Cyclin E controls Drosophila female germline stem cell maintenance independently of its role in proliferation by modulating responsiveness to niche signals.
ABSTRACT: Stem cells must proliferate while maintaining 'stemness'; however, much remains to be learned about how factors that control the division of stem cells influence their identity. Multiple stem cell types display cell cycles with short G1 phases, thought to minimize susceptibility to differentiation factors. Drosophila female germline stem cells (GSCs) have short G1 and long G2 phases, and diet-dependent systemic factors often modulate G2. We previously observed that Cyclin E (CycE), a known G1/S regulator, is atypically expressed in GSCs during G2/M; however, it remained unclear whether CycE has cell cycle-independent roles in GSCs or whether it acts exclusively by modulating the cell cycle. In this study, we detected CycE activity during G2/M, reflecting its altered expression pattern, and showed that CycE and its canonical partner, Cyclin-dependent kinase 2 (Cdk2), are required not only for GSC proliferation, but also for GSC maintenance. In genetic mosaics, CycE- and Cdk2-deficient GSCs are rapidly lost from the niche, remain arrested in a G1-like state, and undergo excessive growth and incomplete differentiation. However, we found that CycE controls GSC maintenance independently of its role in the cell cycle; GSCs harboring specific hypomorphic CycE mutations are not efficiently maintained despite normal proliferation rates. Finally, CycE-deficient GSCs have an impaired response to niche bone morphogenetic protein signals that are required for GSC self-renewal, suggesting that CycE modulates niche-GSC communication. Taken together, these results show unequivocally that the roles of CycE/Cdk2 in GSC division cycle regulation and GSC maintenance are separable, and thus potentially involve distinct sets of phosphorylation targets.
Project description:Whether stem cells have unique cell cycle machineries and how they integrate with niche interactions remains largely unknown. We identified a hypomorphic cyclin E allele WX that strongly impairs the maintenance of follicle stem cells (FSCs) in the Drosophila ovary but does not reduce follicle cell proliferation or germline stem cell maintenance. CycE(WX) protein can still bind to the cyclin-dependent kinase catalytic subunit Cdk2, but forms complexes with reduced protein kinase activity measured in vitro. By creating additional CycE variants with different degrees of kinase dysfunction and expressing these and CycE(WX) at different levels, we found that higher CycE-Cdk2 kinase activity is required for FSC maintenance than to support follicle cell proliferation. Surprisingly, cycE(WX) FSCs were lost from their niches rather than arresting proliferation. Furthermore, FSC function was substantially restored by expressing either excess DE-cadherin or excess E2F1/DP, the transcription factor normally activated by CycE-Cdk2 phosphorylation of retinoblastoma proteins. These results suggest that FSC maintenance through niche adhesion is regulated by inputs that normally control S phase entry, possibly as a quality control mechanism to ensure adequate stem cell proliferation. We speculate that a positive connection between central regulators of the cell cycle and niche retention may be a common feature of highly proliferative stem cells.
Project description:Drosophila female germline stem cells (GSCs) are found inside the cellular niche at the tip of the ovary. They undergo asymmetric divisions to renew the stem cell lineage and to produce sibling cystoblasts that will in turn enter differentiation. GSCs and cystoblasts contain spectrosomes, membranous structures essential for orientation of the mitotic spindle and that, particularly in GSCs, change shape depending on the cell cycle phase. Using live imaging and a fusion protein of GFP and the spectrosome component Par-1, we follow the complete spectrosome cycle throughout GSC division and quantify the relative duration of the different spectrosome shapes. We also determine that the Par-1 kinase shuttles between the spectrosome and the cytoplasm during mitosis and observe the continuous addition of new material to the GSC and cystoblast spectrosomes. Next, we use the Fly-FUCCI tool to define, in live and fixed tissues, that GSCs have a shorter G1 compared with the G2 phase. The observation of centrosomes in dividing GSCs allowed us to determine that centrosomes separate very early in G1, before centriole duplication. Furthermore, we show that the anterior centrosome associates with the spectrosome only during mitosis and that, upon mitotic spindle assembly, it translocates to the cell cortex, where it remains anchored until centrosome separation. Finally, we demonstrate that the asymmetric division of GSCs is not an intrinsic property of these cells, as the spectrosome of GSC-like cells located outside of the niche can divide symmetrically. Thus, GSCs display unique properties during division, a behaviour influenced by the surrounding niche.
