EGFR/Ras/MAPK signaling mediates adult midgut epithelial homeostasis and regeneration in Drosophila

Many tissues in higher animals undergo dynamic homeostatic growth, wherein damaged or aged cells are replaced by the progeny of resident stem cells. To maintain homeostasis, stem cells must respond to tissue needs. Here we show that in response to damage or stress in the intestinal (midgut) epithelium of adult Drosophila, multiple EGFR ligands and rhomboids (intramembrane proteases that activate some EGFR ligands) are induced, leading to the activation of EGFR signaling in intestinal stem cells (ISCs). Activation of EGFR signaling promotes ISC division and midgut epithelium regeneration, thereby maintaining tissue homeostasis. ISCs defective in EGFR signaling cannot grow or divide, are poorly maintained, and cannot support midgut epithelium regeneration after enteric infection by the bacterium Pseudomonas entomophila. Furthermore, ISC proliferation induced by Jak/Stat signaling is dependent upon EGFR signaling. Thus the EGFR/Ras/MAPK signaling pathway plays central, essential roles in ISC maintenance and the feedback system that mediates intestinal homeostasis.     

Heparan sulfate maintains adult midgut homeostasis in Drosophila

Tissue homeostasis is controlled by the differentiated progeny of residential progenitors (stem cells). Adult stem cells constantly adjust their proliferation/differentiation rates to respond to tissue damage and stresses. However, how differentiated cells maintain tissue homeostasis remains unclear. Here, we find that heparan sulfate (HS), a class of glycosaminoglycan (GAG) chains, protects differentiated cells from loss to maintain intestinal homeostasis. HS depletion in enterocytes (ECs) leads to intestinal homeostasis disruption, with accumulation of intestinal stem cell (ISC)‐like cells and mis‐differentiated progeny. HS‐deficient ECs are prone to cell death/stress and induced cytokine and epidermal growth factor (EGF) expression, which, in turn, promote ISC proliferation and differentiation. Interestingly, HS depletion in ECs results in the inactivation of decapentaplegic (Dpp) signaling. Moreover, ectopic Dpp signaling completely rescued the defects caused by HS depletion. Together, our data demonstrate that HS is required for Dpp signal activation in ECs, thereby protecting ECs from ablation to maintain midgut homeostasis. Our data shed light into the regulatory mechanisms of how differentiated cells contribute to tissue homeostasis maintenance.     

Intestinal stem cell,muscular niche and Wingless signaling

Stem cells are typically supported by local tissue microenvironment named niche. Intestinal stem cells (ISCs) in the Drosophila midgut do not seem to be typical: they are scattered along the basement membrane composed of extracellular matrix, and are not associated with any obvious cellular niches. In addition, regulatory mechanisms controlling ISC self-renewal remain unknown. Recently, we have obtained evidence to show that Wingless signaling is critical for ISC self-renewal. Wingless is specifically produced from the underlying circular muscles and is able to transverse through the basement membrane and reach ISCs, where it activates a canonical Wnt signaling pathway to promote ISC self-renewal. Our study reveals a muscular niche for ISCs and Wnt signaling as a conserved mechanism regulating ISC self-renewal from Drosophila to mammals. Here we provide a brief overview of our findings, and discuss future perspectives on the regulatory mechanisms underlying ISC self-renewal and differentiation.     

The UPD3 cytokine couples environmental challenge and intestinal stem cell division through modulation of JAK/STAT signaling in the stem cell microenvironment

In Drosophila, the replacement of spent enterocytes (ECs) relies on division of intestinal stem cells (ISCs) and differentiation of their progeny, the enteroblasts (EBs). Recent studies have revealed a role for JAK/STAT signaling in the modulation of the rate of ISC division in response to environmental challenge. Here, we demonstrate the critical role of the UPD3 cytokine in the JAK/STAT-dependent response to enteric infection. We show that upd3 expression is activated in ECs and in EBs that massively differentiate in response to challenge. We show that the UPD3 cytokine, which is secreted basally and accumulates at the basement membrane, is required for stimulation of JAK/STAT signaling in EBs and visceral muscles (VMs). We further show that stimulation of ISC division requires active JAK/STAT signaling in EBs and VMs, but apparently not in ISCs. Our results suggest that EBs and VMs modulate the rate of the EGFR-dependent ISC division through upd3-dependent production of the EGF ligands Spitz and Vein, respectively. This study therefore supports the notion that the production of the UPD3 cytokine in stem cell progeny (ECs and EBs) stimulates intestinal stem cell division through modulation of JAK/STAT signaling in the stem cell microenvironment (EBs and VMs).     

