbachia significantly reduce the biodiversity of the gut microbiota without changing the total microbial load. Changing the gut microbiota composition with antibiotic treatment enhances Wolbachia density but does not confer greater antiviral protection against Drosophila C virus to the host. In conclusion, there are significant interactions between Wolbachia and gut microbiota, but changing gut microbiota composition is not likely to be a means through which Wolbachia conveys antiviral protection to its host (Ye, 2017).

    Stable host gene expression in the gut of adult Drosophila melanogaster with different bacterial mono-associations

    There is growing evidence that the microbes found in the digestive tracts of animals influence host biology, but how they accomplish this is still not understood. This study evaluated how different microbial species commonly associated with laboratory-reared Drosophila melanogaster impact host biology at the level of gene expression in the dissected adult gut and in the entire adult organism. It was observed that guts from animals associated from the embryonic stage with either zero, one or three bacterial species demonstrated indistinguishable transcriptional profiles. Additionally, it was found that the gut transcriptional profiles of animals reared in the presence of the yeast Saccharomyces cerevisiae alone or in combination with bacteria could recapitulate those of conventionally-reared animals. In contrast, whole body transcriptional profiles of conventionally-reared animals were distinct from all of the treatments tested. These data suggest that adult flies are insensitive to the ingestion of the bacteria found in their gut, but that prior to adulthood, different microbes impact the host in ways that lead to global transcriptional differences observable across the whole adult body (Elya, 2016).

    Drosophila melanogaster establishes a species-specific mutualistic interaction with stable gut-colonizing bacteria

    This study identified bacterial species from wild-caught D. melanogaster that stably associate with the host independently of continuous inoculation. Moreover, it was shown that specific Acetobacter wild isolates can proliferate in the gut. It was further demonstrated that the interaction between D. melanogaster and the wild isolated Acetobacter thailandicus is mutually beneficial and that the stability of the gut association is key to this mutualism. The stable population in the gut of D. melanogaster allows continuous bacterial spreading into the environment, which is advantageous to the bacterium itself. The bacterial dissemination is in turn advantageous to the host because the next generation of flies develops in the presence of this particularly beneficial bacterium. A. thailandicus leads to a faster host development and higher fertility of emerging adults when compared to other bacteria isolated from wild-caught flies. Furthermore, A. thailandicus is sufficient and advantageous when D. melanogaster develops in axenic or freshly collected figs, respectively. This isolate of A. thailandicus colonizes several genotypes of D. melanogaster but not the closely related D. simulans, indicating that the stable association is host specific. This work establishes a new conceptual model to understand D. melanogaster-gut microbiota interactions in an ecological context; stable interactions can be mutualistic through microbial farming, a common strategy in insects. Moreover, these results develop the use of D. melanogaster as a model to study gut microbiota proliferation and colonization (Pais, 2018).

    The Drosophila microbiome has a limited influence on sleep, activity, and courtship behaviors

    In animals, commensal microbes modulate various physiological functions, including behavior. While microbiota exposure is required for normal behavior in mammals, it is not known how widely this dependency is present in other animal species. The hypothesis is proposed that the microbiome has a major influence on the behavior of the vinegar fly (Drosophila melanogaster), a major invertebrate model organism. Several assays were used to test the contribution of the microbiome on some well-characterized behaviors: defensive behavior, sleep, locomotion, and courtship in microbe-bearing, control flies and two generations of germ-free animals. None of the behaviors were largely influenced by the absence of a microbiome, and the small or moderate effects were not generalizable between replicates and/or generations. These results refute the hypothesis, indicating that the Drosophila microbiome does not have a major influence over several behaviors fundamental to the animal's survival and reproduction. The impact of commensal microbes on animal behaviour may not be broadly conserved (Selkrig, 2018).