Project description: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.
Project description:Stem cells depend on intrinsic and local factors to maintain their identity and activity, but they also sense and respond to changing external conditions. We previously showed that germline stem cells (GSCs) and follicle stem cells (FSCs) in the Drosophila ovary respond to diet via insulin signals. Insulin signals directly modulate the GSC cell cycle at the G2 phase, but additional unknown dietary mediators control both G1 and G2. Target of rapamycin, or TOR, is part of a highly conserved nutrient-sensing pathway affecting growth, proliferation, survival and fertility. Here, we show that optimal TOR activity maintains GSCs but does not play a major role in FSC maintenance, suggesting differential regulation of GSCs versus FSCs. TOR promotes GSC proliferation via G2 but independently of insulin signaling, and TOR is required for the proliferation, growth and survival of differentiating germ cells. We also report that TOR controls the proliferation of FSCs but not of their differentiating progeny. Instead, TOR controls follicle cell number by promoting survival, independently of either the apoptotic or autophagic pathways. These results uncover specific TOR functions in the control of stem cells versus their differentiating progeny, and reveal parallels between Drosophila and mammalian follicle growth.
Project description:Establishment and maintenance of functional stem cells is critical for organ development and tissue homeostasis. Little is known about the mechanisms underlying stem establishment during organogenesis. Drosophila testes are among the most thoroughly characterized systems for studying stem cell behavior, with germline stem cells (GSCs) and somatic cyst stem cells (CySCs) cohabiting a discrete stem cell niche at the testis apex. GSCs and CySCs are arrayed around hub cells that also comprise the niche and communication between hub cells, GSCs, and CySCs regulates the balance between stem cell maintenance and differentiation. Recent data has shown that functional, asymmetrically dividing GSCs are first established at ?23 h after egg laying during Drosophila testis morphogenesis (Sheng et al., 2009). This process correlates with coalescence of the hub, but development of CySCs from somatic gonadal precursors (SGPs) was not examined. Here, we show that functional CySCs are present at the time of GSC establishment, and that Jak-STAT signaling is necessary and sufficient for CySC maintenance shortly thereafter. Furthermore, hyper-activation of Jak in CySCs promotes expansion of the GSC population, while ectopic Jak activation in the germline induces GSC gene expression in GSC daughter cells but does not prevent spermatogenic differentiation. Together, these observations indicate that, similar to adult testes, Jak-STAT signaling from the hub acts on both GSCs and CySC to regulate their development and differentiation, and that additional signaling from CySCs to the GSCs play a dominant role in controlling GSC maintenance during niche formation.
Project description:<h4>Background</h4>Drosophila female germline stem cells (GSCs) reside adjacent to a cellular niche that secretes Bone Morphogenetic Protein (BMP) ligands and anchors the GSCs through adherens junctions. The GSCs divide asymmetrically such that one daughter remains in the niche as a GSC, while the other is born away from the niche and differentiates. However, given that the BMP signal can be diffusible, it remains unclear how a local extracellular asymmetry is sufficient to result in a robust pattern of asymmetric division.<h4>Methods and findings</h4>Here we show that GSCs are polarized with respect to the cellular niche. We first use a modified biosensor to demonstrate that the small GTPase Rac is asymmetrically activated within the GSC at the niche-GSC interface. Experiments using loss-of-function and gain-of-function mutations in Rac indicate that asymmetric Rac activity both localizes the microtubule binding protein Apc2 to orient one GSC centrosome at the niche-GSC interface during interphase and activates the Jun N-terminal kinase pathway to increase the ability of the GSC to respond to BMP ligands. Other processes act in concert with each function of Rac. Specifically, we demonstrate that the GSC cell cycle arrests at prometaphase if centrosomes are misoriented.<h4>Conclusions</h4>Thus, the GSCs, an adult stem cell present in a cellular niche, have a niche-associated polarity that couples control of the division plane with increased response to an extracellular maintenance signal. Other processes work in parallel with the Rac-mediated polarity to ensure a robust pattern of asymmetric division. We suggest that all adult stem cells likely employ multiple, independently acting mechanisms to ensure asymmetric division to maintain tissue homeostasis.