Injury-induced BMP signaling negatively regulates Drosophila midgut homeostasis

Although much is known about injury-induced signals that increase rates of Drosophila melanogaster midgut intestinal stem cell (ISC) proliferation, it is largely unknown how ISC activity returns to quiescence after injury. In this paper, we show that the bone morphogenetic protein (BMP) signaling pathway has dual functions during midgut homeostasis. Constitutive BMP signaling pathway activation in the middle midgut mediated regional specification by promoting copper cell differentiation. In the anterior and posterior midgut, injury-induced BMP signaling acted autonomously in ISCs to limit proliferation and stem cell number after injury. Loss of BMP signaling pathway members in the midgut epithelium or loss of the BMP signaling ligand decapentaplegic from visceral muscle resulted in phenotypes similar to those described for juvenile polyposis syndrome, a human intestinal tumor caused by mutations in BMP signaling pathway components. Our data establish a new link between injury and hyperplasia and may provide insight into how BMP signaling mutations drive formation of human intestinal cancers.     

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.     

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

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

Intestinal stem cells in the adult Drosophila midgut

Drosophila has long been an excellent model organism for studying stem cell biology. Notably, studies of Drosophila's germline stem cells have been instrumental in developing the stem cell niche concept. The recent discovery of somatic stem cells in adult Drosophila, particularly the intestinal stem cells (ISCs) of the midgut, has established Drosophila as an exciting model to study stem cell-mediated adult tissue homeostasis and regeneration. Here, we review the major signaling pathways that regulate the self-renewal, proliferation and differentiation of Drosophila ISCs, discussing how this regulation maintains midgut homeostasis and mediates regeneration of the intestinal epithelium after injury.     

JNK activity in somatic stem cells causes loss of tissue homeostasis in the aging Drosophila gut

Metazoans employ cytoprotective and regenerative strategies to maintain tissue homeostasis. Understanding the coordination of these strategies is critical to developing accurate models for aging and associated diseases. Here we show that cytoprotective Jun N-terminal kinase (JNK) signaling influences regeneration in the Drosophila gut by directing proliferation of intestinal stem cells (ISCs). Interestingly, this function of JNK contributes to the loss of tissue homeostasis in old and stressed intestines by promoting the accumulation of misdifferentiated ISC daughter cells. Ectopic Delta/Notch signaling in these cells causes their abnormal differentiation but also limits JNK-induced proliferation. Protective JNK signaling and control of cell proliferation and differentiation by Delta/Notch signaling thus have to be carefully balanced to ensure tissue homeostasis. Our findings suggest that this balance is lost in old animals, increasing the potential for neoplastic transformation.     

BMP signaling is required for controlling somatic stem cell self-renewal in the Drosophila ovary

BMP signaling is essential for promoting self-renewal of mouse embryonic stem cells and Drosophila germline stem cells and for repressing stem cell proliferation in the mouse intestine and skin. However, it remains unknown whether BMP signaling can promote self-renewal of adult somatic stem cells. In this study, we show that BMP signaling is necessary and sufficient for promoting self-renewal and proliferation of somatic stem cells (SSCs) in the Drosophila ovary. BMP signaling is required in SSCs to directly control their maintenance and division, but is dispensable for proliferation of their differentiated progeny. Furthermore, BMP signaling is required to control SSC self-renewal, but not survival. Moreover, constitutive BMP signaling prolongs the SSC lifespan. Therefore, our study clearly demonstrates that BMP signaling directly promotes SSC self-renewal and proliferation in the Drosophila ovary. Our work further suggests that BMP signaling could promote self-renewal of adult stem cells in other systems.     

