Damage‐induced regeneration of the intestinal stem cell pool through enteroblast mitosis in the Drosophila midgut

Many adult tissues and organs including the intestine rely on resident stem cells to maintain homeostasis and regeneration. In mammals, the progenies of intestinal stem cells (ISCs) can dedifferentiate to generate ISCs upon ablation of resident stem cells. However, whether and how mature tissue cells generate ISCs under physiological conditions remains unknown. Here, we show that infection of the Drosophila melanogaster intestine with pathogenic bacteria induces entry of enteroblasts (EBs), which are ISC progenies, into the mitotic cycle through upregulation of epidermal growth factor receptor (EGFR)‐Ras signaling. We also show that ectopic activation of EGFR‐Ras signaling in EBs is sufficient to drive enteroblast mitosis cell autonomously. Furthermore, we find that the dividing enteroblasts do not gain ISC identity as a prerequisite to divide, and the regenerative ISCs are produced through EB mitosis. Taken together, our work uncovers a new role for EGFR‐Ras signaling in driving EB mitosis and replenishing the ISC pool during fly intestinal regeneration, which may have important implications for tissue homeostasis and tumorigenesis in vertebrates.     

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. Our recent work demonstrates that Rac1-dependent ROS production mediates intestinal stem cell (ISC) proliferation in mouse models of colorectal cancer (CRC). Here, we use 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. Our results demonstrate that Rac1 is necessary and sufficient to drive ISC proliferation and regeneration in an ROS-dependent manner. Our 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.     

Dual role of BMP signaling in the regulation of Drosophila intestinal stem cell self-renewal

Many adult organs including Drosophila adult midguts rely on resident stem cells to replenish damaged cells during tissue homeostasis and regeneration. Previous studies have shown that, upon injury, intestinal stem cells (ISCs) in the midguts can increase proliferation and lineage differentiation to meet the demand for tissue repair. Our recent study has demonstrated that, in response to certain injury, midguts can expand ISC population size as an additional regenerative mechanism. We found that injury elicited by bleomycin feeding or bacterial infection increased the production of two BMP ligands (Dpp and Gbb) in enterocytes (ECs), leading to elevated BMP signaling in progenitor cells that drove an expansion of ISCs by promoting their symmetric self-renewing division. Interestingly, we also found that BMP signaling in ECs inhibits the production of Dpp and Gbb, and that this negative feedback mechanism is required to reset ISC pool size to the homeostatic state. Our findings suggest that BMP signaling exerts two opposing influences on stem cell activity depending on where it acts: BMP signaling in progenitor cells promotes ISC self-renewal while BMP signaling in ECs restricts ISC self-renewal by preventing excessive production of BMP ligands. Our results further suggest that transient expansion of ISC population in conjunction with increasing ISC proliferation provides a more effective strategy for tissue regeneration.     

EGFR and Notch signaling respectively regulate proliferative activity and multiple cell lineage differentiation of Drosophila gastric stem cells

Quiescent, multipotent gastric stem cells (GSSCs) in the copper cell region of adult Drosophila midgut can produce all epithelial cell lineages found in the region, including acid-secreting copper cells, interstitial cells and enteroendocrine cells, but mechanisms controlling their quiescence and the ternary lineage differentiation are unknown. By using cell ablation or damage-induced regeneration assays combined with cell lineage tracing and genetic analysis, here we demonstrate that Delta (Dl)-expressing cells in the copper cell region are the authentic GSSCs that can self-renew and continuously regenerate the gastric epithelium after a sustained damage. Lineage tracing analysis reveals that the committed GSSC daughter with activated Notch will invariably differentiate into either a copper cell or an interstitial cell, but not the enteroendocrine cell lineage, and loss-of-function and gain-of-function studies revealed that Notch signaling is both necessary and sufficient for copper cell/interstitial cell differentiation. We also demonstrate that elevated epidermal growth factor receptor (EGFR) signaling, which is achieved by the activation of ligand Vein from the surrounding muscle cells and ligand Spitz from progenitor cells, mediates the regenerative proliferation of GSSCs following damage. Taken together, we demonstrate that Dl is a specific marker for Drosophila GSSCs, whose cell cycle status is dependent on the levels of EGFR signaling activity, and the Notch signaling has a central role in controlling cell lineage differentiation from GSSCs by separating copper/interstitial cell lineage from enteroendocrine cell lineage.     

