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.     

E-Cadherin Is Required for Centrosome and Spindle Orientation in Drosophila Male Germline Stem Cells

Many adult stem cells reside in a special microenvironment known as the niche, where they receive essential signals that specify stem cell identity. Cell-cell adhesion mediated by cadherin and integrin plays a crucial role in maintaining stem cells within the niche. In Drosophila melanogaster, male germline stem cells (GSCs) are attached to niche component cells (i.e., the hub) via adherens junctions. The GSC centrosomes and spindle are oriented toward the hub-GSC junction, where E-cadherin-based adherens junctions are highly concentrated. For this reason, adherens junctions are thought to provide a polarity cue for GSCs to enable proper orientation of centrosomes and spindles, a critical step toward asymmetric stem cell division. However, understanding the role of E-cadherin in GSC polarity has been challenging, since GSCs carrying E-cadherin mutations are not maintained in the niche. Here, we tested whether E-cadherin is required for GSC polarity by expressing a dominant-negative form of E-cadherin. We found that E-cadherin is indeed required for polarizing GSCs toward the hub cells, an effect that may be mediated by Apc2. We also demonstrated that E-cadherin is required for the GSC centrosome orientation checkpoint, which prevents mitosis when centrosomes are not correctly oriented. We propose that E-cadherin orchestrates multiple aspects of stem cell behavior, including polarization of stem cells toward the stem cell-niche interface and adhesion of stem cells to the niche supporting cells.     

Structural polarity and dynamics of male germline stem cells in the milkweed bug (<Emphasis Type="Italic">Oncopeltus fasciatus</Emphasis>)

The male germline stem cells (GSCs) of the milkweed bug present an extraordinary structural polarity that is, to our knowledge, unequalled by any other type of stem cells. They consist of a perikaryon and numerous projections arising from the cell pole directed toward the apical cells, the proposed niche of the GSCs. The projections can traverse a considerable distance until their terminals touch the apical cells. From hatching until death, the GSC projections undergo conspicuous changes, the sequence of which has been deduced from observations of all developmental stages. Projection formation starts from lobular cell protrusions showing trabecular ingrowths of the cell membrane. Finger-like projections result from a process of growth and carving out. The newly formed projections contain mostly only free ribosomes other than a few mitochondria. A stereotyped degradation process commences in the projection terminals: profiles of circular, often concentric, cisternae of rough endoplasmic reticulum appear and turn into myelin bodies, whereas mitochondria become more numerous. The cytoplasm vesiculates, lysosomal bodies appear, and mitochondria become swollen. At the same time, the projection terminals are segregated by transverse ingrowths of the cell membrane. Finally, autophagic vacuoles and myelin bodies fill the segregated terminals, which then rupture. Simultaneously, new projections seem to sprout from the perikaryon of the GSCs. These dynamics, which are not synchronized among the GSCs, indicate that a novel type of signal exchange and transduction between the stem cells and their niche is involved in the regulation of asymmetric versus symmetric division of GSCs.     

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.     

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.     

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.     

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.     

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.     

The miRNA pathway intrinsically controls self-renewal of Drosophila germline stem cells

Stem cells uniquely self-renew and maintain tissue homoeostasis by differentiating into different cell types to replace aged or damaged cells [1]. During oogenesis of Drosophila melanogaster, self-renewal of germline stem cells (GSCs) requires both intrinsic signaling mechanisms and extrinsic signals from neighboring niche cells [2]. Emerging evidence suggests that microRNA (miRNA)-mediated translational regulation may also control Drosophila GSC self-renewal [3, 4]. It is unclear, however, whether the miRNA pathway functions within stem cells or niche cells to maintain GSCs. In Drosophila, Dicer-1 (Dcr-1) and the double-stranded RNA binding protein Loquacious (Loqs) catalyze miRNA biogenesis [3-5]. Here, we generate loqs knockout (loqs(KO)) flies by ends-out homologous recombination and show that loqs is essential for embryonic viability and ovarian GSC maintenance. Both developmental and miRNA processing defects are rescued by transgenic expression of Loqs-PB, but not Loqs-PA. Furthermore, mosaic germline analysis indicates that Loqs is required intrinsically for GSC maintenance. Consistently, GSCs are restored in loqs mutant ovaries by germline expression, but not somatic expression, of Loqs-PB. Together, these results demonstrate that Loqs-PB, but not Loqs-PA, is necessary and sufficient for Drosophila development and the miRNA pathway. Our study strongly suggests that miRNAs play an intrinsic, but not extrinsic, role in Drosophila female GSC self-renewal.     

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.     

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.     

Live imaging of the Drosophila spermatogonial stem cell niche reveals novel mechanisms regulating germline stem cell output

Adult stem cells modulate their output by varying between symmetric and asymmetric divisions, but have rarely been observed in living intact tissues. Germline stem cells (GSCs) in the Drosophila testis are anchored to somatic hub cells and were thought to exclusively undergo oriented asymmetric divisions, producing one stem cell that remains hub-anchored and one daughter cell displaced out of the stem cell-maintaining micro-environment (niche). We developed extended live imaging of the Drosophila testis niche, allowing us to track individual germline cells. Surprisingly, new wild-type GSCs are generated in the niche during steady-state tissue maintenance by a previously undetected event we term 'symmetric renewal', where interconnected GSC-daughter cell pairs swivel such that both cells contact the hub. We also captured GSCs undergoing direct differentiation by detaching from the hub. Following starvation-induced GSC loss, GSC numbers are restored by symmetric renewals. Furthermore, upon more severe (genetically induced) GSC loss, both symmetric renewal and de-differentiation (where interconnected spermatogonia fragment into pairs while moving towards then establishing contact with the hub) occur simultaneously to replenish the GSC pool. Thus, stereotypically oriented stem cell divisions are not always correlated with an asymmetric outcome in cell fate, and changes in stem cell output are governed by altered signals in response to tissue requirements.     

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.