Regulation of Stem Cell Proliferation and Cell Fate Specification by Wingless/Wnt Signaling Gradients Enriched at Adult Intestinal Compartment Boundaries

Intestinal stem cell (ISC) self-renewal and proliferation are directed by Wnt/β-catenin signaling in mammals, whereas aberrant Wnt pathway activation in ISCs triggers the development of human colorectal carcinoma. Herein, we have utilized the Drosophila midgut, a powerful model for ISC regulation, to elucidate the mechanisms by which Wingless (Wg)/Wnt regulates intestinal homeostasis and development. We provide evidence that the Wg signaling pathway, activation of which peaks at each of the major compartment boundaries of the adult intestine, has essential functions. Wg pathway activation in the intestinal epithelium is required not only to specify cell fate near compartment boundaries during development, but also to control ISC proliferation within compartments during homeostasis. Further, in contrast with the previous focus on Wg pathway activation within ISCs, we demonstrate that the primary mechanism by which Wg signaling regulates ISC proliferation during homeostasis is non-autonomous. Activation of the Wg pathway in absorptive enterocytes is required to suppress JAK-STAT signaling in neighboring ISCs, and thereby their proliferation. We conclude that Wg signaling gradients have essential roles during homeostasis and development of the adult intestine, non-autonomously controlling stem cell proliferation inside compartments, and autonomously specifying cell fate near compartment boundaries.     

The intestinal stem cell signature identifies colorectal cancer stem cells and predicts disease relapse

A frequent complication in colorectal cancer (CRC) is regeneration of the tumor after therapy. Here, we report that a gene signature specific for adult intestinal stem cells (ISCs) predicts disease relapse in CRC patients. ISCs are marked by high expression of the EphB2 receptor, which becomes gradually silenced as cells differentiate. Using EphB2 and the ISC marker Lgr5, we have FACS-purified and profiled mouse ISCs, crypt proliferative progenitors, and late transient amplifying cells to define a gene program specific for normal ISCs. Furthermore, we discovered that ISC-specific genes identify a stem-like cell population positioned at the bottom of tumor structures reminiscent of crypts. EphB2 sorted ISC-like tumor cells display robust tumor-initiating capacity in immunodeficient mice as well as long-term self-renewal potential. Taken together, our data suggest that the ISC program defines a cancer stem cell niche within colorectal tumors and plays a central role in CRC relapse.     

BMP and Hh signaling affects primordial germ cell division in Drosophila

The germline is segregated from the remainder of the soma during early embryonic development in metazoan species. In Drosophila, female primordial germ cells (PGCs) continue to proliferate during larval development, and become germline stem cells at the early pupal stage. To elucidate the roles of growth factors in larval PGC division, we examined expression patterns of a bone morphogenetic protein (BMP) growth factor, Decapentaplegic (Dpp), and Hedgehog (Hh), along with factors downstream of each, in the ovary during larval development. Dpp signaling appeared in the ovarian soma from early larval development, and was prominent in the terminal filament cells at late larval stage, whereas Hh appeared in the ovarian soma and PGCs from the third instar larval stage. The number of PGCs decreased when components of these signal transduction pathways were abrogated by RNAi in the PGCs, indicating that both Dpp and Hh signals directly regulate PGC proliferation. Experiments on the up- and down-regulation of Dpp and Hh with a tissue-specific Gal4 driver indicated that Dpp and Hh act as extrinsic and autocrine growth factors. Furthermore, heat-pulse experiments with hs-Gal4 showed that Dpp is active in PGC proliferation throughout larval development, whereas Hh has effects only during late larval development. In addition to Dpp, the reduction of Glass bottom boat (Gbb), another BMP molecule, caused a decrease in the number of PGCs and initiation of larval PGCs differentiation into cystocytes, indicating that Gbb functions to promote PGC division and repress differentiation.