Protocol for evaluating autophagy using LysoTracker staining in the epithelial follicle stem cells of the Drosophila ovary.
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ABSTRACT:
SUBMITTER: Correa I
PROVIDER: S-EPMC8209645 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Adult stem cells must balance self-renewal and differentiation for tissue homeostasis. The Drosophila ovary has provided a wealth of information about the extrinsic niche signals and intrinsic molecular processes required to ensure appropriate germline stem cell renewal and differentiation. The factors controlling behavior of the more recently identified follicle stem cells of the ovary are less well-understood but equally important for fertility. Here we report that translational regulators play a critical role in controlling these cells. Specifically, the translational regulator Caprin (Capr) is required in the follicle stem cell lineage to ensure maintenance of this stem cell population and proper encapsulation of developing germ cells by follicle stem cell progeny. In addition, reduction of one copy of the gene fmr1, encoding the translational regulator Fragile X Mental Retardation Protein, exacerbates the Capr encapsulation phenotype, suggesting Capr and fmr1 are regulating a common process. Caprin was previously characterized in vertebrates as Cytoplasmic Activation/Proliferation-Associated Protein. Significantly, we find that loss of Caprin alters the dynamics of the cell cycle, and we present evidence that misregulation of CycB contributes to the disruption in behavior of follicle stem cell progeny. Our findings support the idea that translational regulators may provide a conserved mechanism for oversight of developmentally critical cell cycles such as those in stem cell populations.
| S-EPMC3320888 | biostudies-literature
Project description:Addressing the complexity of organogenesis at a system-wide level requires a complete understanding of adult cell types, their origin, and precursor relationships. The Drosophila ovary has been a model to study how coordinated stem cell units, germline, and somatic follicle stem cells maintain and renew an organ. However, lack of cell type-specific tools have limited our ability to study the origin of individual cell types and stem cell units. Here, we used a single-cell RNA sequencing approach to uncover all known cell types of the developing ovary, reveal transcriptional signatures, and identify cell type-specific markers for lineage tracing. Our study identifies a novel cell type corresponding to the elusive follicle stem cell precursors and predicts subtypes of known cell types. Altogether, we reveal a previously unanticipated complexity of the developing ovary and provide a comprehensive resource for the systematic analysis of ovary morphogenesis.
| S-EPMC7000915 | biostudies-literature
Project description:Epithelial stem cells are routinely lost or damaged during adult life and must therefore be replaced to maintain homeostasis. Recent studies indicate that stem cell replacement occurs through neutral competition in many types of epithelial tissues, but little is known about the factors that determine competitive outcome. The epithelial follicle stem cells (FSCs) in the Drosophila ovary are regularly lost and replaced during normal homeostasis, and we show that FSC replacement conforms to a model of neutral competition. In addition, we found that FSCs mutant for the basolateral junction genes, lethal giant larvae (lgl) or discs large (dlg), undergo a biased competition for niche occupancy characterized by increased invasion of neighboring FSCs and reduced loss. Interestingly, FSCs mutant for a third basolateral junction gene, scribble (scrib), do not exhibit biased competition, suggesting that Lgl and Dlg regulate niche competition through a Scrib-independent process. Lastly, we found that FSCs have a unique cell polarity characterized by broadly distributed adherens junctions and the lack of a mature apical domain. Collectively, these observations indicate that Lgl and Dlg promote the differentiation of FSC progeny to a state in which they are less prone to invade the neighboring niche. In addition, we demonstrate that the neutral drift model can be adapted to quantify non-neutral behavior of mutant clones.
| S-EPMC4084627 | biostudies-literature
Project description:The follicle stem cells (FSCs) in the Drosophila ovary are an important experimental model for the study of epithelial stem cell biology. Although decades of research support the conclusion that there are two FSCs per ovariole, a recent study used a novel clonal marking system to conclude that there are 15-16 FSCs per ovariole. We performed clonal analysis using both this novel clonal marking system and standard clonal marking systems, and identified several problems that may have contributed to the overestimate of FSC number. In addition, we developed new methods for accurately measuring clone size, and found that FSC clones produce, on average, half of the follicle cells in each ovariole. Our findings provide strong independent support for the conclusion that there are typically two active FSCs per ovariole, though they are consistent with up to four FSCs per germarium.
