Aging and insulin signaling differentially control normal and tumorous germline stem cells

Aging influences stem cells, but the processes involved remain unclear. Insulin signaling, which controls cellular nutrient sensing and organismal aging, regulates the G2 phase of Drosophila female germ line stem cell (GSC) division cycle in response to diet; furthermore, this signaling pathway is attenuated with age. The role of insulin signaling in GSCs as organisms age, however, is also unclear. Here, we report that aging results in the accumulation of tumorous GSCs, accompanied by a decline in GSC number and proliferation rate. Intriguingly, GSC loss with age is hastened by either accelerating (through eliminating expression of Myt1, a cell cycle inhibitory regulator) or delaying (through mutation of insulin receptor (dinR) GSC division, implying that disrupted cell cycle progression and insulin signaling contribute to age‐dependent GSC loss. As flies age, DNA damage accumulates in GSCs, and the S phase of the GSC cell cycle is prolonged. In addition, GSC tumors (which escape the normal stem cell regulatory microenvironment, known as the niche) still respond to aging in a similar manner to normal GSCs, suggesting that niche signals are not required for GSCs to sense or respond to aging. Finally, we show that GSCs from mated and unmated females behave similarly, indicating that female GSC–male communication does not affect GSCs with age. Our results indicate the differential effects of aging and diet mediated by insulin signaling on the stem cell division cycle, highlight the complexity of the regulation of stem cell aging, and describe a link between ovarian cancer and aging.     

Control of germline stem cell division frequency--a novel, developmentally regulated role for epidermal growth factor signaling

Exploring adult stem cell dynamics in normal and disease states is crucial to both better understanding their in vivo role and better realizing their therapeutic potential. Here we address the division frequency of Germline Stem Cells (GSCs) in testes of Drosophila melanogaster. We show that GSC division frequency is under genetic control of the highly conserved Epidermal Growth Factor (EGF) signaling pathway. When EGF signaling was attenuated, we detected a two-fold increase in the percentage of GSCs in mitotic division compared to GSCs in control animals. Ex vivo and in vivo experiments using a marker for cells in S-phase of the cell cycle showed that the GSCs in EGF mutant testes divide faster than GSCs in control testes. The increased mitotic activity of GSCs in EGF mutants was rescued by restoring EGF signaling in the GSCs, and reproduced in testes from animals with soma-depleted EGF-Receptor (EGFR). Interestingly, EGF attenuation specifically increased the GSC division frequency in adult testes, but not in larval testes. Furthermore, GSCs in testes with tumors resulting from the perturbation of other conserved signaling pathways divided at normal frequencies. We conclude that EGF signaling from the GSCs to the CySCs normally regulates GSC division frequency. The EGF signaling pathway is bifurcated and acts differently in adult compared to larval testes. In addition, regulation of GSC division frequency is a specific role for EGF signaling as it is not affected in all tumor models. These data advance our understanding concerning stem cell dynamics in normal tissues and in a tumor model.     

Heparan sulfate regulates the number and centrosome positioning of Drosophila male germline stem cells

Stem cell division is tightly controlled via secreted signaling factors and cell adhesion molecules provided from local niche structures. Molecular mechanisms by which each niche component regulates stem cell behaviors remain to be elucidated. Here we show that heparan sulfate (HS), a class of glycosaminoglycan chains, regulates the number and asymmetric division of germline stem cells (GSCs) in the Drosophila testis. We found that GSC number is sensitive to the levels of 6-O sulfate groups on HS. Loss of 6-O sulfation also disrupted normal positioning of centrosomes, a process required for asymmetric division of GSCs. Blocking HS sulfation specifically in the niche, termed the hub, led to increased GSC numbers and mispositioning of centrosomes. The same treatment also perturbed the enrichment of Apc2, a component of the centrosome-anchoring machinery, at the hub–GSC interface. This perturbation of the centrosome-anchoring process ultimately led to an increase in the rate of spindle misorientation and symmetric GSC division. This study shows that specific HS modifications provide a novel regulatory mechanism for stem cell asymmetric division. The results also suggest that HS-mediated niche signaling acts upstream of GSC division orientation control.     

Notch Signaling Mediates the Age-Associated Decrease in Adhesion of Germline Stem Cells to the Niche

Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs), and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention) is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion.     

