Integrins control epithelial stem cell proliferation in the Drosophila ovary by modulating the Notch pathway.

Cell proliferation and differentiation show a remarkable inverse relationship. The temporal coupling between cell cycle withdrawal and differentiation of stem cells (SCs) is crucial for epithelial tissue growth, homeostasis and regeneration. Proliferation vs. differentiation SC decisions are often controlled by the surrounding microenvironment, of which the basement membrane (BM; a specialized form of extracellular matrix surrounding cells and tissues), is one of its main constituents. Years of research have shown that integrin-mediated SC-BM interactions regulate many aspects of SC biology, including the proliferation-to-differentiation switch. However, these studies have also demonstrated that the SC responses to interactions with the BM are extremely diverse and depend on the cell type and state and on the repertoire of BM components and integrins involved. Here, we show that eliminating integrins from the follicle stem cells (FSCs) of the Drosophila ovary and their undifferentiated progeny increases their proliferation capacity. This results in an excess of various differentiated follicle cell types, demonstrating that cell fate determination can occur in the absence of integrins. Because these phenotypes are similar to those found in ovaries with decreased laminin levels, our results point to a role for the integrin-mediated cell-BM interactions in the control of epithelial cell division and subsequent differentiation. Finally, we show that integrins regulate proliferation by restraining the activity of the Notch/Delta pathway during early oogenesis. Our work increases our knowledge of the effects of cell-BM interactions in different SC types and should help improve our understanding of the biology of SCs and exploit their therapeutic potential.

Visualization and Quantification of Drosophila Larval Ovaries.

The morphogenesis of the ovarian germline stem cell (GSC) niche during larval stages in Drosophila provides the initial cellular and molecular basis for female gamete production in the adult. During larval instars, the Drosophila female gonad matures gradually from a round structure enclosing primordial germ cells (PGCs) and somatic cells into a functional organ containing GSC populations in their niches that later in adult stages support oogenesis. In this chapter, we describe a technique for dissecting, staining, and analyzing gonads from female Drosophila larvae and early pupae, offering the possibility of a direct view of the morphogenesis of an ovarian niche.

Live imaging of the Drosophila ovarian niche shows spectrosome and centrosome dynamics during asymmetric germline stem cell division.

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.

The careful control of Polo kinase by APC/C-Ube2C ensures the intercellular transport of germline centrosomes during Drosophila oogenesis.

A feature of metazoan reproduction is the elimination of maternal centrosomes from the oocyte. In animals that form syncytial cysts during oogenesis, including Drosophila and human, all centrosomes within the cyst migrate to the oocyte where they are subsequently degenerated. The importance and the underlying mechanism of this event remain unclear. Here, we show that, during early Drosophila oogenesis, control of the Anaphase Promoting Complex/Cyclosome (APC/C), the ubiquitin ligase complex essential for cell cycle control, ensures proper transport of centrosomes into the oocyte through the regulation of Polo/Plk1 kinase, a critical regulator of the integrity and activity of the centrosome. We show that novel mutations in the APC/C-specific E2, Vihar/Ube2c, that affect its inhibitory regulation on APC/C cause precocious Polo degradation and impedes centrosome transport, through destabilization of centrosomes. The failure of centrosome migration correlates with weakened microtubule polarization in the cyst and allows ectopic microtubule nucleation in nurse cells, leading to the loss of oocyte identity. These results suggest a role for centrosome migration in oocyte fate maintenance through the concentration and confinement of microtubule nucleation activity into the oocyte. Considering the conserved roles of APC/C and Polo throughout the animal kingdom, our findings may be translated into other animals.

Stem cell niche organization in the Drosophila ovary requires the ECM component Perlecan.

