RESUMEN
Drosophila ovarian germline stem cells (GSCs) are a powerful model for stem cell research. In this study, we use single-cell RNA sequencing (scRNA-seq), an RNAi screen and bioinformatic analysis, to identify genes involved in germ cell differentiation, including 34 genes with upregulated expression during early germ cell development and 19 genes that may regulate germ cell differentiation. Among these, a gene we have named eggplant (eggpl) is highly expressed in GSCs and downregulated in early daughter cells. RNAi knockdown of eggpl causes germ cell proliferation and differentiation defects. In flies fed a rich yeast diet, the expression of eggpl is significantly lower and knockdown or knockout of eggpl phenocopies a rich diet. In addition, eggpl knockdown suppresses the reduction in germ cell proliferation caused by inhibition of the insulin effector PI3K. These findings suggest that downregulation of eggpl links nutritional status to germ cell proliferation and differentiation. Collectively, this study provides new insights into the signaling networks that regulate early germ cell development and identifies eggpl as a key player in this process.
Asunto(s)
Proteínas de Drosophila , Solanum melongena , Animales , Drosophila/genética , Solanum melongena/genética , Solanum melongena/metabolismo , Proteínas de Drosophila/metabolismo , Diferenciación Celular/genética , Células Germinativas/metabolismo , Análisis de Secuencia de ARN , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismoRESUMEN
Adult stem cell niche boundaries must be precisely maintained to facilitate the segregation of stem cell and daughter cell fates. However, the mechanisms that govern this process in epithelial tissues are not fully understood. In this study, we investigated the relationship between two signals, Wnt and EGFR, that are necessary for self-renewal of the epithelial follicle stem cells (FSCs) in the Drosophila ovary, but must be downregulated in cells that have exited the niche to allow for differentiation. We found that Wingless produced by inner germarial sheath (IGS) cells acts over a short distance to activate Wnt signaling in FSCs, and that movement across the FSC niche boundary is limited. In addition, we show that Wnt signaling functions genetically upstream of EGFR signaling by activating the expression of the EGFR ligand, Spitz, and that constitutive activation of EGFR partially rescues the self-renewal defect caused by loss of Wnt signaling. Collectively, our findings support a model in which the Wnt and EGFR pathways operate in a signaling hierarchy to promote FSC self-renewal.
Asunto(s)
Autorrenovación de las Células , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Receptores ErbB/metabolismo , Folículo Ovárico/citología , Receptores de Péptidos de Invertebrados/metabolismo , Células Madre/citología , Proteína Wnt1/metabolismo , Animales , Proteínas de Drosophila/genética , Factor de Crecimiento Epidérmico/genética , Factor de Crecimiento Epidérmico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosforilación , Receptores Notch/metabolismo , Transducción de Señal , Nicho de Células Madre , Células Madre/metabolismo , Transcripción GenéticaRESUMEN
Understanding how cell fate decisions are regulated is a central question in stem cell biology. Recent studies have demonstrated that intracellular pH (pHi) dynamics contribute to this process. Indeed, the pHi of cells within a tissue is not simply a consequence of chemical reactions in the cytoplasm and other cellular activity, but is actively maintained at a specific setpoint in each cell type. We found previously that the pHi of cells in the follicle stem cell (FSC) lineage in the Drosophila ovary increases progressively during differentiation from an average of 6.8 in the FSCs, to 7.0 in newly produced daughter cells, to 7.3 in more differentiated cells. Two major regulators of pHi in this lineage are Drosophila sodium-proton exchanger 2 (dNhe2) and a previously uncharacterized gene, CG8177, that is homologous to mammalian anion exchanger 2 (AE2). Based on this homology, we named the gene anion exchanger 2 (ae2). Here, we generated null alleles of ae2 and found that homozygous mutant flies are viable but have severe defects in ovary development and adult oogenesis. Specifically, we find that ae2 null flies have smaller ovaries, reduced fertility, and impaired follicle formation. In addition, we find that the follicle formation defect can be suppressed by a decrease in dNhe2 copy number and enhanced by the overexpression of dNhe2, suggesting that this phenotype is due to the dysregulation of pHi. These findings support the emerging idea that pHi dynamics regulate cell fate decisions and our studies provide new genetic tools to investigate the mechanisms by which this occurs.