Rab11 maintains connections between germline stem cells and niche cells in the Drosophila ovary

All stem cells have the ability to balance their production of self-renewing and differentiating daughter cells. The germline stem cells (GSCs) of the Drosophila ovary maintain such balance through physical attachment to anterior niche cap cells and stereotypic cell division, whereby only one daughter remains attached to the niche. GSCs are attached to cap cells via adherens junctions, which also appear to orient GSC division through capture of the fusome, a germline-specific organizer of mitotic spindles. Here we show that the Rab11 GTPase is required in the ovary to maintain GSC-cap cell junctions and to anchor the fusome to the anterior cortex of the GSC. Thus, rab11-null GSCs detach from niche cap cells, contain displaced fusomes and undergo abnormal cell division, leading to an early arrest of GSC differentiation. Such defects are likely to reflect a role for Rab11 in E-cadherin trafficking as E-cadherin accumulates in Rab11-positive recycling endosomes (REs) and E-cadherin and Armadillo (beta-catenin) are both found in reduced amounts on the surface of rab11-null GSCs. The Rab11-positive REs through which E-cadherin transits are tightly associated with the fusome. We propose that this association polarizes the trafficking by Rab11 of E-cadherin and other cargoes toward the anterior cortex of the GSC, thus simultaneously fortifying GSC-niche junctions, fusome localization and asymmetric cell division. These studies bring into focus the important role of membrane trafficking in stem cell biology.     

Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands

Adult stem cells reside in specialized microenvironments, or niches, that are essential for their function in vivo. Stem cells are physically attached to the niche, which provides secreted factors that promote their self-renewal and proliferation. Despite intense research on the role of the niche in regulating stem cell function, much less is known about how the niche itself is controlled. We previously showed that insulin signals directly stimulate germline stem cell (GSC) division and indirectly promote GSC maintenance via the niche in Drosophila. Insulin-like peptides are required for maintenance of cap cells (a major component of the niche) via modulation of Notch signaling, and they also control attachment of GSCs to cap cells and E-cadherin levels at the cap cell–GSC junction. Here, we further dissect the molecular and cellular mechanisms underlying these processes. We show that insulin and Notch ligands directly stimulate cap cells to maintain their numbers and indirectly promote GSC maintenance. We also report that insulin signaling, via phosphoinositide 3-kinase and FOXO, intrinsically controls the competence of cap cells to respond to Notch ligands and thereby be maintained. Contrary to a previous report, we also find that Notch ligands originated in GSCs are not required either for Notch activation in the GSC niche, or for cap cell or GSC maintenance. Instead, the niche itself produces ligands that activate Notch signaling within cap cells, promoting stability of the GSC niche. Finally, insulin signals control cap cell–GSC attachment independently of their role in Notch signaling. These results are potentially relevant to many systems in which Notch signaling modulates stem cells and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function.     

Age-related changes of germline stem cell activity, niche signaling activity and egg production in Drosophila

Adult stem cells are important in replenishing aged cells to maintain tissue homeostasis. Aging in turn may exert profound effects on stem cell's regenerative potential, but to date the mechanisms of such stem cell aging are poorly understood, and it is not clear to what extent stem cell aging contributes to tissue or organ aging. Here we show in female Drosophila that germline stem cell (GSC) division rate progressively declines with age, which is accompanied by reduced decapentaplegic (dpp) niche signaling pathway activation within GSCs. Egg production also rapidly declines with age, which is accompanied by both decreased stem cell division and increased incidence of cell death of developing eggs, especially in the oldest females. Genetically increasing dpp expression delays GSC activity decline and transiently increases egg production. We conclude that age-related decline of reproduction is caused by both decreased GSC activity and increased incidence of cell death during oogenesis, while decreased GSC activity is attributed to declined signaling from the regulatory niche. We suggest that niche functional decay may be an important mechanism for stem cell aging and system failure.     

The division of Drosophila germline stem cells and their precursors requires a specific cyclin

A fundamental yet essentially unexplored question in stem cell biology is whether the stem cell cycle has specific features. Three B-cyclins in Drosophila, Cyclins (Cyc) A, B, and B3, associate with CDK1 and play partially redundant roles in embryogenic mitosis . Here, we show that the division of Drosophila GSCs and their precursors, the primordial germ cells (PGCs), specifically requires CycB. CycB is ubiquitously expressed in both germline and somatic lineages. However, CycB mutation does not have obvious effect on somatic development but causes PGCs to severely under proliferate. Moreover, both female and male CycB mutant GSCs fail to be maintained properly. Removing Cyclin B specifically from female GSCs causes the same defect, confirming the direct and cell-autonomous function of Cyclin B for GSC division. In contrast, two other G2 cyclins, CycA and CycB3, are also expressed in PGCs and GSCs, but overexpressing CycA cannot rescue the CycB mutant defects. These results indicate that the requirement of CycB for PGC and GSC divisions unlikely reflects the insufficient level of G2 cyclins in the CycB mutant but is in favor of a distinct function of CycB in these cells. Our results indicate that stem cells may use specific cell cycle regulators for their division.     