Stem cells reside in specialized microenvironments or niches that balance stem cell proliferation and differentiation.1,2 The extracellular matrix (ECM) is an essential component of most niches, because it controls niche homeostasis, provides physical support, and conveys extracellular signals.3-11 Basement membranes (BMs) are thin ECM sheets that are constituted mainly by Laminins, Perlecan, Collagen IV, and Entactin/Nidogen and surround epithelia and other tissues.12 Perlecans are secreted proteoglycans that interact with ECM proteins, ligands, receptors, and growth factors such as FGF, PDGF, VEGF, Hedgehog, and Wingless.13-18 Thus, Perlecans have structural and signaling functions through the binding, storage, or sequestering of specific ligands. We have used the Drosophila ovary to assess the importance of Perlecan in the functioning of a stem cell niche. Ovarioles in the adult ovary are enveloped by an ECM sheath and possess a tapered structure at their anterior apex termed the germarium. The anterior tip of the germarium hosts the germline niche, where two to four germline stem cells (GSCs) reside together with a few somatic cells: terminal filament cells (TFCs), cap cells (CpCs), and escort cells (ECs).19 We report that niche architecture in the developing gonad requires trol, that niche cells secrete an isoform-specific Perlecan-rich interstitial matrix, and that DE-cadherin-dependent stem cell-niche adhesion necessitates trol. Hence, we provide evidence to support a structural role for Perlecan in germline niche establishment during larval stages and in the maintenance of a normal pool of stem cells in the adult niche.

mastermind regulates niche ageing independently of the Notch pathway in the Drosophila ovary.

Proper stem cell activity in tissues ensures the correct balance between proliferation and differentiation, thus allowing tissue homeostasis and repair. The Drosophila ovary develops well-defined niches that contain on average 2-4 germline stem cells (GSCs), whose maintenance depends on systemic signals and local factors. A known player in the decline of tissue homeostasis is ageing, which correlates with the waning of resident stem cell populations. In Drosophila, ovaries from old females contain fewer GSCs than those from young flies. We isolated niche cells of aged ovaries, performed a transcriptomic analysis and identified mastermind (mam) as a factor for Drosophila ovarian niche functionality during ageing. We show that mam is upregulated in aged niche cells and that we can induce premature GSC loss by overexpressing mam in otherwise young niche cells. High mam levels in niche cells induce reduced Hedgehog amounts, a decrease in cadherin levels and a likely increase in reactive oxygen species, three scenarios known to provoke GSC loss. Mam is a canonical co-activator of the Notch pathway in many Drosophila tissues. However, we present evidence to support a Notch-independent role for mam in the ovarian germline niche.

EXD2 governs germ stem cell homeostasis and lifespan by promoting mitoribosome integrity and translation.

Mitochondria are subcellular organelles that are critical for meeting the bioenergetic and biosynthetic needs of the cell. Mitochondrial function relies on genes and RNA species encoded both in the nucleus and mitochondria, and on their coordinated translation, import and respiratory complex assembly. Here, we characterize EXD2 (exonuclease 3'-5' domain-containing 2), a nuclear-encoded gene, and show that it is targeted to the mitochondria and prevents the aberrant association of messenger RNAs with the mitochondrial ribosome. Loss of EXD2 results in defective mitochondrial translation, impaired respiration, reduced ATP production, increased reactive oxygen species and widespread metabolic abnormalities. Depletion of the Drosophila melanogaster EXD2 orthologue (CG6744) causes developmental delays and premature female germline stem cell attrition, reduced fecundity and a dramatic extension of lifespan that is reversed with an antioxidant diet. Our results define a conserved role for EXD2 in mitochondrial translation that influences development and ageing.

Laminin Levels Regulate Tissue Migration and Anterior-Posterior Polarity during Egg Morphogenesis in Drosophila.

Basement membranes (BMs) are specialized extracellular matrices required for tissue organization and organ formation. We study the role of laminin and its integrin receptor in the regulation of tissue migration during Drosophila oogenesis. Egg production in Drosophila involves the collective migration of follicle cells (FCs) over the BM to shape the mature egg. We show that laminin content in the BM increases with time, whereas integrin amounts in FCs do not vary significantly. Manipulation of integrin and laminin levels reveals that a dynamic balance of integrin-laminin amounts determines the onset and speed of FC migration. Thus, the interplay of ligand-receptor levels regulates tissue migration in vivo. Laminin depletion also affects the ultrastructure and biophysical properties of the BM and results in anterior-posterior misorientation of developing follicles. Laminin emerges as a key player in the regulation of collective cell migration, tissue stiffness, and the organization of anterior-posterior polarity in Drosophila.