Asunto(s)
Antiportadores/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Oogénesis , Ovario/embriología , Ovario/metabolismo , Animales , Drosophila melanogaster/genética , Epistasis Genética , Femenino , Fertilidad , Mutación/genética , Tamaño de los Órganos , Folículo Ovárico/embriología , Interferencia de ARN , Homología de Secuencia de AminoácidoRESUMEN
In the epithelial follicle stem cells (FSCs) of the Drosophila ovary, Epidermal Growth Factor Receptor (EGFR) signaling promotes self-renewal, whereas Notch signaling promotes differentiation of the prefollicle cell (pFC) daughters. We have identified two proteins, Six4 and Groucho (Gro), that link the activity of these two pathways to regulate the earliest cell fate decision in the FSC lineage. Our data indicate that Six4 and Gro promote differentiation towards the polar cell fate by promoting Notch pathway activity. This activity of Gro is antagonized by EGFR signaling, which inhibits Gro-dependent repression via p-ERK mediated phosphorylation. We have found that the phosphorylated form of Gro persists in newly formed pFCs, which may delay differentiation and provide these cells with a temporary memory of the EGFR signal. Collectively, these findings demonstrate that phosphorylated Gro labels a transition state in the FSC lineage and describe the interplay between Notch and EGFR signaling that governs the differentiation processes during this period.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Receptores ErbB/metabolismo , Proteínas de Homeodominio/genética , Proteínas del Tejido Nervioso/genética , Folículo Ovárico/embriología , Receptores de Péptidos de Invertebrados/metabolismo , Receptores Notch/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Diferenciación Celular/genética , Células Epiteliales/citología , Femenino , Proteínas de Homeodominio/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Folículo Ovárico/citología , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/genética , Transducción de Señal/genética , Células Madre/citología , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Drosophila melanogaster/citología , Folículo Ovárico/citología , Nicho de Células Madre , Animales , Linaje de la Célula , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Femenino , Proteínas Hedgehog/metabolismo , Transducción de Señal , Células del Estroma/citología , Células del Estroma/metabolismo , Transgenes , Proteína Wnt1/metabolismoRESUMEN
Measurements of Drosophila fecundity are used in a wide variety of studies, such as investigations of stem cell biology, nutrition, behavior, and toxicology. In addition, because fecundity assays are performed on live flies, they are suitable for longitudinal studies such as investigations of aging or prolonged chemical exposure. However, standard Drosophila fecundity assays have been difficult to perform in a high-throughput manner because experimental factors such as the physiological state of the flies and environmental cues must be carefully controlled to achieve consistent results. In addition, exposing flies to a large number of different experimental conditions (such as chemical additives in the diet) and manually counting the number of eggs laid to determine the impact on fecundity is time-consuming. We have overcome these challenges by combining a new multiwell fly culture strategy with a novel 3D-printed fly transfer device to rapidly and accurately transfer flies from one plate to another; the RoboCam, a low-cost, custom built robotic camera to capture images of the wells automatically; and an image segmentation pipeline to automatically identify and quantify eggs. We show that this method is compatible with robust and consistent egg laying throughout the assay period; and demonstrate that the automated pipeline for quantifying fecundity is very accurate (r2 = 0.98 for the correlation between the automated egg counts and the ground truth) In addition, we show that this method can be used to efficiently detect the effects on fecundity induced by dietary exposure to chemicals. Taken together, this strategy substantially increases the efficiency and reproducibility of high throughput egg laying assays that require exposing flies to multiple different media conditions.
