Stem cell dynamics in response to nutrient availability

When nutrient availability becomes limited, animals must actively adjust their metabolism to allocate limited resources and maintain tissue homeostasis. However, it is poorly understood how tissues maintained by adult stem cells respond to chronic changes in metabolism. To begin to address this question, we fed flies a diet lacking protein (protein starvation) and assayed both germline and intestinal stem cells. Our results revealed a decrease in stem cell proliferation and a reduction in stem cell number; however, a small pool of active stem cells remained. Upon refeeding, stem cell number increased dramatically, indicating that the remaining stem cells are competent to respond quickly to changes in nutritional status. Stem cell maintenance is critically dependent upon intrinsic and extrinsic factors that act to regulate stem cell behavior. Activation of the insulin/IGF signaling pathway in stem cells and adjacent support cells in the germline was sufficient to suppress stem cell loss during starvation. Therefore, our data indicate that stem cells can directly sense changes in the systemic environment to coordinate their behavior with the nutritional status of the animal, providing a paradigm for maintaining tissue homeostasis under metabolic stress.     

Regulation of adult stem cell behavior by nutrient signaling

Adult stem cells play an essential role?throughout life, maintaining tissue and organ function by providing a reservoir of cells for homeostasis and repair. Maintenance and activity of adult stem cells have been the focus of numerous studies that have revealed stem cell-intrinsic factors and signals from the local microenvironment that regulate stem cell behavior. A growing body of work has provided evidence that circulating, systemic factors also contribute to the regulation of stem cell behavior in numerous tissues. We have demonstrated that Drosophila male germline stem cells (GSCs) and intestinal stem cells (ISCs) respond to changes in nutrient availability, specifically amino acids. Furthermore, we have shown that insulin signaling plays an important role in mediating the effects of changes in nutritional conditions. Notably, insulin signaling is cell-autonomously required within male GSCs for maintenance. Here we discuss our data regarding the effects and mechanisms by which changes in systemic nutritional conditions may influence the maintenance and activity of adult stem cells via insulin signaling.     

Sensory regulation of the C. elegans germline through TGF-β-dependent signaling in the niche

The proliferation/differentiation balance of stem and progenitor cell populations must respond to the physiological needs of the organism [1, 2]. Mechanisms underlying this plasticity are not well understood. The C. elegans germline provides a tractable system to study the influence of the environment on progenitor cells (stem cells and their proliferative progeny). Germline progenitors accumulate during larval stages to form an adult pool from which gametes are produced. Notch pathway signaling from the distal tip cell (DTC) niche to the germline maintains the progenitor pool [3-5], and the larval germline cell cycle is boosted by insulin/IGF-like receptor signaling [6]. Here we show that, independent of its role in the dauer decision, TGF-β regulates the balance of proliferation versus differentiation in the C. elegans germline in response to sensory cues that report population density and food abundance. Ciliated ASI sensory neurons are required for TGF-β-mediated expansion of the larval germline progenitor pool, and the TGF-β receptor pathway acts in the germline stem cell niche. TGF-β signaling thereby couples germline development to the quality of the environment, providing a novel cellular and molecular mechanism linking sensory experience of the environment to reproduction.     

Regulation of adult stem cell behavior by nutrient signaling

Adult stem cells play an essential role throughout life, maintaining tissue and organ function by providing a reservoir of cells for homeostasis and repair. Maintenance and activity of adult stem cells have been the focus of numerous studies that have revealed stem cell-intrinsic factors and signals from the local microenvironment that regulate stem cell behavior. A growing body of work has provided evidence that circulating, systemic factors also contribute to the regulation of stem cell behavior in numerous tissues. We have demonstrated that Drosophila male germline stem cells (GSCs) and intestinal stem cells (ISCs) respond to changes in nutrient availability, specifically amino acids. Furthermore, we have shown that insulin signaling plays an important role in mediating the effects of changes in nutritional conditions. Notably, insulin signaling is cell-autonomously required within male GSCs for maintenance. Here we discuss our data regarding the effects and mechanisms by which changes in systemic nutritional conditions may influence the maintenance and activity of adult stem cells via insulin signaling.Key words: Drosophila, stem cells, nutrition, insulin, niche     

Role of the insulin/Tor signaling network in starvation-induced programmed cell death in Drosophila oogenesis

