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
A major goal in the study of adult stem cells is to understand how cell fates are specified at the proper time and place to facilitate tissue homeostasis. Here, we found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles in the Drosophila ovarian epithelial follicle stem cell (FSC) lineage. First, Ben is part of the JNK signaling pathway, and we found that it, as well as other JNK pathway genes, are essential for differentiation of FSC daughter cells. Our data suggest that JNK signaling promotes differentiation by suppressing the activation of the EGFR effector, ERK. Also, we found that loss of ben, but not the JNK kinase hemipterous, resulted in an upregulation of hedgehog signaling, increased proliferation and increased niche competition. Lastly, we demonstrate that the hypercompetition phenotype caused by loss of ben is suppressed by decreasing the rate of proliferation or knockdown of the hedgehog pathway effector, Smoothened (Smo). Taken together, our findings reveal a new layer of regulation in which a single gene influences cell signaling at multiple stages of differentiation in the early FSC lineage.
Asunto(s)
Proteínas de Drosophila/genética , Receptores ErbB/genética , Proteínas Hedgehog/genética , Folículo Ovárico/crecimiento & desarrollo , Receptores de Péptidos de Invertebrados/genética , Receptor Smoothened/genética , Enzimas Ubiquitina-Conjugadoras/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Proliferación Celular/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Células Epiteliales/citología , Femenino , Sistema de Señalización de MAP Quinasas/genética , Folículo Ovárico/metabolismo , Nicho de Células Madre/genética , Células Madre/citologíaRESUMEN
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
Understanding how cell fate decisions are regulated is a fundamental goal of developmental and stem cell biology. Most studies on the control of cell fate decisions address the contributions of changes in transcriptional programming, epigenetic modifications, and biochemical differentiation cues. However, recent studies have found that other aspects of cell biology also make important contributions to regulating cell fate decisions. These cues can have a permissive or instructive role and are integrated into the larger network of signaling, functioning both upstream and downstream of developmental signaling pathways. Here, we summarize recent insights into how cell fate decisions are influenced by four aspects of cell biology: metabolism, reactive oxygen species (ROS), intracellular pH (pHi), and cell morphology. For each topic, we discuss how these cell biological cues interact with each other and with protein-based mechanisms for changing gene transcription. In addition, we highlight several questions that remain unanswered in these exciting and relatively new areas of the field.
Asunto(s)
Diferenciación Celular/genética , Transducción de Señal/genética , Células Madre/metabolismo , Biología Celular , Concentración de Iones de Hidrógeno , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/fisiología , Transcripción GenéticaRESUMEN
In the germarium of the Drosophila ovary, germline cysts are encapsulated one at a time by a follicular epithelium derived from two follicle stem cells (FSCs). Ovaries in flies mutant for the serine/threonine kinase Pak exhibit a novel phenotype, in which two side-by-side cysts are encapsulated at a time, generating paired egg chambers. This striking phenotype originates in the pupal ovary, where the developing germarium is shaped by the basal stalk, a stack of cells formed by cell intercalation. The process of basal stalk formation is not well understood, and we provide evidence that the cell intercalation is driven by actomyosin contractility of DE-Cadherin-adhered cells, leading to a column of disk-shaped cells exhibiting a novel radial cell polarity. Cell intercalation fails in Pak mutant ovaries, leading to abnormally wide basal stalks and consequently wide germaria with side-by-side cysts. We present evidence that Pak mutant germaria have extra FSCs, and we propose that contact of a germline cyst with the basal stalk in the pupal ovary contributes to FSC niche formation. The wide basal stalk in Pak mutants enables the formation of extra FSC niches which are mispositioned and yet functional, indicating that the FSC niche can be established in diverse locations.
Asunto(s)
Polaridad Celular , Drosophila melanogaster/citología , Drosophila melanogaster/enzimología , Folículo Ovárico/citología , Folículo Ovárico/enzimología , Nicho de Células Madre , Animales , Proteínas de Drosophila , Drosophila melanogaster/anatomía & histología , Femenino , Modelos Biológicos , Mutación/genética , Óvulo/citología , Óvulo/metabolismo , Fenotipo , Quinasas p21 ActivadasRESUMEN
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
The production of eggs in the Drosophila ovary requires complex interactions between multiple cell types that coexist within the same solid tissue. This cellular heterogeneity makes the ovary a rich subject of study, but also makes it challenging to identify transcriptional differences between individual cell types using methods such as bulk RNA sequencing. The development of single-cell RNA sequencing (scRNA-seq) techniques addresses this limitation by providing an avenue to profile genetic and functional heterogeneity at a cellular resolution. Here, we describe the isolation and preparation of the Drosophila ovary for scRNA-seq. This protocol emphasizes a short preparation time, high cell viability, prevention of RNA-degradation, and reduction of technical variation to achieve highly reproducible single-cell profiles.
