Increased centrosome amplification in aged stem cells of the Drosophila midgut

Age-related changes in long-lived tissue-resident stem cells may be tightly linked to aging and age-related diseases such as cancer. Centrosomes play key roles in cell proliferation, differentiation and migration. Supernumerary centrosomes are known to be an early event in tumorigenesis and senescence. However, the age-related changes of centrosome duplication in tissue-resident stem cells in vivo remain unknown. Here, using anti-γ-tubulin and anti-PH3, we analyzed mitotic intestinal stem cells with supernumerary centrosomes in the adult Drosophila midgut, which may be a versatile model system for stem cell biology. The results showed increased centrosome amplification in intestinal stem cells of aged and oxidatively stressed Drosophila midguts. Increased centrosome amplification was detected by overexpression of PVR, EGFR, and AKT in intestinal stem cells/enteroblasts, known to mimic age-related changes including hyperproliferation of intestinal stem cells and hyperplasia in the midgut. Our data show the first direct evidence for the age-related increase of centrosome amplification in intestinal stem cells and suggest that the Drosophila midgut is an excellent model for studying molecular mechanisms underlying centrosome amplification in aging adult stem cells in vivo.     

Pathogenic stimulation of intestinal stem cell response in drosophila

Stem cell‐mediated tissue repair is a promising approach for many diseases. Mammalian intestine is an actively regenerating tissue such that epithelial cells are constantly shedding and underlying precursor cells are constantly replenishing the loss of cells. An imbalance of these processes will lead to intestinal diseases including inflammation and cancer. Mammalian intestinal stem cells (ISCs) are located in bases of crypts but at least two groups of cells have been cited as stem cells. Moreover, precursor cells in the transit amplifying zone can also proliferate. The involvement of multiple cell types makes it more difficult to examine tissue damage response in mammalian intestine. In adult Drosophila midgut, the ISCs are the only cells that can go through mitosis. By feeding pathogenic bacteria and stress inducing chemicals to adult flies, we demonstrate that Drosophila ISCs in the midgut can respond by increasing their division. The resulting enteroblasts, precursor cells for enterocytes and enteroendocrine cells, also differentiate faster to become cells resembling enterocyte lineage. These results are consistent with the idea that Drosophila midgut stem cells can respond to tissue damage induced by pathogens and initiate tissue repair. This system should allow molecular and genetic analyses of stem cell‐mediated tissue repair. J. Cell. Physiol. 220: 664–671, 2009. © 2009 Wiley‐Liss, Inc.     

Another notch in stem cell biology: Drosophila intestinal stem cells and the specification of cell fates

Previous work has suggested that many stem cells can be found in microanatomic niches, where adjacent somatic cells of the niche control the differentiation and proliferation states of their resident stem cells. Recently published work examining intestinal stem cells (ISCs) in the adult Drosophila midgut suggests a new paradigm where some stem cells actively control the cell fate decisions of their daughters. Here, we review recent literature((1)) demonstrating that, in the absence of a detectable stem cell niche, multipotent Drosophila ISCs modulate the Notch signaling pathway in their adjacent daughter cells in order to specify the differentiated lineages of their descendants. These observations made in Drosophila are challenging and advancing our understanding of stem cell biology.     

Tuberous sclerosis complex and Myc coordinate the growth and division of Drosophila intestinal stem cells

Intestinal stem cells (ISCs) in the adult Drosophila melanogaster midgut can respond to damage and support repair. We demonstrate in this paper that the tuberous sclerosis complex (TSC) plays a critical role in balancing ISC growth and division. Previous studies have shown that imaginal disc cells that are mutant for TSC have increased rates of growth and division. However, we report in this paper that loss of TSC in the adult Drosophila midgut results in the formation of much larger ISCs that have halted cell division. These mutant ISCs expressed proper stem cell markers, did not differentiate, and had defects in multiple steps of the cell cycle. Slowing the growth by feeding rapamycin or reducing Myc was sufficient to rescue the division defect. The TSC mutant guts had a thinner epithelial structure than wild-type tissues, and the mutant flies were more susceptible to tissue damage. Therefore, we have uncovered a context-dependent phenotype of TSC mutants in adult ISCs, such that the excessive growth leads to inhibition of division.     

Redox regulation by Keap1 and Nrf2 controls intestinal stem cell proliferation in Drosophila

In Drosophila, intestinal stem cells (ISCs) respond to oxidative challenges and inflammation by increasing proliferation rates. This phenotype is part of a regenerative response, but can lead to hyperproliferation and epithelial degeneration in the aging animal. Here we show that Nrf2, a master regulator of the cellular redox state, specifically controls the proliferative activity of ISCs, promoting intestinal homeostasis. We find that Nrf2 is constitutively active in ISCs and that repression of Nrf2 by its negative regulator Keap1 is required for ISC proliferation. We further show that Nrf2 and Keap1 exert this function in ISCs by regulating the intracellular redox balance. Accordingly, loss of Nrf2 in ISCs causes accumulation of reactive oxygen species and accelerates age-related degeneration of the intestinal epithelium. Our findings establish Keap1 and Nrf2 as a critical redox management system that regulates stem cell function in high-turnover tissues.     