Polyinosinic-polycytidylic acid accelerates intestinal stem cell proliferation via modulating Myc expression

It is well known that exposure of double-stranded RNA (dsRNA) to intestine immediately induces villus damage with severe diarrhea, which is mediated by toll-like receptor 3 signaling activation. However, the role of intestinal stem cells (ISCs) remains obscure during the pathology. In the present study, polyinosinic-polycytidylic acid (poly[I:C]), mimicking viral dsRNA, was used to establish intestinal damage model. Mice were acutely and chronically exposed to poly(I:C), and ISCs in jejunum were analyzed. The results showed that the height of villus was shorter 48 hr after acute poly(I:C) exposure compared with that of controls, while chronic poly(I:C) treatment increased both villus height and crypt depth in jejunum compared with control animals. The numbers of ISCs in jejunum were significantly increased after acute and chronic poly(I:C) exposure. Poly (I:C)-stimulated ISCs have stronger capacities to differentiate into intestine endocrine cells. Mechanistically, poly(I:C) treatment increased expression of Stat1 and Axin2 in the intestinal crypt, which was along with increased expression of Myc, Bcl2, and ISC proliferation. These findings suggest that dsRNA exposure could induce ISC proliferation to ameliorate dsRNA-induced intestinal injury.     

Major signaling pathways in intestinal stem cells

Background

The discovery of markers to identify the intestinal stem cell population and the generation of powerful transgenic mouse models to study stem cell physiology have led to seminal discoveries in stem cell biology.

Scope of review

In this review we give an overview of the current knowledge in the field of intestinal stem cells (ISCs) highlighting the most recent progress on markers defining the ISC population and pathways governing intestinal stem cell maintenance and differentiation. Furthermore we review their interaction with other stem cell related pathways. Finally we give an overview of alteration of these pathways in human inflammatory gastrointestinal diseases.

Major conclusions

We highlight the complex network of interactions occurring among different pathways and put in perspective the many layers of regulation that occur in maintaining the intestinal homeostasis.

General significance

Understanding the involvement of ISCs in inflammatory diseases can potentially lead to new therapeutic approaches to treat inflammatory GI pathologies such as IBD and celiac disease and could reveal the molecular mechanisms leading to the pathogenesis of dysplasia and cancer in inflammatory chronic conditions. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Lymphatics act as a signaling hub to regulate intestinal stem cell activity

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Intestinal stem cell proliferation and epithelial homeostasis in the adult Drosophila midgut

Adult tissue homeostasis requires a tight balance between the removal of old or damaged cells and the production of new ones. Such processes are usually driven by dedicated stem cells that reside within specific tissue locations or niches.The intestinal epithelium has a remarkable regenerative capacity, which has made it a prime paradigm for the study of stem cell-driven tissue self-renewal. The discovery of the presence of stem cells in the adult midgut of the fruit fly Drosophila melanogaster has significantly impacted our understanding of the role of stem cells in intestinal homeostasis. Here we will review the current knowledge of the main mechanisms involved in the regulation of tissue homeostasis in the adult Drosophila midgut, with a focus on the role of stem cells in this process. We will also discuss processes involving acute or chronic disruption of normal intestinal homeostasis such as damage-induced regeneration and ageing.     

Interplay of cell proliferation and cell death in Drosophila tissue regeneration

Regeneration is a fascinating process that allows some organisms to reconstruct damaged tissues. In addition to the classical regeneration model of the Drosophila larval imaginal discs, the genetically induced tissue ablation model has promoted the understanding of molecular mechanisms underlying cell death, proliferation, and remodeling for tissue regeneration. Recent studies have also revealed that tissue injury responses occur not only locally but also systemically, even in the uninjured region. Genetic studies in Drosophila have demonstrated the dynamic role of the cell death‐induced tissue response in the reconstruction of damaged tissues.     

Mesenchymal to epithelial transition during tissue homeostasis and regeneration: Patching up the Drosophila midgut epithelium

Stem cells are responsible for preserving morphology and function of adult tissues. Stem cells divide to self-renew and to generate progenitor cells to sustain cell demand from the tissue throughout the organism''s life. Unlike stem cells, the progenitor cells have limited proliferation potential but have the capacity to terminally differentiate and thereby to substitute older or damaged mature cells. Recent findings indicate that adult stem cells can adapt their division kinetics dynamically to match changes in tissue demand during homeostasis and regeneration. However, cell turnover not only requires stem cell division but also needs timed differentiation of the progenitor cells, which has been much less explored. In this Extra View article, we discuss the ability of progenitor cells to actively postpone terminal differentiation in the absence of a local demand and how tissue demand activates terminal differentiation via a conserved mesenchymal-epithelial transition program revealed in our recent EMBO J paper and other published and unpublished data. The extent of the significance of these results is discussed for models of tissue dynamics during both homeostasis and regeneration.     