Asunto(s)
Drosophila , Análisis de la Célula Individual , Animales , Femenino , Drosophila/genética , Análisis de la Célula Individual/métodos , Análisis de Secuencia de ARN/métodos , Ovario/metabolismo , Secuencia de Bases , ARN/genética , ARN/metabolismo , Perfilación de la Expresión Génica/métodosRESUMEN
Intracellular pH dynamics is increasingly recognized to regulate myriad cell behaviors. We report a finding that intracellular pH dynamics also regulates adult stem cell lineage specification. We identify an intracellular pH gradient in mouse small intestinal crypts, lowest in crypt stem cells and increasing along the crypt column. Disrupting this gradient by inhibiting H+ efflux by Na+/H+ exchanger 1 abolishes crypt budding and blocks differentiation of Paneth cells, which are rescued with exogenous WNT. Using single-cell RNA sequencing and lineage tracing we demonstrate that intracellular pH dynamics acts downstream of ATOH1, with increased pH promoting differentiation toward the secretory lineage. Our findings indicate that an increase in pH is required for the lineage specification that contributes to crypt maintenance, establishing a role for intracellular pH dynamics in cell fate decisions within an adult stem cell lineage.
Asunto(s)
Intestinos , Células Madre , Ratones , Animales , Linaje de la Célula , Diferenciación Celular/fisiología , Concentración de Iones de Hidrógeno , Mucosa IntestinalRESUMEN
A central factor in the maintenance of tissue integrity is the response of stem cells to variations in the levels of niche signals. In the gut, intestinal stem cells (ISCs) depend on Wnt ligands for self-renewal and proliferation. Transient increases in Wnt signaling promote regeneration after injury or in inflammatory bowel diseases, whereas constitutive activation of this pathway leads to colorectal cancer. Here, we report that Discs large 1 (Dlg1), although dispensable for polarity and cellular turnover during intestinal homeostasis, is required for ISC survival in the context of increased Wnt signaling. RNA sequencing (RNA-seq) and genetic mouse models demonstrated that DLG1 regulates the cellular response to increased canonical Wnt ligands. This occurs via the transcriptional regulation of Arhgap31, a GTPase-activating protein that deactivates CDC42, an effector of the non-canonical Wnt pathway. These findings reveal a DLG1-ARHGAP31-CDC42 axis that is essential for the ISC response to increased niche Wnt signaling.
Asunto(s)
Mucosa Intestinal , Vía de Señalización Wnt , Animales , Ratones , Proliferación Celular , Proteínas Activadoras de GTPasa/metabolismo , Mucosa Intestinal/metabolismo , Intestinos , Nicho de Células Madre , Células Madre , Vía de Señalización Wnt/genéticaRESUMEN
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 follicle stem cell (FSC) lineage in the Drosophila ovary is a highly informative model of in vivo epithelial stem cell biology. Studies over the past 30 years have identified roles for every major signaling pathway in the early FSC lineage. These pathways regulate a wide variety of cell behaviors, including self-renewal, proliferation, survival and differentiation. Studies of cell signaling in the follicle epithelium have provided new insights into how these cell behaviors are coordinated within an epithelial stem cell lineage and how signaling pathways interact with each other in the native, in vivo context of a living tissue. Here, we review these studies, with a particular focus on how these pathways specify differences between the FSCs and their daughter cells. We also describe common themes that have emerged from these studies, and highlight new research directions that have been made possible by the detailed understanding of the follicle epithelium.
Asunto(s)
Folículo Ovárico/citología , Transducción de Señal , Células Madre/metabolismo , Animales , Drosophila/crecimiento & desarrollo , Femenino , Folículo Ovárico/metabolismo , Nicho de Células Madre , Células Madre/citologíaRESUMEN
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
Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and differentiation. Typically, pHi dynamics are determined in cultured cells, which are amenable to measuring and experimentally manipulating pHi. However, the recent development of new tools and methodologies has made it possible to study pHi dynamics within intact, live tissue. For Drosophila research, one important development was the generation of a transgenic line carrying a pHi biosensor, mCherry::pHluorin. Here, we describe a protocol that we routinely use for imaging live Drosophila ovarioles to measure pHi in the epithelial follicle stem cell (FSC) lineage in mCherry::pHluorin transgenic wild type lines; however, the methods described here can be easily adapted for other tissues, including the wing discs and eye epithelium. We describe techniques for expressing mCherry::pHluorin in the FSC lineage, maintaining ovarian tissue during live imaging, and acquiring and analyzing images to obtain pHi values.