RESUMEN
For more than 100 years, the fruit fly Drosophila melanogaster has been one of the most studied model organisms. Here, we present a single-cell atlas of the adult fly, Tabula Drosophilae, that includes 580,000 nuclei from 15 individually dissected sexed tissues as well as the entire head and body, annotated to >250 distinct cell types. We provide an in-depth analysis of cell type-related gene signatures and transcription factor markers, as well as sexual dimorphism, across the whole animal. Analysis of common cell types between tissues, such as blood and muscle cells, reveals rare cell types and tissue-specific subtypes. This atlas provides a valuable resource for the Drosophila community and serves as a reference to study genetic perturbations and disease models at single-cell resolution.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/genética , Transcriptoma , Animales , Núcleo Celular/metabolismo , Bases de Datos Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Femenino , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Genes de Insecto , Masculino , RNA-Seq , Caracteres Sexuales , Análisis de la Célula Individual , Factores de Transcripción/genéticaRESUMEN
During the past several years, it has become increasingly possible to study adult stem cells in their native territories within tissues. These studies have provided new evidence for the existence of stem cells in the breast, muscle, lung and kidney and have led to a deeper understanding of the best-known stem cells in Drosophila and mice. Tissue stem cells are turning out to be diverse, with varying division rates, lineage lengths, and mechanisms of regulation. In addition, stem cells are now known to engage in a wide variety of interactions with neighboring cells and extracellular matrices, and to respond to various neural and hormonal signals. Stem cell niches are also diverse, sometimes harboring multiple stem cell types. Internally, a stem cell's chromatin and cytoskeletal organization play key roles. Understanding how stem cells and their progeny are controlled will illuminate fundamental biological mechanisms that govern the construction and maintenance of tissues within metazoan animals.
Asunto(s)
Envejecimiento , Células Madre/citología , Células Madre/metabolismo , Animales , Cromatina/genética , HumanosRESUMEN
The Drosophila ovary is a widely used model for germ cell and somatic tissue biology. Here we use single-cell RNA-sequencing (scRNA-seq) to build a comprehensive cell atlas of the adult Drosophila ovary that contains transcriptional profiles for every major cell type in the ovary, including the germline stem cells and their niche cells, follicle stem cells, and previously undescribed subpopulations of escort cells. In addition, we identify Gal4 lines with specific expression patterns and perform lineage tracing of subpopulations of escort cells and follicle cells. We discover that a distinct subpopulation of escort cells is able to convert to follicle stem cells in response to starvation or upon genetic manipulation, including knockdown of escargot, or overactivation of mTor or Toll signalling.
Asunto(s)
Drosophila/citología , Ovario/citología , Animales , Linaje de la Célula , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Folículo Ovárico/citología , Folículo Ovárico/metabolismo , Ovario/metabolismo , Análisis de la Célula IndividualRESUMEN
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.
Asunto(s)
Drosophila melanogaster/citología , Células Epiteliales/citología , Folículo Ovárico/citología , Células Madre/citología , Células Madre/fisiología , Animales , Femenino , OvarioRESUMEN
Metazoan physiology depends on intricate patterns of gene expression that remain poorly known. Using transposon mutagenesis in Drosophila, we constructed a library of 7404 protein trap and enhancer trap lines, the Carnegie collection, to facilitate gene expression mapping at single-cell resolution. By sequencing the genomic insertion sites, determining splicing patterns downstream of the enhanced green fluorescent protein (EGFP) exon, and analyzing expression patterns in the ovary and salivary gland, we found that 600-900 different genes are trapped in our collection. A core set of 244 lines trapped different identifiable protein isoforms, while insertions likely to act as GFP-enhancer traps were found in 256 additional genes. At least 8 novel genes were also identified. Our results demonstrate that the Carnegie collection will be useful as a discovery tool in diverse areas of cell and developmental biology and suggest new strategies for greatly increasing the coverage of the Drosophila proteome with protein trap insertions.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Regulación del Desarrollo de la Expresión Génica , Biblioteca de Genes , Genes de Insecto/genética , Animales , Secuencia de Bases , Cartilla de ADN , Elementos Transponibles de ADN/genética , Proteínas de Drosophila/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Proteínas Fluorescentes Verdes/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Ovario/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Glándulas Salivales/metabolismo , Análisis de Secuencia de ADNRESUMEN