    A Mesh-Duox pathway regulates homeostasis in the insect gut

    The metazoan gut harbours complex communities of commensal and symbiotic bacterial microorganisms. The quantity and quality of these microorganisms fluctuate dynamically in response to physiological changes. The mechanisms that hosts have developed to respond to and manage such dynamic changes and maintain homeostasis remain largely unknown. This study identified a dual oxidase (Duox)-regulating pathway that contributes to maintaining homeostasis in the gut of both Aedes aegypti and Drosophila melanogaster. A gut-membrane-associated protein, named Mesh, plays an important role in controlling the proliferation of gut bacteria by regulating Duox expression through an Arrestin-mediated MAPK JNK/ERK phosphorylation cascade. Expression of both Mesh and Duox is correlated with the gut bacterial microbiome, which, in mosquitoes, increases dramatically soon after a blood meal. Ablation of Mesh abolishes Duox induction, leading to an increase of the gut microbiome load. This study reveals that the Mesh-mediated signalling pathway is a central homeostatic mechanism of the insect gut (Xiao, 2017).

    Probabilistic invasion underlies natural gut microbiome stability

    Species compositions of gut microbiomes impact host health, but the processes determining these compositions are largely unknown. An unexplained observation is that gut species composition varies widely between individuals but is largely stable over time within individuals. Stochastic factors during establishment may drive these alternative stable states (colonized versus non-colonized), which can influence susceptibility to pathogens, such as Clostridium difficile. A precise, high-throughput technique revealed stable between-host variation in colonization when individual germ-free flies were fed their own natural commensals (including the probiotic Lactobacillus plantarum). Some flies were colonized while others remained germ-free even at extremely high bacterial doses. Thus, alternative stable states of colonization exist even in this low-complexity model of host-microbe interactions. These alternative states are driven by a fundamental asymmetry between the inoculum population and the stably colonized population that is mediated by spatial localization and a population bottleneck, which makes stochastic effects important by lowering the effective population size. Prior colonization with other bacteria reduced the chances of subsequent colonization, thus increasing the stability of higher-diversity guts. Therefore, stable gut diversity may be driven by inherently stochastic processes, which has important implications for combatting infectious diseases and for stably establishing probiotics in the gut (Obadia, 2017).

    Gut microbiota modifies olfactory-guided microbial preferences and foraging decisions in Drosophila

    The gut microbiota affects a wide spectrum of host physiological traits, including development, germline, immunity, nutrition, and longevity. Association with microbes also influences fitness-related behaviors such as mating and social interactions. Although the gut microbiota is evidently important for host wellbeing, how hosts become associated with particular assemblages of microbes from the environment remains unclear. This study presents evidence that the gut microbiota can modify microbial and nutritional preferences of Drosophila melanogaster. By experimentally manipulating the gut microbiota of flies subjected to behavioral and chemosensory assays, fly-microbe attractions were found to be shaped by the identity of the host microbiota. Conventional flies exhibit preference for their associated Lactobacillus, a behavior also present in axenic flies as adults and marginally as larvae. By contrast, fly preference for Acetobacter is primed by early-life exposure and can override the innate preference. These microbial preferences are largely olfactory guided and have profound impact on host foraging, as flies continuously trade off between acquiring beneficial microbes and balancing nutrients from food. This study shows a role of animal microbiota in shaping host fitness-related behavior through their chemosensory responses, opening a research theme on the interrelationships between the microbiota, host sensory perception, and behavior (Wong, 2017).

    How gut transcriptional function of Drosophila melanogaster varies with the presence and composition of the gut microbiota

    Despite evidence from laboratory experiments that perturbation of the gut microbiota affects many traits of the animal host, understanding of the effect of variation in microbiota composition on animals in natural populations is very limited. This study used Drosophila to identify the impact of natural variation in the taxonomic composition of gut bacterial communities on host traits, with the gut transcriptome as a molecular index of microbiota-responsive host traits. Use of the gut transcriptome was validated by demonstrating significant transcriptional differences between the guts of laboratory flies colonized with bacteria and maintained under axenic conditions. Wild Drosophila from six field collections made over two years had gut bacterial communities of diverse composition, dominated to varying extents by Acetobacteraceae and Enterobacteriaceae. The gut transcriptomes also varied among collections and differed markedly from those of laboratory flies. However, no overall relationship between variation in the wild fly transcriptome and taxonomic composition of the gut microbiota was evident at all taxonomic scales of bacteria tested for both individual fly genes and functional categories in Gene Ontology. It is concluded that the interaction between microbiota composition and host functional traits may be confounded by uncontrolled variation in both ecological circumstance and host traits (e.g., genotype, age physiological condition) under natural conditions, and that microbiota effects on host traits identified in the laboratory should, therefore, be extrapolated to field population with great caution (Bost, 2017).

