methods-for-imaging-intracellular-ph-follicle-stem-cell-lineage-live

Methods for Imaging Intracellular pH of the Follicle Stem Cell Lineage in Live <em>Drosophila</em> Ovarian Tissue

Changes in intracellular pH (pHi) play important roles in the regulation of many cellular functions, including metabolism, proliferation, and ...

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.

Dephosphorylation of cell cycle-regulated proteins correlates with anoxia-induced suspended animation in Caenorhabditis elegans.

Einige Metazoen haben die Kapazit&auml;t, schweren Sauerstoffmangel zu &uuml;berleben entwickelt. Die Fadenw&uuml;rmer, Caenorhabditis Elegans, ausgesetzt Anoxie (0 kPa, 0 %, die O(2)) in einem wiederherstellbaren Zustand der Leblosigkeit in allen Phasen des Lebenszyklus tritt. D. h. alle mikroskopisch wahrnehmbaren Bewegung h&ouml;rt, einschlie&szlig;lich Zellteilung, entwicklungsbedingte Fortschreiten, F&uuml;tterung und Motilit&auml;t. Um zu verstehen, Leblosigkeit, verglichen wir Sauerstoff beraubt Embryonen zu unbehandelten Embryonen in Wildtyp und Hif-1-Mutanten. Wir fanden, dass Hif-1 Mutanten Anoxie &uuml;berleben, darauf hindeutet, dass die Mechanismen zum Anoxie &Uuml;berleben von denen f&uuml;r Hypoxie erforderlich sind. Pr&uuml;fung der Wildtyp Embryonen ausgesetzt Anoxie zeigen diese Blastomeren Verhaftung in Interphase, Prophase, Metaphase, und Telophase aber nicht Anaphase. Analyse des energetischen Zustands der anoxischen Embryonen angegeben eine reversible Depression in der ATP zu ADP-Verh&auml;ltnis. Angesichts der Tatsache, dass eine Abnahme der ATP Konzentrationen wahrscheinlich eine Vielzahl zellul&auml;rer Prozesse, einschlie&szlig;lich der Signaltransduktion, eingreift, verglichen wir den Zustand der Phosphorylierung von mehreren Proteinen anoxischen Embryonen und Normoxic Embryonen. Wir fanden, dass die Phosphorylierung des Histon H3 und Zellzyklus-regulierten Proteine von der MPM-2-Antik&ouml;rper erkannt nicht nachweisbar in anoxischen Embryonen. So dephosphorylation der spezifischen Proteine korrelieren mit der Einrichtung und die Wartung einen Zustand induziert durch Anoxie Suspended Animation.

Dephosphorylierung von Zellzyklus-regulierten Proteinen korreliert mit induziert durch Anoxie Leblosigkeit in Caenorhabditis Elegans.

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.

Suspended animation in C. elegans requires the spindle checkpoint.

Reaktion auf Umweltsignale wie Anoxie geben Sie viele Organismen einen Status Suspended Animation, eine extreme Form der Ruhe in die mikroskopisch sichtbare Bewegung wird aufgehoben. Wir haben ein Gen, San-1, identifiziert, die f&uuml;r die Leblosigkeit in Caenorhabditis Elegans Embryonen erforderlich ist. Wir zeigen, dass San-1 als eine Spindel Pr&uuml;fpunkt Komponente in C. Elegans fungiert. W&auml;hrend induziert durch Anoxie Suspended Animation Embryonen ohne funktionale SAN-1 oder eine zweite Spindel Pr&uuml;fpunkt-Komponente, MDF-2, gelang es, Zellzyklus, festzunehmen Chromosom Missegregation ausgestellt, und zeigten reduzierte Lebensf&auml;higkeit. Diese Daten stellen ein Modell f&uuml;r wie ein dynamischer biologischer Prozess in Leblosigkeit verhaftet ist.

Leblosigkeit in C. Elegans erfordert der Spindel-Checkpoint.

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.

Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans.

Buying time in suspended animation.

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.

Breaking out of the mold: diversity within adult stem cells and their niches.

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.

The carnegie protein trap library: a versatile tool for Drosophila developmental studies.

Adult epithelial stem cells are thought to reside in specific niches, where they are maintained by adhesion to stromal cells and by intercellular signals. In niches that harbor multiple adjacent stem cells, such as those maintaining Drosophila germ cells, lost stem cells are replaced by division of neighboring stem cells or reversion of transit cells. We have characterized the Drosophila follicle stem cell (FSC) niche as a model of the epithelial niche to learn whether nonneighboring cells can also generate stem cell replacements. Exactly two stroma-free FSC niches holding single FSCs are located in fixed locations on opposite edges of the Drosophila ovariole. FSC daughters regularly migrate across the width of the ovariole to the other niche before proliferating and contributing to the follicle cell monolayer. Crossmigrating FSC daughters compete with the resident FSC for niche occupancy and are the source of replacement FSCs. The ability of stem cell daughters to target a distant niche and displace its resident stem cell suggests that precancerous mutations might spread from niche to niche within stem cell-based tissues.

An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement.

Though much has been learned about the process of ovarian follicle maturation through studies of oogenesis in both vertebrate and invertebrate systems, less is known about how follicles form initially. In Drosophila, two somatic follicle stem cells (FSCs) in each ovariole give rise to all polar cells, stalk cells, and main body cells needed to form each follicle. We show that one daughter from each FSC founds most follicles but that cell type specification is independent of cell lineage, in contrast to previous claims of an early polar/stalk lineage restriction. Instead, key intercellular signals begin early and guide cell behavior. An initial Notch signal from germ cells is required for FSC daughters to migrate across the ovariole and on occasion to replace the opposite stem cell. Both anterior and posterior polar cells arise in region 2b at a time when approximately 16 cells surround the cyst. Later, during budding, stalk cells and additional polar cells are specified in a process that frequently transfers posterior follicle cells onto the anterior surface of the next older follicle. These studies provide new insight into the mechanisms that underlie stem cell replacement and follicle formation during Drosophila oogenesis.

Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary.

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.

Drosophila models of epithelial stem cells and their niches.

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.

A dynamic population of stromal cells contributes to the follicle stem cell niche in the Drosophila ovary.

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.

Basolateral junction proteins regulate competition for the follicle stem cell niche in the Drosophila ovary.

The Drosophila melanogaster genetic tool box includes many stocks for generating genetically mosaic tissue in which a clone of cells, related by lineage, contain a common genetic alteration. These tools have made it possible to study the postembryonic function of essential genes and to better understand how individual cells interact within intact tissues. We have screened through 201 enhancer-trap flippase lines to identify lines that produce useful clone patterns in the adult ovary. We found that approximately 70% of the lines produced clones that were present in the adult ovary and that many ovarian cell types were represented among the different clone patterns produced by these lines. We have also identified and further characterized five particularly useful enhancer-trap flippase lines. These lines make it possible to generate clones specifically in germ cells, escort cells, prefollicle cells, or terminal filament cells. In addition, we have found that chickadee is specifically upregulated in the posterior escort cells, follicle stem cells, and prefollicle cells that comprise the follicle stem cell niche region. Collectively, these studies provide several new tools for genetic mosaic analysis in the Drosophila ovary.

Enhancer-trap flippase lines for clonal analysis in the Drosophila ovary.

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.

EGFR signaling promotes self-renewal through the establishment of cell polarity in Drosophila follicle stem cells.

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.

A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary.

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.

Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation.

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.

Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche.

The process of selecting for cellular fitness through competition plays a critical role in both development and disease. The germarium, a structure at the tip of the ovariole of a Drosophila ovary, contains two follicle stem cells (FSCs) that undergo neutral competition for the stem cell niche. Using the FSCs as a model, we performed a genetic screen through a collection of 126 mutants in essential genes on the X chromosome to identify candidates that increase or decrease competition for the FSC niche. We identified ∼55 and 6% of the mutations screened as putative FSC hypo- or hyper-competitors, respectively. We found that a large majority of mutations in vesicle trafficking genes (11 out of the 13 in the collection of mutants) are candidate hypo-competition alleles, and we confirmed the hypo-competition phenotype for four of these alleles. We also show that Sec16 and another COPII vesicle trafficking component, Sar1, are required for follicle cell differentiation. Lastly, we demonstrate that, although some components of vesicle trafficking are also required for neutral competition in the cyst stem cells of the testis, there are important tissue-specific differences. Our results demonstrate a critical role for vesicle trafficking in stem cell niche competition and differentiation, and we identify a number of putative candidates for further exploration.

Article	Year
Dephosphorylation of cell cycle-regulated proteins correlates with anoxia-induced suspended animation in Caenorhabditis elegans. Molecular biology of the cell\| PubMed ID: 12006646	2002
Suspended animation in C. elegans requires the spindle checkpoint. Science (New York, N.Y.)\| PubMed ID: 14605367	2003
Carbon monoxide-induced suspended animation protects against hypoxic damage in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 15184665	2004
Buying time in suspended animation. Scientific American\| PubMed ID: 15934652	2005
Breaking out of the mold: diversity within adult stem cells and their niches. Current opinion in genetics & development\| PubMed ID: 16919446	2006
The carnegie protein trap library: a versatile tool for Drosophila developmental studies. Genetics\| PubMed ID: 17194782	2007
An epithelial niche in the Drosophila ovary undergoes long-range stem cell replacement. Cell stem cell\| PubMed ID: 18371362	2007
Regulation of epithelial stem cell replacement and follicle formation in the Drosophila ovary. Genetics\| PubMed ID: 19948890	2010
Drosophila models of epithelial stem cells and their niches. Wiley interdisciplinary reviews. Developmental biology\| PubMed ID: 23801493	2012
A dynamic population of stromal cells contributes to the follicle stem cell niche in the Drosophila ovary. Development (Cambridge, England)\| PubMed ID: 24131631	2013
Basolateral junction proteins regulate competition for the follicle stem cell niche in the Drosophila ovary. PloS one\| PubMed ID: 24991805	2014
Enhancer-trap flippase lines for clonal analysis in the Drosophila ovary. G3 (Bethesda, Md.)\| PubMed ID: 25024257	2014
EGFR signaling promotes self-renewal through the establishment of cell polarity in Drosophila follicle stem cells. eLife\| PubMed ID: 25437306	2014
A Pak-regulated cell intercalation event leading to a novel radial cell polarity is involved in positioning of the follicle stem cell niche in the Drosophila ovary. Development (Cambridge, England)\| PubMed ID: 25516970	2015
Increased intracellular pH is necessary for adult epithelial and embryonic stem cell differentiation. The Journal of cell biology\| PubMed ID: 27821494	2016
Phosphorylated Groucho delays differentiation in the follicle stem cell lineage by providing a molecular memory of EGFR signaling in the niche. Development (Cambridge, England)\| PubMed ID: 27836963	2016
Neutral Competition for Drosophila Follicle and Cyst Stem Cell Niches Requires Vesicle Trafficking Genes. Genetics\| PubMed ID: 28512187	2017

Todd Nystul

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