Amino-acid starvation leads to an inhibition of cellular proliferation and the induction of programmed cell death (PCD) in the Drosophila ovary. Disruption of insulin signaling has been shown to inhibit the progression of oogenesis, but it is unclear whether this phenotype mimics starvation. Here, we investigate whether the insulin-mediated phosphoinositide kinase-3 pathway regulates PCD in mid oogenesis. We reasoned that under well-fed conditions, disruption of positive components of the insulin signaling pathway within the germline would mimic starvation and produce degenerating egg chambers. Surprisingly, mutants did not mimic starvation but instead produced many abnormal egg chambers in which the somatic follicle cells disappeared and the germline persisted. These abnormal egg chambers did not show an induction of caspases and lysosomes like that observed in wild-type (WT) degenerating egg chambers. Egg chambers from insulin signaling mutants were resistant to starvation-induced PCD, indicating that a complete block in insulin-signaling prevents the proper response to starvation. However, target of rapamycin (Tor) mutants did show a phenotype that mimicked WT starvation-induced PCD, indicating an insulin independent regulation of PCD via Tor signaling. These results suggest that inhibition of the insulin signaling pathway is not sufficient to regulate starvation-induced PCD in mid oogenesis. Furthermore, starvation-induced PCD is regulated by Tor signaling converging with the canonical insulin signaling pathway.     

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.     

Autophagy in Drosophila ovaries is induced by starvation and is required for oogenesis

Autophagy, an evolutionarily conserved lysosome-mediated degradation, promotes cell survival under starvation and is controlled by insulin/target of rapamycin (TOR) signaling. In Drosophila, nutrient depletion induces autophagy in the fat body. Interestingly, nutrient availability and insulin/TOR signaling also influence the size and structure of Drosophila ovaries, however, the role of nutrient signaling and autophagy during this process remains to be elucidated. Here, we show that starvation induces autophagy in germline cells (GCs) and in follicle cells (FCs) in Drosophila ovaries. This process is mediated by the ATG machinery and involves the upregulation of Atg genes. We further demonstrate that insulin/TOR signaling controls autophagy in FCs and GCs. The analysis of chimeric females reveals that autophagy in FCs, but not in GCs, is required for egg development. Strikingly, when animals lack Atg gene function in both cell types, ovaries develop normally, suggesting that the incompatibility between autophagy-competent GCs and autophagy-deficient FCs leads to defective egg development. As egg morphogenesis depends on a tightly linked signaling between FCs and GCs, we propose a model in which autophagy is required for the communication between these two cell types. Our data establish an important function for autophagy during oogenesis and contributes to the understanding of the role of autophagy in animal development.     

The oogenic germline starvation response in C. elegans

Many animals alter their reproductive strategies in response to environmental stress. Here we have investigated how L4 hermaphrodites of Caenorhabditis elegans respond to starvation. To induce starvation, we removed food at 2 h intervals from very early- to very late-stage L4 animals. The starved L4s molted into adulthood, initiated oogenesis, and began producing embryos; however, all three processes were severely delayed, and embryo viability was reduced. Most animals died via 'bagging,' because egg-laying was inhibited, and embryos hatched in utero, consuming their parent hermaphrodites from within. Some animals, however, avoided bagging and survived long term. Long-term survival did not rely on embryonic arrest but instead upon the failure of some animals to produce viable progeny during starvation. Regardless of the bagging fate, starved animals showed two major changes in germline morphology: All oogenic germlines were dramatically reduced in size, and these germlines formed only a single oocyte at a time, separated from the remainder of the germline by a tight constriction. Both changes in germline morphology were reversible: Upon re-feeding, the shrunken germlines regenerated, and multiple oocytes formed concurrently. The capacity for germline regeneration upon re-feeding was not limited to the small subset of animals that normally survive starvation: When bagging was prevented ectopically by par-2 RNAi, virtually all germlines still regenerated. In addition, germline shrinkage strongly correlated with oogenesis, suggesting that during starvation, germline shrinkage may provide material for oocyte production. Finally, germline shrinkage and regeneration did not depend upon crowding. Our study confirms previous findings that starvation uncouples germ cell proliferation from germline stem cell maintenance. Our study also suggests that when nutrients are limited, hermaphrodites scavenge material from their germlines to reproduce. We discuss our findings in light of the recently proposed state of dormancy, termed Adult Reproductive Diapause.     

