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.     

Clonal expansion of ovarian germline stem cells during niche formation in Drosophila

Stem cell niches are specific regulatory microenvironments formed by neighboring stromal cells. Owing to difficulties in identifying stem cells and their niches in many systems, mechanisms that control niche formation and stem cell recruitment remain elusive. In the Drosophila ovary, two or three germline stem cells (GSCs) have recently been shown to reside in a niche, in which terminal filaments (TFs) and cap cells are two major components. We report that signals from newly formed niches promote clonal expansion of GSCs during niche formation in the Drosophila ovary. After the formation of TFs and cap cells, anterior primordial germ cells (PGCs) adjacent to TFs/cap cells can develop into GSCs at the early pupal stage while the rest directly differentiate. The anterior PGCs are very mitotically active and exhibit two division patterns with respect to cap cells. One of these patterns generates two daughters that both contact cap cells and potentially become GSCs. Our lineage tracing study confirms that one PGC can generate two or three GSCs to occupy a whole niche ('clonal expansion'). decapentaplegic (dpp), the Drosophila homolog of human bone morphogenetic protein 2/4, is expressed in anterior somatic cells of the gonad, including TFs/cap cells. dpp overexpression promotes PGC proliferation and causes the accumulation of more PGCs in the gonad. A single PGC mutant for thick veins, encoding an essential dpp receptor, loses the ability to clonally populate a niche. Therefore, dpp is probably one of the mitotic signals that promote the clonal expansion of GSCs in a niche. This study also suggests that signals from newly formed niche cells are important for expanding stem cells and populating niches.     

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.     

Restricting self-renewal signals within the stem cell niche: multiple levels of control

Germline stem cells (GSCs) were the first stem cells demonstrated to be regulated by the microenvironment or niche in the Drosophila ovary a decade ago. In the Drosophila ovary, as a stem cell divides, one daughter remaining in the niche continues to self-renew, and the other daughter positioned outside the niche undergoes differentiation. The niche produces bone morphogenetic proteins (BMPs) that only act within one cell diameter to ensure that at every division only one of two GSC daughters self-renews and thus maintains a stable GSC pool. Within the past decade, great progress has been made toward understanding how functions of BMP niche signals are restricted to GSCs. In this review, we have discussed multiple levels of control underlying the restriction of BMP signals within the niche. Because the niche mechanism has been shown to regulate stem cells in various organisms including mammals, the knowledge gained from the Drosophila GSC niche should help gain a better understanding of how niche signals are controlled in other stem cell systems.     

Development of the male germline stem cell niche in Drosophila

Stem cells are found in specialized microenvironments, or "niches", which regulate stem cell identity and behavior. The adult testis and ovary in Drosophila contain germline stem cells (GSCs) with well-defined niches, and are excellent models for studying niche development. Here, we investigate the formation of the testis GSC niche, or "hub", during the late stages of embryogenesis. By morphological and molecular criteria, we identify and follow the development of an embryonic hub that forms from a subset of anterior somatic gonadal precursors (SGPs) in the male gonad. Embryonic hub cells form a discrete cluster apart from other SGPs, express several molecular markers in common with the adult hub and organize anterior-most germ cells in a rosette pattern characteristic of GSCs in the adult. The sex determination genes transformer and doublesex ensure that hub formation occurs only in males. Interestingly, hub formation occurs in both XX and XY gonads mutant for doublesex, indicating that doublesex is required to repress hub formation in females. This work establishes the Drosophila male GSC niche as a model for understanding the mechanisms controlling niche formation and initial stem cell recruitment, as well as the development of sexual dimorphism in the gonad.     

Drosophila glypicans regulate the germline stem cell niche

Stem cells are maintained in vivo by short-range signaling systems in specialized microenvironments called niches, but the molecular mechanisms controlling the physical space of the stem cell niche are poorly understood. In this study, we report that heparan sulfate (HS) proteoglycans (HSPGs) are essential regulators of the germline stem cell (GSC) niches in the Drosophila melanogaster gonads. GSCs were lost in both male and female gonads of mutants deficient for HS biosynthesis. dally, a Drosophila glypican, is expressed in the female GSC niche cells and is responsible for maintaining the GSC niche. Ectopic expression of dally in the ovary expanded the niche area, showing that dally is required for restriction of the GSC niche space. Interestingly, the other glypican, dally-like, plays a major role in regulating male GSC niche maintenance. We propose that HSPGs define the physical space of the niche by serving as trans coreceptors, mediating short-range signaling by secreted factors.     