Cell Signalling: Combining Pathways for Diversification and Reproducibility.

How a given signalling pathway can generate diverse outcomes is an open question. A new study shows that EGFR signalling in combination with JAK/STAT or BMP pathways induces different cell fates. Antagonistic interactions between downstream targets further stabilizes epithelial patterning.

Myosin light-chain phosphatase regulates basal actomyosin oscillations during morphogenesis.

Contractile actomyosin networks generate forces that drive tissue morphogenesis. Actomyosin contractility is controlled primarily by reversible phosphorylation of the myosin-II regulatory light chain through the action of myosin kinases and phosphatases. While the role of myosin light-chain kinase in regulating contractility during morphogenesis has been largely characterized, there is surprisingly little information on myosin light-chain phosphatase (MLCP) function in this context. Here, we use live imaging of Drosophila follicle cells combined with mathematical modelling to demonstrate that the MLCP subunit flapwing (flw) is a key regulator of basal myosin oscillations and cell contractions underlying egg chamber elongation. Flw expression decreases specifically on the basal side of follicle cells at the onset of contraction and flw controls the initiation and periodicity of basal actomyosin oscillations. Contrary to previous reports, basal F-actin pulsates similarly to myosin. Finally, we propose a quantitative model in which periodic basal actomyosin oscillations arise in a cell-autonomous fashion from intrinsic properties of motor assemblies.

ECM-Regulator timp Is Required for Stem Cell Niche Organization and Cyst Production in the Drosophila Ovary.

The extracellular matrix (ECM) is a pivotal component adult tissues and of many tissue-specific stem cell niches. It provides structural support and regulates niche signaling during tissue maintenance and regeneration. In many tissues, ECM remodeling depends on the regulation of MMP (matrix metalloproteinase) activity by inhibitory TIMP (tissue inhibitors of metalloproteinases) proteins. Here, we report that the only Drosophila timp gene is required for maintaining the normal organization and function of the germline stem cell niche in adult females. timp mutant ovaries show reduced levels of both Drosophila Collagen IV α chains. In addition, tissue stiffness and the cellular organization of the ovarian niche are affected in timp mutants. Finally, loss of timp impairs the ability of the germline stem cell niche to generate new cysts. Our results demonstrating a crucial role for timp in tissue organization and gamete production thus provide a link between the regulation of ECM metabolism and tissue homeostasis.

Genetics and mechanisms of ovarian cancer: parallels between Drosophila and humans.

Considering the degree of detail available at the genetic and cellular levels, the Drosophila ovary stands out as a powerful system to identify new players in the regulation of key aspects of cancer progression. In this review, we will comment on how the use of the Drosophila ovary has helped to elucidate some of the molecular bases of ovarian malignancies and to identify and characterize critical tumour suppressor genes and oncogenes with an impact in human pathologies.

Concise review: The plasticity of stem cell niches: a general property behind tissue homeostasis and repair.

Stem cell activity is tightly regulated during development and in adult tissues through the combined action of local and systemic effectors. While stem cells and their microenvironments are capable of sustaining homeostasis in normal physiological circumstances, they also provide host tissues with a remarkable plasticity to respond to perturbations. Here, we review recent discoveries that shed light on the adaptive response of niches to systemic signals and aging, and on the ability of niches to modulate signaling upon local perturbations. These characteristics of stem cells and their niches give organs an essential advantage to deal with aging, injury or pathological conditions.

Cytoneme-mediated delivery of hedgehog regulates the expression of bone morphogenetic proteins to maintain germline stem cells in Drosophila.