Some metazoans have evolved the capacity to survive severe oxygen deprivation. The nematode, Caenorhabditis elegans, exposed to anoxia (0 kPa, 0% O(2)) enters into a recoverable state of suspended animation during all stages of the life cycle. That is, all microscopically observable movement ceases including cell division, developmental progression, feeding, and motility. To understand suspended animation, we compared oxygen-deprived embryos to nontreated embryos in both wild-type and hif-1 mutants. We found that hif-1 mutants survive anoxia, suggesting that the mechanisms for anoxia survival are different from those required for hypoxia. Examination of wild-type embryos exposed to anoxia show that blastomeres arrest in interphase, prophase, metaphase, and telophase but not anaphase. Analysis of the energetic state of anoxic embryos indicated a reversible depression in the ATP to ADP ratio. Given that a decrease in ATP concentrations likely affects a variety of cellular processes, including signal transduction, we compared the phosphorylation state of several proteins in anoxic embryos and normoxic embryos. We found that the phosphorylation state of histone H3 and cell cycle-regulated proteins recognized by the MPM-2 antibody were not detectable in anoxic embryos. Thus, dephosphorylation of specific proteins correlate with the establishment and/or maintenance of a state of anoxia-induced suspended animation.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Proteínas Nucleares/genética , Oxígeno/metabolismo , Factores de Transcripción , Adaptación Fisiológica , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Anafase/fisiología , Animales , Blastómeros/citología , Blastómeros/metabolismo , Caenorhabditis elegans/citología , Ciclo Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Epítopos/metabolismo , Factor 1 Inducible por Hipoxia , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismoRESUMEN
Despite extensive knowledge about the transcriptional regulation of stem cell differentiation, less is known about the role of dynamic cytosolic cues. We report that an increase in intracellular pH (pHi) is necessary for the efficient differentiation of Drosophila adult follicle stem cells (FSCs) and mouse embryonic stem cells (mESCs). We show that pHi increases with differentiation from FSCs to prefollicle cells (pFCs) and follicle cells. Loss of the Drosophila Na+-H+ exchanger DNhe2 lowers pHi in differentiating cells, impairs pFC differentiation, disrupts germarium morphology, and decreases fecundity. In contrast, increasing pHi promotes excess pFC cell differentiation toward a polar/stalk cell fate through suppressing Hedgehog pathway activity. Increased pHi also occurs with mESC differentiation and, when prevented, attenuates spontaneous differentiation of naive cells, as determined by expression of microRNA clusters and stage-specific markers. Our findings reveal a previously unrecognized role of pHi dynamics for the differentiation of two distinct types of stem cell lineages, which opens new directions for understanding conserved regulatory mechanisms.
Asunto(s)
Envejecimiento/fisiología , Diferenciación Celular , Drosophila melanogaster/citología , Células Epiteliales/citología , Espacio Intracelular/metabolismo , Células Madre Embrionarias de Ratones/citología , Animales , Linaje de la Célula , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliales/metabolismo , Femenino , Proteínas Hedgehog/metabolismo , Concentración de Iones de Hidrógeno , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Folículo Ovárico/citología , Transducción de SeñalRESUMEN
Epithelial stem cells divide asymmetrically, such that one daughter replenishes the stem cell pool and the other differentiates. We found that, in the epithelial follicle stem cell (FSC) lineage of the Drosophila ovary, epidermal growth factor receptor (EGFR) signaling functions specifically in the FSCs to promote the unique partially polarized state of the FSC, establish apical-basal polarity throughout the lineage, and promote FSC maintenance in the niche. In addition, we identified a novel connection between EGFR signaling and the cell-polarity regulator liver kinase B1 (LKB1), which indicates that EGFR signals through both the Ras-Raf-MEK-Erk pathway and through the LKB1-AMPK pathway to suppress apical identity. The development of apical-basal polarity is the earliest visible difference between FSCs and their daughters, and our findings demonstrate that the EGFR-mediated regulation of apical-basal polarity is essential for the segregation of stem cell and daughter cell fates.