    Misato underlies visceral myopathy in Drosophila

    Genetic mechanisms for the pathogenesis of visceral myopathy (VM) have been rarely demonstrated. This study reports the visceral role of misato (mst) in Drosophila and its implications for the pathogenesis of VM. Depletion of mst using three independent RNAi lines expressed by a pan-muscular driver elicited characteristic symptoms of VM, such as abnormal dilation of intestinal tracts, reduced gut motility, feeding defects, and decreased life span. By contrast, exaggerated expression of mst reduced intestine diameters, but increased intestinal motilities along with thickened muscle fibers, demonstrating a critical role of mst in the visceral muscle. Mst expression was detected in the adult intestine with its prominent localization to actin filaments and was required for maintenance of intestinal tubulin and actomyosin structures. Consistent with the subcellular localization of Mst, the intestinal defects induced by mst depletion were dramatically rescued by exogenous expression of an actin member. Upon ageing the intestinal defects were deteriorative with marked increase of apoptotic responses in the visceral muscle. Taken together, it is proposed that the impairment of actomyosin structures induced by mst depletion in the visceral muscle as a pathogenic mechanism for VM (Min, 2017).

    Inflammation-modulated metabolic reprogramming is Required for DUOX-dependent gut immunity in Drosophila

    DUOX, a member of the NADPH xidase family, acts as the first line of defense against enteric pathogens by producing microbicidal reactive oxygen species. DUOX is activated upon enteric infection, but the mechanisms regulating DUOX activity remain incompletely understood. Using Drosophila genetic tools, this study shows that enteric infection results in "pro-catabolic" signaling that initiates metabolic reprogramming of enterocytes toward lipid catabolism, which ultimately governs DUOX homeostasis. Infection induces signaling cascades involving TRAF3 and kinases AMPK and WTS, which regulate TOR kinase to control the balance of lipogenesis versus lipolysis. Enhancing lipogenesis blocks DUOX activity, whereas stimulating lipolysis via ATG1-dependent lipophagy is required for DUOX activation. Drosophila with altered activity in TRAF3-AMPK/WTS-ATG1 pathway components exhibit abolished infection-induced lipolysis, reduced DUOX activation, and enhanced susceptibility to enteric infection. Thus, this work uncovers signaling cascades governing inflammation-induced metabolic reprogramming and provides insight into the pathophysiology of immune-metabolic interactions in the microbe-laden gut epithelia (Lee, 2018).

    Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging in Drosophila

    Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells' environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. This study showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. Flies were generated with knockdown of Mre11, Rad50, Nbs1, ATM, ATR, Chk1, and Chk2, which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly's survival. These data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. These findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging (Park, 2018).

    The Drosophila melanogaster gut microbiota provisions thiamine to its host

    The microbiota of Drosophila melanogaster has a substantial impact on host physiology and nutrition. Some effects may involve vitamin provisioning, but the relationships between microbe-derived vitamins, diet, and host health remain to be established systematically. This study explored the contribution of microbiota in supplying sufficient dietary thiamine (vitamin B1) to support D. melanogaster at different stages of its life cycle. Using chemically defined diets with different levels of available thiamine, it was found that the interaction of thiamine concentration and microbiota did not affect the longevity of adult D. melanogaster Likewise, this interplay did not have an impact on egg production. However, it was determined that thiamine availability has a large impact on offspring development, as axenic offspring were unable to develop on a thiamine-free diet. Offspring survived on the diet only when the microbiota was present or added back, demonstrating that the microbiota was able to provide enough thiamine to support host development. Through gnotobiotic studies, it was determined that Acetobacter pomorum, a common member of the microbiota, was able to rescue development of larvae raised on the no-thiamine diet. Further, it was the only microbiota member that produced measurable amounts of thiamine when grown on the thiamine-free fly medium. Its close relative Acetobacter pasteurianus also rescued larvae; however, a thiamine auxotrophic mutant strain was unable to support larval growth and development. The results demonstrate that the D. melanogaster microbiota functions to provision thiamine to its host in a low-thiamine environment (Sannino, 2018).