Physiological and molecular characterisation of cadmium stress in Schmidtea mediterranea

The planarian Schmidtea mediterranea is a well-studied model organism for developmental research, because of its stem cell system. This characteristic also provides a unique opportunity to study stress management and the effect of stress on stem cells. In this study, we characterised the stress signature at different levels of biological organization. The carcinogenic metal cadmium was used as a model chemical stressor. We focused on stem cell activity and its interaction with other known stress parameters. Here, we have found that S. mediterranea is able to cope with high internal levels of cadmium. At endpoints such as size and mobility, cadmium-related stress effects were detected but all of these responses were transient. Correspondingly, cadmium exposure led to an elevated mitotic activity of the neoblasts, at the same time points when the other responses disappeared. At the molecular level, we observed redox-related responses that can be linked with both repair as well as proliferation mechanisms. Together, our results suggest that these animals have a high plasticity. The induction of stem cell activity may underlie this 'restoring' effect, although a carcinogenic outcome after longer exposure times cannot be excluded.     

Loss of DNA mismatch repair imparts a selective advantage in planarian adult stem cells

Lynch syndrome (LS) leads to an increased risk of early-onset colorectal and other types of cancer and is caused by germline mutations in DNA mismatch repair (MMR) genes. Loss of MMR function results in a mutator phenotype that likely underlies its role in tumorigenesis. However, loss of MMR also results in the elimination of a DNA damage-induced checkpoint/apoptosis activation barrier that may allow damaged cells to grow unchecked. A fundamental question is whether loss of MMR provides pre-cancerous stem cells an immediate selective advantage in addition to establishing a mutator phenotype. To test this hypothesis in an in vivo system, we utilized the planarian Schmidtea mediterranea which contains a significant population of identifiable adult stem cells. We identified a planarian homolog of human MSH2, a MMR gene which is mutated in 38% of LS cases. The planarian Smed-msh2 is expressed in stem cells and some progeny. We depleted Smed-msh2 mRNA levels by RNA-interference and found a striking survival advantage in these animals treated with a cytotoxic DNA alkylating agent compared to control animals. We demonstrated that this tolerance to DNA damage is due to the survival of mitotically active, MMR-deficient stem cells. Our results suggest that loss of MMR provides an in vivo survival advantage to the stem cell population in the presence of DNA damage that may have implications for tumorigenesis.     

Nutrient control of neural stem cells

The physiological status of an organism is able to influence stem cell behaviour to ensure that stem cells meet the needs of the organism during growth, and in response to injury and environmental changes. In particular, the brain is sensitive to metabolic fluctuations. Here we discuss how nutritional status is able to regulate systemic and local insulin/IGF signalling so as to control aspects of neural stem behaviour. Recent results have begun to reveal how systemic signals are relayed to neural stem cells through local interactions with a glial niche. Although much still remains to be discovered, emerging parallels between the regulation of Drosophila and mammalian stem cells suggest a conserved mechanism for how the brain responds to changes in nutritional state.     

smedinx-11 is a planarian stem cell gap junction gene required for regeneration and homeostasis

The largely unknown mechanisms that regulate adult stem cells probably involve signals from neighboring differentiated cells. Gap junction channels providing direct cell-cell communication via small molecules are a crucial component of morphogenesis and normal physiology. However, no specific gap junction protein has yet been functionally linked to adult/somatic stem cell behavior in vivo or to organ regeneration. We report the identification and characterization of smedinx-11--an innexin gap junction channel gene expressed in the adult stem cells (neoblasts) of the planarian Schmidtea mediterranea. smedinx-11 RNAi treatment inhibits regeneration and abrogates neoblast maintenance. Moreover, smedinx-11 expression is enriched in an irradiation-sensitive subpopulation (;X2') and is required for proper expression of other stem cell-specific markers. Analyses of the smedinx-11 downregulation phenotype revealed a striking anterior-posterior neoblast gradient. Our data demonstrate a novel role for gap junction proteins and suggest gap junction-mediated signaling as a new and tractable control point for adult, somatic stem cell regulation.     

Ectopic activation of Dpp signalling in the male Drosophila germline inhibits germ cell differentiation

The mechanisms that control differentiation of stem cells to specialised cell types probably include factors intrinsic to stem cells as well as extrinsic factors produced by the microenvironment of the stem cell niche. The Drosophila male germline is renewed from a population of stem cells located in the apical tip of the adult testis. The morphological relationship between germline stem cells and their surrounding somatic cells is well understood but the factors that regulate stem cell proliferation and differentiation are still being uncovered. This study examined the effect of stimulating Dpp signalling directly in male germ cells. Ectopic Dpp or Activin signalling resulted in overproliferation of both stem cell-like and spermatogonial-like cells in the apical region of the testis. A third cell population that expressed stem cell markers was seen to proliferate in the distal testis when Dpp signalling was either stimulated or repressed in germline stem cells.     