Differentiation-defective stem cells outcompete normal stem cells for niche occupancy in the Drosophila ovary

Rapid progress has recently been made regarding how the niche controls stem cell function, but little is yet known about how stem cells in the same niche interact with one another. In this study, we show that differentiation-defective Drosophila ovarian germline stem cells (GSCs) can outcompete normal ones for niche occupancy in a cadherin-dependent manner. The differentiation-defective bam or bgcn mutant GSCs invade the niche space of neighboring wild-type GSCs and gradually push them out of the niche by upregulating E-cadherin expression. Furthermore, the bam/bgcn-mediated GSC competition requires E-cadherin and normal GSC division, but not the self-renewal-promoting BMP niche signal, while different E-cadherin levels can sufficiently stimulate GSC competition. Therefore, we propose that GSCs have a competitive relationship for niche occupancy, which may serve as a quality control mechanism to ensure that accidentally differentiated stem cells are rapidly removed from the niche and replaced by functional ones.     

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.     

Jak-STAT regulation of male germline stem cell establishment during Drosophila embryogenesis

Germline stem cells (GSCs) in Drosophila are descendants of primordial germ cells (PGCs) specified during embryogenesis. The precise timing of GSC establishment in the testis has not been determined, nor is it known whether mechanisms that control GSC maintenance in the adult are involved in GSC establishment. Here, we determine that PGCs in the developing male gonad first become GSCs at the embryo to larval transition. This coincides with formation of the embryonic hub; the critical signaling center that regulates adult GSC behavior within the stem cell microenvironment (niche). We find that the Jak-STAT signaling pathway is activated in a subset of PGCs that associate with the newly-formed embryonic hub. These PGCs express GSC markers and function like GSCs, while PGCs that do not associate with the hub begin to differentiate. In the absence of Jak-STAT activation, PGCs adjacent to the hub fail to exhibit the characteristics of GSCs, while ectopic activation of the Jak-STAT pathway prevents differentiation. These findings show that stem cell formation is closely linked to development of the stem cell niche, and suggest that Jak-STAT signaling is required for initial establishment of the GSC population in developing testes.     

Diet controls normal and tumorous germline stem cells via insulin-dependent and -independent mechanisms in Drosophila

The external environment influences stem cells, but this process is poorly understood. Our previous work showed that germline stem cells (GSCs) respond to diet via neural insulin-like peptides (DILPs) that act directly on the germ line to upregulate stem cell division and cyst growth under a protein-rich diet in Drosophila. Here, we report that DILPs specifically control the G2 phase of the GSC cell cycle via phosphoinositide-3 kinase (PI3K) and dFOXO, and that a separate diet mediator regulates the G1 phase. Furthermore, GSC tumors, which escape the normal stem cell regulatory microenvironment, or niche, still respond to diet via both mechanisms, indicating that niche signals are not required for GSCs to sense or respond to diet. Our results document the effects of diet and insulin-like signals on the cell cycle of stem cells within an intact organism and demonstrate that the response to diet requires multiple signals. Moreover, the retained ability of GSC tumors to respond to diet parallels the long known connections between diet, insulin signaling, and cancer risk in humans.     

Centrosome misorientation mediates slowing of the cell cycle under limited nutrient conditions in Drosophila male germline stem cells

Drosophila male germline stem cells (GSCs) divide asymmetrically, balancing self-renewal and differentiation. Although asymmetric stem cell division balances between self-renewal and differentiation, it does not dictate how frequently differentiating cells must be produced. In male GSCs, asymmetric GSC division is achieved by stereotyped positioning of the centrosome with respect to the stem cell niche. Recently we showed that the centrosome orientation checkpoint monitors the correct centrosome orientation to ensure an asymmetric outcome of the GSC division. When GSC centrosomes are not correctly oriented with respect to the niche, GSC cell cycle is arrested/delayed until the correct centrosome orientation is reacquired. Here we show that induction of centrosome misorientation upon culture in poor nutrient conditions mediates slowing of GSC cell proliferation via activation of the centrosome orientation checkpoint. Consistently, inactivation of the centrosome orientation checkpoint leads to lack of cell cycle slowdown even under poor nutrient conditions. We propose that centrosome misorientation serves as a mediator that transduces nutrient information into stem cell proliferation, providing a previously unappreciated mechanism of stem cell regulation in response to nutrient conditions.     