    RNA polymerase III limits longevity downstream of TORC1

    Three distinct RNA polymerases transcribe different classes of genes in the eukaryotic nucleus. RNA polymerase (Pol) III is the essential, evolutionarily conserved enzyme that generates short, non-coding RNAs, including tRNAs and 5S rRNA. The historical focus on transcription of protein-coding genes has left the roles of Pol III in organismal physiology relatively unexplored. Target of rapamycin kinase complex 1 (TORC1) regulates Pol III activity, and is also an important determinant of longevity. This raises the possibility that Pol III is involved in ageing. This study shows that Pol III limits lifespan downstream of TORC1. A reduction in Pol III extends chronological lifespan in yeast and organismal lifespan in worms and flies. Inhibiting the activity of Pol III in the gut of adult worms or flies is sufficient to extend lifespan; in flies, longevity can be achieved by Pol III inhibition specifically in intestinal stem cells. The longevity phenotype is associated with amelioration of age-related gut pathology and functional decline, dampened protein synthesis and increased tolerance of proteostatic stress. Pol III acts on lifespan downstream of TORC1, and limiting Pol III activity in the adult gut achieves the full longevity benefit of systemic TORC1 inhibition. Hence, Pol III is a pivotal mediator of this key nutrient-signalling network for longevity; the growth-promoting anabolic activity of Pol III mediates the acceleration of ageing by TORC1. The evolutionary conservation of Pol III affirms its potential as a therapeutic target (Filer, 2017).

    The task of carrying out transcription in the eukaryotic nucleus is divided among RNA Pol I, II and III. This specialization is evident in the biogenesis of the translation machinery, a task that requires the co-ordinated activity of all three polymerases: Pol I generates the 45S pre-rRNA that is subsequently processed into mature rRNAs, Pol II transcribes various RNAs including mRNAs encoding ribosomal proteins, while Pol III provides the tRNAs and 5S rRNA. This costly process of generating protein synthetic capacity is tightly regulated to match the extrinsic conditions and the intrinsic need for protein synthesis by the key driver of cellular anabolism, TORC1. The central position of TORC1 in the control of fundamental cellular processes is mirrored by the notable effect of its activity on organismal physiology: following its initial discovery in worms, inhibition of TORC1 has been demonstrated to extend lifespan in all tested organisms, from yeast to mice, with beneficial effects on a range of age-related diseases and dysfunctions. TORC1 strongly activates Pol III transcription and this relationship suggests the possibility that inhibition of Pol III promotes longevity (Filer, 2017).

    In Saccharomyces cerevisiae, each of the 17 Pol III subunits is encoded by an essential gene. This study generated a yeast strain in which the largest Pol III subunit (C160, encoded by RPC160, also known as RPO31) is fused to the auxin-inducible degron (AID). The fusion protein can be targeted for degradation by the ectopically expressed E3 ubiquitin ligase (OsTir) in the presence of indole-3-acetic acid (IAA) to achieve conditional inhibition of Pol III. It was confirmed that IAA treatment triggered degradation of the fusion protein, and it was observed that IAA treatment also improved the survival of the RPC160-AID strain upon prolonged culture. In addition, IAA treatment of the control strain lacking the AID fusion reduced its survival relative to both the same strain in the absence of IAA and to the RPC160-AID strain in the presence of IAA. Hence, Pol III depletion appears to extend the chronological lifespan in yeast. While IAA had no substantial effect on the survival of a strain carrying the AID domain fused to the largest subunit of Pol II (RPB220, also known as RP021), this strain appeared to survive better than the control strain did in the presence of IAA, indicating that inhibition of Pol II may also extend chronological lifespan. Chronological lifespan of yeast is a measure of survival in a nutritionally limited, quiescent population, whereas replicative lifespan measures the number of daughters produced by a single mother cell in its lifetime. No evidence was found that inhibition of Pol III causes an increase in the replicative lifespan in yeast (Filer, 2017).