Mesenchymal to epithelial transition during tissue homeostasis and regeneration: Patching up the Drosophila midgut epithelium

Stem cells are responsible for preserving morphology and function of adult tissues. Stem cells divide to self-renew and to generate progenitor cells to sustain cell demand from the tissue throughout the organism's life. Unlike stem cells, the progenitor cells have limited proliferation potential but have the capacity to terminally differentiate and thereby to substitute older or damaged mature cells. Recent findings indicate that adult stem cells can adapt their division kinetics dynamically to match changes in tissue demand during homeostasis and regeneration. However, cell turnover not only requires stem cell division but also needs timed differentiation of the progenitor cells, which has been much less explored. In this Extra View article, we discuss the ability of progenitor cells to actively postpone terminal differentiation in the absence of a local demand and how tissue demand activates terminal differentiation via a conserved mesenchymal-epithelial transition program revealed in our recent EMBO J paper and other published and unpublished data. The extent of the significance of these results is discussed for models of tissue dynamics during both homeostasis and regeneration.     

Multiple signaling pathways regulate yeast cell death during the response to mating pheromones

Mating pheromones promote cellular differentiation and fusion of yeast cells with those of the opposite mating type. In the absence of a suitable partner, high concentrations of mating pheromones induced rapid cell death in approximately 25% of the population of clonal cultures independent of cell age. Rapid cell death required Fig1, a transmembrane protein homologous to PMP-22/EMP/MP20/Claudin proteins, but did not require its Ca2+ influx activity. Rapid cell death also required cell wall degradation, which was inhibited in some surviving cells by the activation of a negative feedback loop involving the MAP kinase Slt2/Mpk1. Mutants lacking Slt2/Mpk1 or its upstream regulators also underwent a second slower wave of cell death that was independent of Fig1 and dependent on much lower concentrations of pheromones. A third wave of cell death that was independent of Fig1 and Slt2/Mpk1 was observed in mutants and conditions that eliminate calcineurin signaling. All three waves of cell death appeared independent of the caspase-like protein Mca1 and lacked certain "hallmarks" of apoptosis. Though all three waves of cell death were preceded by accumulation of reactive oxygen species, mitochondrial respiration was only required for the slowest wave in calcineurin-deficient cells. These findings suggest that yeast cells can die by necrosis-like mechanisms during the response to mating pheromones if essential response pathways are lacking or if mating is attempted in the absence of a partner.     

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. Here we show 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, our 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.     

Expanded and fat regulate growth and differentiation in the Drosophila eye through multiple signaling pathways

Mutations in the expanded gene act as hyperplastic tumor suppressors, interfere with cell competition and elevate Dpp signaling. Unlike Dpp overexpression, ex causes few patterning defects. Our data suggest that patterning effects are partly masked by antagonistic roles of other signaling pathways that are also activated. ex causes proliferation of cells in the posterior eye disc that are normally postmitotic. ex mutations elevate Wg signaling, but Dpp signaling antagonizes patterning effects of Wg. By contrast, if Dpp signaling is blocked in ex mutant cells, the elevated Wg signaling preserves an immature developmental state and prevents retinal differentiation. An effect of ex mutations on vesicle transport is suggested by evidence for altered sterol distribution. Mutations in ft show effects on proliferation, Wg signaling and sterols very similar to those of ex mutations. During disc growth, ex was largely epistatic to ft, and the Warts pathway mutation hippo largely epistatic to ex. Our data suggest that ft and ex act partially through the Warts pathway.     

mTORC1 signaling activation increases intestinal stem cell activity and promotes epithelial cell proliferation

The crypt-villus axis of the intestine undergoes a continuous renewal process that is driven by intestinal stem cells (ISCs). However, the homeostasis is disturbed under constant exposure to high ambient temperatures, and the precise mechanism is unclear. We found that both EdU⁺ and Ki67⁺ cell ratios were significantly reduced after exposure to 41°C, as well as the protein synthesis rate of IPEC-J2 cells, and the expression of ubiquitin and heat shock protein 60, 70, and 90 were significantly increased. Additionally, heat exposure decreased enteroid expansion and budding efficiency, as well as induced apoptosis after 48 hr; however, no significant difference was observed in the apoptosis ratio after 24 hr. In the process of heat exposure, the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway was significantly inhibited in both IPEC-J2 cells and enteroids. Correspondingly, treatment of IPEC-J2 and enteroids with the mTORC1 agonist MHY1485 at 41°C significantly attenuated the inhibition of proliferation and protein synthesis, increased the ISC activity, and promoted expansion and budding of enteroid. In summary, we conclude that the mTORC1 signaling pathway regulates intestinal epithelial cell and stem cell activity during heat exposure-induced injury.