| S-EPMC6946398 | biostudies-literature
Project description:Epithelial stem cells are maintained within niches that promote self-renewal by providing signals that specify the stem cell fate. In the Drosophila ovary, epithelial follicle stem cells (FSCs) reside in niches at the anterior tip of the tissue and support continuous growth of the ovarian follicle epithelium. Here, we demonstrate that a neighboring dynamic population of stromal cells, called escort cells, are FSC niche cells. We show that escort cells produce both Wingless and Hedgehog ligands for the FSC lineage, and that Wingless signaling is specific for the FSC niche whereas Hedgehog signaling is active in both FSCs and daughter cells. In addition, we show that multiple escort cells simultaneously encapsulate germ cell cysts and contact FSCs. Thus, FSCs are maintained in a dynamic niche by a non-dedicated population of niche cells.
| S-EPMC3817939 | biostudies-literature
Project description:The Hippo pathway is conserved and plays important roles in organ size control. The core components of the Hippo pathway are two kinases Hippo (Hpo), Warts (Wts), and a transcription-co-activator Yorkie (Yki). Yki activity is regulated by phosphorylation, which affects its nuclear localization and stability. To determine the role of the Hippo pathway in stem cells, we examine follicle stem cells (FSCs) in the Drosophila ovary. Yki is detected in the nucleus of FSCs. Knockdown of yki in the follicle cell lineage leads to a disruption of the follicular epithelium. Mitotic clones of FSCs mutant for hpo or wts are maintained in the niche and tend to replace the other FSCs, and FSCs mutant for yki are rapidly lost, demonstrating that the Hippo pathway is both required and sufficient for FSC maintenance. Using genetic interaction analyses, we demonstrate that the Hedgehog pathway acts upstream of the Hippo pathway in regulating FSC maintenance. The nuclear localization of Yki is enhanced when the Hedgehog signaling is activated. Furthermore, a constitutively active but not a wild-type Yki promotes FSC maintenance as activation of the Hedgehog signaling does, suggesting that the Hedgehog pathway regulates Yki through a post-translational mechanism in maintaining FSCs.
| S-EPMC5493701 | biostudies-literature
Project description: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.
| S-EPMC10011466 | biostudies-literature
Project description:Programmed cell death and cell corpse clearance are an essential part of organismal health and development. Cell corpses are often cleared away by professional phagocytes such as macrophages. However, in certain tissues, neighboring cells known as nonprofessional phagocytes can also carry out clearance functions. Here, we use the Drosophila melanogaster ovary to identify novel genes required for clearance by nonprofessional phagocytes. In the Drosophila ovary, germline cells can die at multiple time points. As death proceeds, the epithelial follicle cells act as phagocytes to facilitate the clearance of these cells. We performed an unbiased kinase screen to identify novel proteins and pathways involved in cell clearance during two death events. Of 224 genes examined, 18 demonstrated severe phenotypes during developmental death and clearance while 12 demonstrated severe phenotypes during starvation-induced cell death and clearance, representing a number of pathways not previously implicated in phagocytosis. Interestingly, it was found that several genes not only affected the clearance process in the phagocytes, but also non-autonomously affected the process by which germline cells died. This kinase screen has revealed new avenues for further exploration and investigation.
| S-EPMC8022946 | biostudies-literature
Project description:We use single cell RNA sequencing approach to identify and characterize all cell types in developing Drosophila ovaries at late third instar larval stage (LL3).
2020-02-04 | GSE131971 | GEO
Project description:Adult stem cells reside in specialized microenvironments called niches, which provide signals for stem cells to maintain their undifferentiated and self-renewing state. To maintain stem cell quality, several types of stem cells are known to be regularly replaced by progenitor cells through niche competition. However, the cellular and molecular bases for stem cell competition for niche occupancy are largely unknown. Here, we show that two Drosophila members of the glypican family of heparan sulfate proteoglycans (HSPGs), Dally and Dally-like (Dlp), differentially regulate follicle stem cell (FSC) maintenance and competitiveness for niche occupancy. Lineage analyses of glypican mutant FSC clones showed that dally is essential for normal FSC maintenance. In contrast, dlp is a hypercompetitive mutation: dlp mutant FSC progenitors often eventually occupy the entire epithelial sheet. RNA interference knockdown experiments showed that Dally and Dlp play both partially redundant and distinct roles in regulating Jak/Stat, Wg, and Hh signaling in FSCs. The Drosophila FSC system offers a powerful genetic model to study the mechanisms by which HSPGs exert specific functions in stem cell replacement and competition.
| S-EPMC5972425 | biostudies-literature