    The observed increase in chronological lifespan may simply indicate increased stress resistance and hence be of limited relevance to organismal ageing. To examine the role of Pol III in organismal ageing directly, animal models were examined. RNA-mediated interference (RNAi) was initiated against rpc-1, the Caenorhabditis elegans orthologue of RPC160, in worms from the L4 stage, causing a partial knockdown of rpc-1 mRNA. This consistently extended the lifespan of worms at both 20°C and 25°C. To reduce Pol III activity in Drosophila melanogaster, a P-element insertion that deletes the transcriptional start site of the gene encoding the Pol III-specific subunit C53 (CG5147EY22749, henceforth called dC53EY, was backcrossed into a healthy, outbred population of flies. Homozygous dC53EY/EY mutants were not viable, but heterozygous females had reduced dC53 mRNA levels and lived longer than controls. Taken together, these data strongly indicate that Pol III limits lifespan in multiple model organisms and conversely, that partial inhibition of its activity is an intervention that increases longevity in multiple species (Filer, 2017).

    The longevity of an animal can be governed from a single organ. In the worm, this role is often played by the gut. To restrict the rpc-1 knockdown to the gut, worms were used that were deficient in rde-1, in which the RNAi machinery deficiency is restored in the gut by gut-specific rde-1 rescue. rpc-1 RNAi extended the lifespan of this strain, both at 20°C and 25°C. Similarly, in the adult fly, driving an RNAi construct targeting the RPC160 orthologue (CG17209, henceforth called dC160,with the mid-gut-specific, RU486-inducible driver TIGS extended the lifespan of females, while the presence of the inducer (RU486) did not affect survival of the control strains. The longevity phenotype could also be recapitulated with RNAi against dC53, another Pol III subunit, indicating that the phenotype was not subunit-specific or due to off-target effects. As well as the gut, longevity can also be associated with the fat body and neurons in flies. However, the longevity phenotype caused by dC160 RNAi appears to be specific to the gut, since no significant lifespan extension was observed upon induction of dC160 RNAi in the fat body of the adult fly, and only a modest, albeit significant, extension resulted from neuronal induction of dC160 RNAi (Filer, 2017).

    The worm gut is composed of only post-mitotic cells. In flies, as in mammals, the adult gut epithelium contains mitotically active intestinal stem cells, and the mid-gut-specific driver TIGS appears to be active in at least some ISCs, prompting restricting of dC160 RNAi induction to this cell type. ISC-specific dC160 RNAi, achieved with the GS5961 driver, was sufficient to promote longevity. In summary, Pol III activity in the gut limits survival in worms and flies, and in the fly, Pol III can drive ageing specifically from the gut stem-cell compartment (Filer, 2017).

    The consequences of Pol III inhibition in the fly gut was assessed. Pol III acts to generate precursor tRNAs (pre-tRNAs) that are processed rapidly to mature tRNAs. Owing to their short half-lives, pre-tRNAs are useful as readouts of in vivo Pol III activity. Profiling the levels of specific pre-tRNAs, pre-tRNAHis, pre-tRNAAla and pre-tRNALeu, relative to the levels of U3 (a small nucleolar RNA transcribed by Pol II) revealed a moderate but significant reduction in Pol III activity upon gut-specific induction of dC160 RNAi. The three polymerases can be directly coordinated to generate the translation machinery. Indeed, Pol III inhibition had knock-on effects on Pol I- but not Pol II-generated transcripts, revealing partial cross-talk. dC160 RNAi also reduced protein synthesis in the gut, consistent with reduced Pol III activity. These effects (reduction in pre-tRNAs or protein synthesis) were not observed after feeding RU486 to the driver-only control. The reduction in protein synthesis was not pathological: total protein content of the gut was unaltered; fecundity, a sensitive readout of a female's nutritional status, was unaffected; and the flies' weight, triacylglycerol and protein levels remained unchanged. Reduced protein synthesis can liberate protein-folding machinery from protein production and increase homeostatic capacity. Indeed, induction of dC160 RNAi in the gut increased the resistance of adult flies to proteostatic challenge with tunicamycin for TIGS-only control. Hence, Pol III can fine-tune the rate of protein synthesis in the adult fly gut without obvious detrimental outcomes, while increasing resistance to proteotoxic stress (Filer, 2017).