Stem cell aging is controlled both intrinsically and extrinsically in the Drosophila ovary

It is widely postulated that tissue aging could be, at least partially, caused by reduction of stem cell number, activity, or both. However, the mechanisms of controlling stem cell aging remain largely a mystery. Here, we use Drosophila ovarian germline stem cells (GSCs) as a model to demonstrate that age-dependent decline in the functions of stem cells and their niche contributes to overall stem cell aging. BMP signaling activity from the niche significantly decreases with age, and increasing BMP signaling can prolong GSC life span and promote their proliferation. In addition, the age-dependent E-cadherin decline in the stem cell-niche junction also contributes to stem cell aging. Finally, overexpression of SOD, an enzyme that helps eliminate free oxygen species, in either GSCs or their niche alone can prolong GSC life span and increase GSC proliferation. Therefore, this study demonstrates that stem cell aging is controlled extrinsically and intrinsically in the Drosophila ovary.     

Aging shifts mitochondrial dynamics toward fission to promote germline stem cell loss

Changes in mitochondrial dynamics (fusion and fission) are known to occur during stem cell differentiation; however, the role of this phenomenon in tissue aging remains unclear. Here, we report that mitochondrial dynamics are shifted toward fission during aging of Drosophila ovarian germline stem cells (GSCs), and this shift contributes to aging‐related GSC loss. We found that as GSCs age, mitochondrial fragmentation and expression of the mitochondrial fission regulator, Dynamin‐related protein (Drp1), are both increased, while mitochondrial membrane potential is reduced. Moreover, preventing mitochondrial fusion in GSCs results in highly fragmented depolarized mitochondria, decreased BMP stemness signaling, impaired fatty acid metabolism, and GSC loss. Conversely, forcing mitochondrial elongation promotes GSC attachment to the niche. Importantly, maintenance of aging GSCs can be enhanced by suppressing Drp1 expression to prevent mitochondrial fission or treating with rapamycin, which is known to promote autophagy via TOR inhibition. Overall, our results show that mitochondrial dynamics are altered during physiological aging, affecting stem cell homeostasis via coordinated changes in stemness signaling, niche contact, and cellular metabolism. Such effects may also be highly relevant to other stem cell types and aging‐induced tissue degeneration.     

小分子RNA决定果蝇生殖干细胞命运的分子机制研究

生殖干细胞是具有自我更新能力的一群生殖细胞,充当配子生成的源泉。果蝇生殖干细胞的特征在于通过不对称分裂产生两个子代细胞,一个通过自我更新维持干细胞特性,另一个则进行分化。生殖干细胞的命运受其周围的微环境——"干细胞niche"控制,而"niche"的功能又通过干细胞的外源和内源信号间的相互作用来完成。小分子RNA通过复杂的RNAi途径调控基因的表达。大量证据表明生殖干细胞的维持和分化需要小分子RNA参与,小分子RNA生成的紊乱会导致干细胞的"丢失"或"未分化"。该文综述了小分子RNA对果蝇生殖干细胞命运调控的研究进展,并讨论新发现的小分子RNA在生殖干细胞命运决定中的相关功能。     

Notch signaling controls germline stem cell niche formation in the Drosophila ovary

Stem cells, which can self-renew and generate differentiated cells, have been shown to be controlled by surrounding microenvironments or niches in several adult tissues. However, it remains largely unknown what constitutes a functional niche and how niche formation is controlled. In the Drosophila ovary, germline stem cells (GSCs), which are adjacent to cap cells and two other cell types, have been shown to be maintained in the niche. In this study, we show that Notch signaling controls formation and maintenance of the GSC niche and that cap cells help determine the niche size in the Drosophila ovary. Expanded Notch activation causes the formation of more cap cells and bigger niches, which support more GSCs, whereas compromising Notch signaling during niche formation decreases the cap cell number and niche size and consequently the GSC number. Furthermore, the niches located away from their normal location can still sufficiently sustain GSC self-renewal by maintaining high local BMP signaling and repressing bam as in normal GSCs. Finally, loss of Notch function in adults results in rapid loss of the GSC niche, including cap cells and thus GSCs. Our results indicate that Notch signaling is important for formation and maintenance of the GSC niche, and that cap cells help determine niche size and function.