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.     

Coordinated regulation of niche and stem cell precursors by hormonal signaling

Stem cells and their niches constitute units that act cooperatively to achieve adult body homeostasis. How such units form and whether stem cell and niche precursors might be coordinated already during organogenesis are unknown. In fruit flies, primordial germ cells (PGCs), the precursors of germ line stem cells (GSCs), and somatic niche precursors develop within the larval ovary. Together they form the 16-20 GSC units of the adult ovary. We show that ecdysone receptors are required to coordinate the development of niche and GSC precursors. At early third instar, ecdysone receptors repress precocious differentiation of both niches and PGCs. Early repression is required for correct morphogenesis of the ovary and for protecting future GSCs from differentiation. At mid-third instar, ecdysone signaling is required for niche formation. Finally, and concurrent with the initiation of wandering behavior, ecdysone signaling initiates PGC differentiation by allowing the expression of the differentiation gene bag of marbles in PGCs that are not protected by the newly formed niches. All the ovarian functions of ecdysone receptors are mediated through early repression, and late activation, of the ecdysone target gene broad. These results show that, similar to mammals, a brain-gland-gonad axis controls the initiation of oogenesis in insects. They further exemplify how a physiological cue coordinates the formation of a stem cell unit within an organ: it is required for niche establishment and to ensure that precursor cells to adult stem cells remain undifferentiated until the niches can accommodate them. Similar principles might govern the formation of additional stem cell units during organogenesis.     

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.     

Gbb/Bmp signaling is essential for maintaining germline stem cells and for repressing bam transcription in the Drosophila testis

Stem cells are responsible for replacing damaged or dying cells in various adult tissues throughout a lifetime. They possess great potential for future regenerative medicine and gene therapy. However, the mechanisms governing stem cell regulation are poorly understood. Germline stem cells (GSCs) in the Drosophila testis have been shown to reside in niches, and thus these represent an excellent system for studying relationships between niches and stem cells. Here we show that Bmp signals from somatic cells are essential for maintaining GSCs in the Drosophila testis. Somatic cyst cells and hub cells express two Bmp molecules, Gbb and Dpp. Our genetic analysis indicates that gbb functions cooperatively with dpp to maintain male GSCs, although gbb alone is essential for GSC maintenance. Furthermore, mutant clonal analysis shows that Bmp signals directly act on GSCs and control their maintenance. In GSCs defective in Bmp signaling, expression of bam is upregulated, whereas forced bam expression in GSCs causes the GSCs to be lost. This study demonstrates that Bmp signals from the somatic cells maintain GSCs, at least in part, by repressing bam expression in the Drosophila testis. dpp signaling is known to be essential for maintaining GSCs in the Drosophila ovary. This study further suggests that both Drosophila male and female GSCs use Bmp signals to maintain GSCs.     

Self-maintained escort cells form a germline stem cell differentiation niche

Stem cell self-renewal is controlled by concerted actions of niche signals and intrinsic factors in a variety of systems. In the Drosophila ovary, germline stem cells (GSCs) in the niche continuously self-renew and generate differentiated germ cells that interact physically with escort cells (ECs). It has been proposed that escort stem cells (ESCs), which directly contact GSCs, generate differentiated ECs to maintain the EC population. However, it remains unclear whether the differentiation status of germ cells affects EC behavior and how the interaction between ECs and germ cells is regulated. In this study, we have found that ECs can undergo slow cell turnover regardless of their positions, and the lost cells are replenished by their neighboring ECs via self-duplication rather than via stem cells. ECs extend elaborate cellular processes that exhibit extensive interactions with differentiated germ cells. Interestingly, long cellular processes of ECs are absent when GSC progeny fail to differentiate, suggesting that differentiated germ cells are required for the formation or maintenance of EC cellular processes. Disruption of Rho functions leads to the disruption of long EC cellular processes and the accumulation of ill-differentiated single germ cells by increasing BMP signaling activity outside the GSC niche, and also causes gradual EC loss. Therefore, our findings indicate that ECs interact extensively with differentiated germ cells through their elaborate cellular processes and control proper germ cell differentiation. Here, we propose that ECs form a niche that controls GSC lineage differentiation and is maintained by a non-stem cell mechanism.     