    Having demonstrated the relevance of Pol III for ageing, whether it acts on lifespan downstream of TORC1 was investigated in Drosophila. Numerous observations in several organisms support the model in which TORC1 localizes on Pol III-transcribed loci and promotes phosphorylation of the components of the Pol III transcriptional machinery to activate transcription, in part by inhibition of the Pol III repressor, Maf1. Using chromatin immunoprecipitation (ChIP) with two independently generated antibodies against Drosophila TOR (target of rapamycin), TOR enrichment was observed on Pol III-target genes in the adult fly, relative to Pol II targets. Inhibition of TORC1 by feeding rapamycin to flies reduced the levels of pre-tRNAs in whole flies. Rapamycin also reduced pre-tRNA levels specifically in the gut relative to U3. Since rapamycin results in re-scaling of the gut, evidenced by the reduction in the total RNA content of the organ, it was also confirmed that the drug reduced pre-tRNA levels relative to total RNA. Interestingly, rapamycin did not cause a decrease in 45S pre-rRNA in the gut, suggesting a lack of sustained Pol I inhibition. Additionally, gut-specific overexpression of Maf1 reduced the levels of pre-tRNAs and extended lifespan, confirming that Maf1 acts on Pol III in the adult gut. These data are consistent with TORC1 driving systemic and gut-specific Pol III activity in the adult fruitfly (Filer, 2017).

    To examine whether the lifespan effects of Pol III are downstream of TORC1, adult-onset Pol III inhibition was combined with rapamycin treatment. Rapamycin feeding or gut-specific dC160 RNAi resulted in the same magnitude of lifespan extension. The two treatments were not additive, consistent with their acting on the same longevity pathway. The same effect was observed with RNAi against dC53 in the gut, as well as when dC160 RNAi was restricted to the ISCs. Importantly, rapamycin feeding also inhibited phosphorylation of the TORC1 substrate, S6 kinase (S6K), in both the gut and the whole fly, and decreased fecundity, while gut-specific C160 RNAi did not have these effects. This confirms that Pol III inhibition does not impact TORC1 activity locally or systemically, and therefore, Pol III acts downstream of TORC1 in ageing (Filer, 2017).

    TORC1 inhibition is known to ameliorate age-related pathology and functional decline of the gut. Whether inhibition of Pol III was sufficient to block the dysplasia resulting from hyperproliferation and aberrant differentiation of ISCs was examined by assessing the characteristic, age-dependent increase in dividing phospho-histone H3 (pH3)-positive cells. Inducing dC160 RNAi in the fly gut or solely in the ISCs ameliorated this pathology. These treatments also counteracted the age-related loss of gut barrier function, decreasing the number of flies displaying extra-intestinal accumulation of a blue food dye (the 'Smurf' phenotype). It as also found that rpc-1 RNAi reduced the severity of age-related loss of gut-barrier function in worms. In Drosophila, gut health and TORC1 inhibition are specifically linked to female survival. Indeed, induction of dC160 RNAi in the gut had a sexually dimorphic effect on lifespan, as the effect on males, although significant, was lower in magnitude relative to the effect on females. Overall, the data show that gut- or ISC-specific inhibition of Pol III, which extends lifespan, is sufficient to ameliorate age-related impairments in gut health, which may be causative of or correlate with this longevity (Filer, 2017).

    This study demonstrates that the adult-onset decrease in the growth-promoting anabolic function mediated by Pol III in the gut, and specifically in the intestinal stem-cell compartment, is sufficient to recapitulate the longevity benefits of rapamycin treatment. Pol III activity is essential for growth; its detrimental effects on ageing suggest an antagonistic pleiotropy in which wild-type levels of Pol III activity are optimised for growth and reproductive fitness in early life but prove detrimental for later health. This study reveals a fundamental role for Pol III in adult physiology, implicating wild-type Pol III activity in age-related stem-cell dysfunction, declining gut health and organismal survival downstream of nutrient signalling pathways. The longevity resulting from partial Pol III inhibition in adulthood is likely to result from the reduced provision of protein synthetic machinery; however, differential regulation of tRNA genes or Pol III-mediated changes to chromatin organization may also be involved, as has been suggested in other contexts (Arimbasseri, 2016). The strong structural and functional conservation of Pol III in eukaryotes suggests that studies of its influence on mammalian ageing are warranted and could lead to important therapies (Filer, 2017).

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