Stem cell function and maintenance – ends that matter: Role of telomeres and telomerase

Stem cell research holds a promise to treat and prevent age-related degenerative changes in humans. Literature is replete with studies showing that stem cell function declines with aging, especially in highly proliferative tissues/organs. Among others, telomerase and telomere damage is one of the intrinsic physical instigators that drive age-related degenerative changes. In this review we provide brief overview of telomerase-deficient aging affects in diverse stem cells populations. Furthermore, potential disease phenotypes associated with telomerase dysregulation in a specific stem cell population is also discussed in this review. Additionally, the role of telomerase in stem cell driven cancer is also briefly touched upon.     

Mathematical modelling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells II

The Drosophila posterior midgut epithelium mainly consists of intestinal stem cells (ISCs); semi-differentiated cells, i.e. enteroblasts (EBs); and two types of fully differentiated cells, i.e. enteroendocrine cells (EEs) and enterocytes (ECs), which are controlled by signalling pathways. In [M. Kuwamura, K. Maeda, and T. Adachi-Yamada, Mathematical modeling and experiments for the proliferation and differentiation of Drosophila intestinal stem cells I, J. Biol. Dyn. 4 (2009), pp. 248-257], on the basis of the functions of the Wnt and Notch signalling pathways, we studied the regulatory mechanism for the proliferation and differentiation of ISCs under the assumption that the Wnt proteins are supplied from outside the cellular system of ISCs. In this paper, we experimentally show that the Wnt proteins are specifically expressed in ISCs, EBs, and EEs, and theoretically show that the cellular system of ISCs can be self-maintained under the assumption that the Wnt proteins are produced in the cellular system of ISCs. These results provide a useful basis for determining whether an environmental niche is required for maintaining the cellular system of tissue stem cells.     

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.     

Autocrine Platelet-derived Growth Factor-Vascular Endothelial Growth Factor Receptor-related (Pvr) Pathway Activity Controls Intestinal Stem Cell Proliferation in the Adult Drosophila Midgut

A dynamic pool of undifferentiated somatic stem cells proliferate and differentiate to replace dead or dying mature cell types and maintain the integrity and function of adult tissues. Intestinal stem cells (ISCs) in the Drosophila posterior midgut are a well established model to study the complex genetic circuitry that governs stem cell homeostasis. Exposure of the intestinal epithelium to environmental toxins results in the expression of cytokines and growth factors that drive the rapid proliferation and differentiation of ISCs. In the absence of stress signals, ISC homeostasis is maintained through intrinsic pathways. In this study, we uncovered the PDGF- and VEGF-receptor related (Pvr) pathway as an essential regulator of ISC homeostasis under unstressed conditions in the posterior midgut. We found that Pvr is coexpressed with its ligand Pvf2 in ISCs and that hyperactivation of the Pvr pathway distorts the ISC developmental program and drives intestinal dysplasia. In contrast, we show that mutant ISCs in the Pvf/Pvr pathway are defective in homeostatic proliferation and differentiation, resulting in a failure to generate mature cell types. Additionally, we determined that extrinsic stress signals generated by enteropathogenic infection are epistatic to the hypoplasia generated in Pvf/Pvr mutants, making the Pvr pathway unique among all previously studied intrinsic pathways. Our findings illuminate an evolutionarily conserved signal transduction pathway with essential roles in metazoan embryonic development and direct involvement in numerous disease states.     

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/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.     

Regulation of intestinal stem cell proliferation by human methyl-CpG-binding protein-2 in Drosophila

Recent studies have suggested the involvement of epigenetic factors such as methyl-CpG-binding protein-2 (MeCP2) in tumorigenesis. In addition, cancer may represent a stem cell-based disease, suggesting that understanding of stem cell regulation could provide valuable insights into the mechanisms of tumorigenesis. However, the function of epigenetic factors in stem cell regulation in adult tissues remains poorly understood. In the present study, we investigated the role of human MeCP2 (hMeCP2), a bridge factor linked to DNA modification and histone modification, in stem cell proliferation using adult Drosophila midgut, which appears to be an excellent model system to study stem cell biology. Results show that enterocyte (EC)-specific expression of hMeCP2 in adult midgut using an exogenous GAL4/UAS expression system induced intestinal stem cell (ISC) proliferation marked by staining with anti-phospho-histone H3 antibody and BrdU incorporation assays. In addition, hMeCP2 expression in ECs activated extracellular stress-response kinase signals in ISCs. Furthermore, expression of hMeCP2 modulated the distribution of heterochromatin protein-1 in ECs. Our data suggests the hypothesis that the expression of hMeCP2 in differentiated ECs stimulates ISC proliferation, implying a role of MeCP2 as a stem cell regulator.