Asunto(s)
Polaridad Celular , Drosophila/metabolismo , Receptores ErbB/metabolismo , Transducción de Señal , Células Madre/metabolismo , Animales , Regulación hacia Abajo , Drosophila/citología , Células Epiteliales/citología , Células Epiteliales/metabolismo , Células Madre/citologíaRESUMEN
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.
Asunto(s)
Células Madre Adultas/citología , Drosophila/citología , Folículo Ovárico/citología , Nicho de Células Madre , Células Madre Adultas/metabolismo , Animales , Diferenciación Celular , Polaridad Celular , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Mutación , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Folículo Ovárico/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Epithelial stem cells are regulated through a complex interplay of signals from diffusible ligands, cellular interactions, and attachment to the extracellular matrix. The development of Drosophila models of epithelial stem cells and their associated niche has made it possible to dissect the contribution of each of these factors in vivo, during both basal homeostasis and in response to acute damage such as infection. Studies of Drosophila epithelial stem cells have also provided insight into the mechanisms by which a healthy population of stem cells are maintained throughout adulthood by demonstrating, for example, that stem cells have a finite lifespan and may be displaced by replacement cells competing for niche occupancy. Here, we summarize the literature on each of the known Drosophila epithelial stem cells, with a focus on the two most well-characterized types, the follicle stem cells (FSCs) in the ovary and the intestinal stem cells (ISCs) in the posterior midgut. Several themes have emerged from these studies, which suggest that there may be a common set of features among niches in a variety of epithelia. For example, unlike the simpler Drosophila germline stem cell niches, both the FSC and ISC niches produce multiple, partially redundant, niche signals, some of which activate pathways such as Wnt/Wingless, Hedgehog, and epidermal growth factor (EGF) that also regulate mammalian epithelial tissue renewal. Further study into these relatively new stem cell models will be of use in understanding both the specifics of epithelial regeneration and the diversity of mechanisms that regulate adult stem cells in general.
Asunto(s)
Drosophila/citología , Células Epiteliales/citología , Modelos Animales , Nicho de Células Madre , Células Madre/citología , Animales , RegeneraciónRESUMEN
Oxygen deprivation is a major cause of cellular damage and death. Here we demonstrate that Caenorhabditis elegans embryos, which can survive both in anoxia (<0.001 kPa O(2)) by entering into suspended animation and in mild hypoxia (0.25-1 kPa O(2)) through a hypoxia-inducible factor 1-mediated response, cannot survive in intermediate concentrations of oxygen, between 0.01 and 0.1 kPa O(2). Moreover, we show that carbon monoxide can protect C. elegans embryos against hypoxic damage in this sensitive range. Carbon monoxide can also rescue the hypoxia-sensitive mutant hif-1(ia04) from lethality in hypoxia. This work defines the oxygen tensions over which hypoxic damage occurs in C. elegans embryos and demonstrates that carbon monoxide can prevent this damage by inducing suspended animation.
Asunto(s)
Caenorhabditis elegans/fisiología , Monóxido de Carbono/farmacología , Factores de Transcripción , Animales , Caenorhabditis elegans/embriología , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Proteínas de Unión al ADN/metabolismo , Factor 1 Inducible por Hipoxia , Proteínas Nucleares/metabolismo , Oxígeno/toxicidad , Análisis de SupervivenciaRESUMEN
In response to environmental signals such as anoxia, many organisms enter a state of suspended animation, an extreme form of quiescence in which microscopically visible movement ceases. We have identified a gene, san-1, that is required for suspended animation in Caenorhabditis elegans embryos. We show that san-1 functions as a spindle checkpoint component in C. elegans. During anoxia-induced suspended animation, embryos lacking functional SAN-1 or a second spindle checkpoint component, MDF-2, failed to arrest the cell cycle, exhibited chromosome missegregation, and showed reduced viability. These data provide a model for how a dynamic biological process is arrested in suspended animation.