Project description:Germline stem cells (GSCs) self-renew and differentiate to sustain a continuous production of gametes. In the female Drosophila germ line, two differentiation factors, bag of marbles ( bam) and benign gonial cell neoplasm ( bgcn), work in concert in the stem cell daughter to promote the generation of eggs. In GSCs, bam transcription is repressed by signaling from the niche and is activated in stem cell daughters. In contrast, bgcn is transcribed in both the GSCs and stem cell daughters, but little is known about how bgcn is transcriptionally modulated. Here we find that the conserved protein Nipped-A acts through the Tat interactive protein 60-kDa (Tip60) histone acetyl transferase complex in the germ line to promote GSC daughter differentiation. We find that Nipped-A is required for efficient exit from the gap phase 2 (G2) of cell cycle of the GSC daughter and for expression of a differentiation factor, bgcn. Loss of Nipped-A results in accumulation of GSC daughters . Forced expression of bgcn in Nipped-A germline-depleted ovaries rescues this differentiation defect. Together, our results indicate that Tip60 complex coordinates cell cycle progression and expression of bgcn to help drive GSC daughters toward a differentiation program.
Project description:In the Drosophila ovary, bone morphogenetic protein (BMP) signaling activated by the niche promotes germline stem cell (GSC) self-renewal and proliferation, whereas E-cadherin-mediated cell adhesion anchors GSCs in the niche for their continuous self-renewal. Here we show that Lissencephaly-1 (Lis1) regulates BMP signaling and E-cadherin-mediated adhesion between GSCs and their niche and thereby controls GSC self-renewal. Lis1 mutant GSCs are lost faster than control GSCs because of differentiation but not because of cell death, indicating that Lis1 controls GSC self-renewal. The Lis1 mutant GSCs exhibit reduced BMP signaling activity, and Lis1 interacts genetically with the BMP pathway components in the regulation of GSC maintenance. Mechanistically, Lis1 binds directly to and stabilizes the SMAD protein Mothers against decapentaplegic (Mad), facilitates its phosphorylation, and thereby regulates BMP signaling. Finally, the Lis1 mutant GSCs accumulate less E-cadherin in the stem cell-niche junction than do their wild-type counterparts. Germline-specific expression of an activated BMP receptor thickveins (Tkv) or E-cadherin can partially rescue the loss phenotype of Lis1 mutant GSCs. Therefore, this study has revealed a role of Lis1 in the control of Drosophila ovarian GSC self-renewal, at least partly by regulating niche signal transduction and niche adhesion. It has been known that Lis1 controls neural precursor/stem cell proliferation in the developing mammalian brain; this study further suggests that Lis1, which is widely expressed in adult mammalian tissues, could regulate adult tissue stem cells through modulating niche signaling and adhesion.
Project description:Adult <i>C. elegans</i> germline stem cells (GSCs) and mouse embryonic stem cells (mESCs) exhibit a non-canonical cell cycle structure with an abbreviated G1 phase and phase-independent expression of Cdk2 and cyclin E. Mechanisms that promote the abbreviated cell cycle remain unknown, as do the consequences of not maintaining an abbreviated cell cycle in these tissues. In GSCs, we discovered that loss of <i>gsk-3</i> results in reduced GSC proliferation without changes in differentiation or responsiveness to GLP-1/Notch signaling. We find that DPL-1 transcriptional activity inhibits CDK-2 mRNA accumulation in GSCs, which leads to slower S-phase entry and progression. Inhibition of <i>dpl-1</i> or transgenic expression of CDK-2 via a heterologous germline promoter rescues the S-phase entry and progression defects of the <i>gsk-3</i> mutants, demonstrating that transcriptional regulation rather than post-translational control of CDK-2 establishes the abbreviated cell cycle structure in GSCs. This highlights an inhibitory cascade wherein GSK-3 inhibits DPL-1 and DPL-1 inhibits <i>cdk-2</i> transcription. Constitutive GSK-3 activity through this cascade maintains an abbreviated cell cycle structure to permit the efficient proliferation of GSCs necessary for continuous tissue output.
Project description: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.