Female germline stem cell niches of earwigs are structurally simple and different from those of Drosophila melanogaster

Stem cells function in niches, which consist of somatic cells that control the stem cells' self‐renewal, proliferation, and differentiation. Drosophila ovary germline niche consists of the terminal filament (TF) cells, cap cells, and escort stem cells; signaling from the TF cells and the cap cells is essential for maintenance of germline stem cells (GSCs). Here, we show that in the earwig Opisthocosmia silvestris, the female GSC niche is morphologically simple and consist of the TF cells and several structurally uniform escort cells. The most posterior cell of the TF (the basal cell of the TF) differs from remaining TF cells and is separated from the anterior region of the germarium by the processes of the escort cells, and consequently, does not contact the GSCs directly. We also show that between somatic cells of earwig niche argosome‐like vesicles and cytoneme‐like extensions are present. J. Morphol., 2010. © 2009 Wiley‐Liss, Inc.     

Pelota controls self-renewal of germline stem cells by repressing a Bam-independent differentiation pathway

In the Drosophila ovary, germline stem cell (GSC) self-renewal is controlled by both extrinsic and intrinsic factors. The Bmp signal from niche cells controls GSC self-renewal by directly repressing a Bam-dependent differentiation pathway in GSCs. pelota (pelo), which has been previously shown to be required for Drosophila male meiosis, was identified in our genetic screen as a dominant suppressor of the dpp overexpression-induced GSC tumor phenotype. In this study, we reveal the unexpected new role of Pelo in controlling GSC self-renewal by repressing a Bam-independent differentiation pathway. In pelo mutant ovaries, GSCs are lost rapidly owing to differentiation. Results from genetic mosaic analysis and germ cell-specific rescue show that it functions as an intrinsic factor to control GSC self-renewal. In pelo mutant GSCs, Bmp signaling activity detected by Dad-lacZ expression is downregulated, but bam expression is still repressed. Furthermore, bam mutant germ cells are still able to differentiate into cystocytes without pelo function, indicating that Pelo is involved in repressing a Bam-independent differentiation pathway. Consistent with its homology to the eukaryotic translation release factor 1alpha, we show that Pelo is localized to the cytoplasm of the GSC. Therefore, Pelo controls GSC self-renewal by repressing a Bam-independent differentiation pathway possibly through regulating translation. As Pelo is highly conserved from Drosophila to mammals, it may also be involved in the regulation of adult stem cell self-renewal in mammals, including humans.     

Stem cells signal to the niche through the Notch pathway in the Drosophila ovary

Stem cells are maintained and retain their capacity to continue dividing because of the influence of a niche. Although niches are important to maintain "stemness" in a wide variety of tissues, control of these niches is poorly understood. The Drosophila germline stem cells (GSCs) reside in a somatic cell niche. We show that Notch activation can induce the expression of niche-cell markers even in an adult fly; overexpression of Delta in the germline, or activated Notch in the somatic cells, results in extra niche cells, up to 10-fold over the normal number. In turn, these ectopic niche cells induce ectopic GSCs. Conversely, when GCSs do not produce functional Notch ligands, Delta and Serrate, the TGF-beta pathway is not activated in the GSCs, and they differentiate and subsequently leave the niche. Importantly, clonal analysis reveals that the receiving end of the Notch pathway is required in the somatic cells. These data show that a feedback loop exists between the stem cells and niche cells. Demonstration that stem cells can contribute to niche function has far-reaching consequences for stem cell therapies and may provide insight into how cancer can spread throughout an organism via populations of cancer stem cells.     

The hypoxic peri-arteriolar glioma stem cell niche,an integrated concept of five types of niches in human glioblastoma

Glioblastoma is the most lethal primary brain tumor and poor survival of glioblastoma patients is attributed to the presence of glioma stem cells (GSCs). These therapy-resistant, quiescent and pluripotent cells reside in GSC niches, which are specific microenvironments that protect GSCs against radiotherapy and chemotherapy. We previously showed the existence of hypoxic peri-arteriolar GSC niches in glioblastoma tumor samples. However, other studies have described peri-vascular niches, peri-hypoxic niches, peri-immune niches and extracellular matrix niches of GSCs. The aim of this review was to critically evaluate the literature on these five different types of GSC niches. In the present review, we describe that the five niche types are not distinct from one another, but should be considered to be parts of one integral GSC niche model, the hypoxic peri-arteriolar GSC niche. Moreover, hypoxic peri-arteriolar GSC niches are structural and functional look-alikes of hematopoietic stem cell (HSC) niches in the bone marrow. GSCs are maintained in peri-arteriolar niches by the same receptor-ligand interactions as HSCs in bone marrow. Our concept should be rigidly tested in the near future and applied to develop therapies to expel and keep GSCs out of their protective niches to render them more vulnerable to standard therapies.