Stem cells reside in specialised microenvironments, or niches, which often contain support cells that control stem cell maintenance and proliferation. Hedgehog (Hh) proteins mediate homeostasis in several adult niches, but a detailed understanding of Hh signalling in stem cell regulation is lacking. Studying the Drosophila female germline stem cell (GSC) niche, we show that Hh acts as a critical juxtacrine signal to maintain the normal GSC population of the ovary. Hh production in cap cells, a type of niche support cells, is regulated by the Engrailed transcription factor. Hh is then secreted to a second, adjacent population of niche cells, the escort cells, where it activates transcription of the GSC essential factors Decapentaplegic (Dpp) and Glass bottom boat (Gbb). In wild-type niches, Hh protein decorates short filopodia that originate in the support cap cells and that are functionally relevant, as they are required to transduce the Hh pathway in the escort cells and to maintain a normal population of GSCs. These filopodia, reminiscent of wing disc cytonemes, grow several fold in length if Hh signalling is impaired within the niche. Because these long cytonemes project directionally towards the signalling-deficient region, cap cells sense and react to the strength of Hh pathway transduction in the niche. Thus, the GSC niche responds to insufficient Hh signalling by increasing the range of Hh spreading. Although the signal(s) perceived by the cap cells and the receptor(s) involved are still unknown, our results emphasise the integration of signals necessary to maintain a functional niche and the plasticity of cellular niches to respond to challenging physiological conditions.

The Ste20 kinase misshapen is essential for the invasive behaviour of ovarian epithelial cells in Drosophila.

Stationary-to-migratory transitions of epithelial cells have a key role in development and tumour progression. Border cell migration is a powerful system in which to investigate this transition in living organisms. Here, we identify the Ste20-like kinase misshapen (msn) as a novel regulator of border-cell migration in Drosophila. Expression of msn in border cells is independent of the transcription factor slow border cells and of inputs from all pathways that are known to control border-cell migration. The msn gene functions to modulate the levels and/or distribution of Drosophila E-cadherin to promote the invasive migratory behaviour of border cells.

Recent advances in Drosophila stem cell biology.

Stem cells possess the unique properties of self-renewal and the ability to give rise to multiple types of differentiated tissue. The fruit fly Drosophila melanogaster retains several populations of stem cells during adulthood as well as transient populations of stem cells during development. Studies of these different populations of stem cells using the genetic tools available to Drosophila researchers have played an important role in understanding many conserved stem cell characteristics. This review aims highlight some of the recent contributions from this important model system to our understanding of the myriad of processes that interact to control stem cell biology.

Integrins contribute to the establishment and maintenance of cell polarity in the follicular epithelium of the Drosophila ovary.

The generation of epithelial cell polarity is a key process during development. Although the induction and orientation of cell polarity by cell-cell and cell-extracellular matrix (ECM) interactions is well established, the molecular mechanisms by which signals from the ECM control cell polarity in developing epithelial tissues remain poorly understood. Here, we have used the follicular epithelium of the Drosophila ovary to investigate the role that integrins, the main cell-ECM receptors, play in the establishment of apicobasal polarity. Mature follicle cells have an apical side facing the germ line and a basal side in contact with a basement membrane. Our results show that integrins - presumably via interactions with the basement membrane - play a reinforcing role in follicle cell polarization, as they are required to establish and/or maintain follicle cell membrane asymmetry only when contact with the germ line is prevented. We suggest that the primary cue for polarization of the follicular epithelium is contact with the germline cells. In addition, while interfering with apical and lateral polarization cues leads to apoptosis, we show here that inhibition of contact with the basement membrane mediated by integrins does not affect cell survival. Finally, we provide evidence to suggest that integrins are required to orientate epithelial polarity in vivo.

A role for the chaperone Hsp70 in the regulation of border cell migration in the Drosophila ovary.

Unravelling the molecular mechanisms that govern cell migration is of great importance towards understanding both normal embryogenesis and physiological and pathological processes occurring in the adult. Migration of border cells (BCs) during Drosophila oogenesis provides a simple and attractive model in which to address this problem. Here, we show that the molecular chaperone Hsp70 is required for BC migration. Thus, BCs lacking all Hsp70 genes present in the fly genome fail to reorganize their actin cytoskeleton, resulting in migration defects. Similar defects are found when the Hsp70 co-chaperone DnaJ-1, the Drosophila homolog of the human Hsp40, is overexpressed specifically in BCs. In addition, we provide biochemical and genetic evidence for an interaction between DnaJ-1 and PDGF/VEGF receptor (PVR), which is also required for actin-mediated BC migration. Furthermore, our results showing that PVR also interacts genetically with Hsp70 suggest that a mechanism by which the DnaJ-1/Hsp70 chaperone complex regulates BC migration is by modulating PVR function.