Drosophila's insulin/PI3-kinase pathway coordinates cellular metabolism with nutritional conditions

Studies in Drosophila have characterized insulin receptor/phosphoinositide 3-kinase (Inr/PI3K) signaling as a potent regulator of cell growth, but its function during development has remained uncertain. Here we show that inhibiting Inr/PI3K signaling phenocopies the cellular and organismal effects of starvation, whereas activating this pathway bypasses the nutritional requirement for cell growth, causing starvation sensitivity at the organismal level. Consistent with these findings, studies using a pleckstrin homology domain-green fluorescent protein (PH-GFP) fusion as an indicator for PI3K activity show that PI3K is regulated by the availability of dietary protein in vivo. Hence we surmise that an essential function of insulin/PI3K signaling in Drosophila is to coordinate cellular metabolism with nutritional conditions.     

The voyage of stem cell toward terminal differentiation: a brief overview

Presently, worldwide attempts are being made to apply stem cells and stem cell-derived products to a wide range of clinical applications and for the development of cell-based therapies. In order to harness stem cells and manipulate them for therapeutic application, it is very important to understand the basic biology of stem cells and identify the factors that govern the dynamics of these cells in the body. Several signaling pathways have emerged as key regulators of stem cells. Some of these signaling pathways regulate the stem cell's proliferative capacity and therefore act as direct regulators of the stem cell, whereas others are involved in shaping and maintaining the stem cell niche and therefore act as indirect regulators of the stem cell. It is difficult to identify which signaling pathways critically affect the stem cell's behavior and which are important for maintaining the quiescent population. A stem cell receives different extrinsic signals compared with the bulk population and responds to them differently. In order to manipulate these adult cells for therapeutic approaches it is crucial to identify how signaling pathways regulate stem cells either directly by regulating proliferative status or indirectly by influencing the niche. The main challenge is to identify whether different factors provide diverse extrinsic signals to the stem cell and its daughter cell population, or whether there are intrinsic differences in stem cell and daughter cell populations that is reflected in their behavior. In this study, we will focus on the various aspects of stem cell biology and differentiation, as well as exploring the potential strategies to intervene the differentiation process in order to obtain the desired yield of cells applicable in regenerative medicine.     

Stem cells are differentially regulated during development, regeneration and homeostasis in flatworms

The flatworm stem cell system is exceptional within the animal kingdom, as totipotent stem cells (neoblasts) are the only dividing cells within the organism. In contrast to most organisms, piwi-like gene expression in flatworms is extended from germ cells to somatic stem cells. We describe the isolation and characterization of the piwi homologue macpiwi in the flatworm Macrostomum lignano. We use in situ hybridization, antibody staining and RNA interference to study macpiwi expression and function in adults, during postembryonic development, regeneration and upon starvation. We found novelties regarding piwi function and observed differences to current piwi functions in flatworms. First, macpiwi was essential for the maintenance of somatic stem cells in adult animals. A knock-down of macpiwi led to a complete elimination of stem cells and death of the animals. Second, the regulation of stem cells was different in adults and regenerates compared to postembryonic development. Third, sexual reproduction of M. lignano allowed to follow germline formation during postembryonic development, regeneration, and starvation. Fourth, piwi expression in hatchlings further supports an embryonic formation of the germline in M. lignano. Our findings address new questions in flatworm stem cell research and provide a basis for comparison with higher organisms.     

Egfr signaling controls the size of the stem cell precursor pool in the Drosophila ovary

In many animals, germline progenitors are kept undifferentiated to give rise to germline stem cells (GSCs), enabling continuous production of gametes throughout animal life. In the Drosophila ovary, GSCs arise from a subset of primordial germ cells (PGCs) that stay undifferentiated even after gametogenesis has started. How a certain population of PGCs is protected against differentiation, and the significance of its regulatory mechanisms on GSC establishment remain elusive. Here we show that epidermal growth factor receptor (Egfr) signaling in somatic stromal intermingled cells (ICs), activated by its ligand produced in germ cells, controls the size of the PGC pool at the onset of gametogenesis. Egfr signaling in ICs limits the number of cells that express the heparan sulfate proteoglycan Dally, which is required for the movement and stability of the locally-produced stromal morphogen, Decapentaplegic (Dpp, a BMP2/4 homologue). Dpp is received by PGCs and maintains them in an undifferentiated state. Altering Egfr signaling levels changes the size of the PGC pool and affects the number of GSCs established during development. While excess GSC formation is compensated by the adult stage, insufficient GSC formation can lead to adult ovarioles that completely lack GSCs, suggesting that ensuring an absolute size of the PGC pool is crucial for the GSC system.