Excess dietary sugar impairs Drosophila adult stem cells via elevated reactive oxygen species-induced JNK signaling.

High-sugar diets (HSDs) often lead to obesity and type 2 diabetes, both metabolic syndromes associated with stem cell dysfunction. However, it is unclear whether excess dietary sugar affects stem cells. Here, we report that HSD impairs stem cell function in the intestine and ovaries of female Drosophila prior to the onset of insulin resistance, a hallmark of type 2 diabetes. Although 1 week of HSD leads to obesity, impaired oogenesis and altered lipid metabolism, insulin resistance does not occur. HSD increases glucose uptake by germline stem cells (GSCs) and triggers reactive oxygen species-induced JNK signaling, which reduces GSC proliferation. Removal of excess sugar from the diet reverses these HSD-induced phenomena. A similar phenomenon is found in intestinal stem cells (ISCs), except that HSD disrupts ISC maintenance and differentiation. Interestingly, tumor-like GSCs and ISCs are less responsive to HSD, which may be because of their dependence on glycolytic metabolism and high energy demand, respectively. This study suggests that excess dietary sugar induces oxidative stress and damages stem cells before insulin resistance develops, a mechanism that may also occur in higher organisms.

The RNA helicase Ddx52 functions as a growth switch in juvenile zebrafish.

Vertebrate animals usually display robust growth trajectories during juvenile stages, and reversible suspension of this growth momentum by a single genetic determinant has not been reported. Here, we report a single genetic factor that is essential for juvenile growth in zebrafish. Using a forward genetic screen, we recovered a temperature-sensitive allele, pan (after Peter Pan), that suspends whole-organism growth at juvenile stages. Remarkably, even after growth is halted for a full 8-week period, pan mutants are able to resume a robust growth trajectory after release from the restrictive temperature, eventually growing into fertile adults without apparent adverse phenotypes. Positional cloning and complementation assays revealed that pan encodes a probable ATP-dependent RNA helicase (DEAD-Box Helicase 52; ddx52) that maintains the level of 47S precursor ribosomal RNA. Furthermore, genetic silencing of ddx52 and pharmacological inhibition of bulk RNA transcription similarly suspend the growth of flies, zebrafish and mice. Our findings reveal evidence that safe, reversible pauses of juvenile growth can be mediated by targeting the activity of a single gene, and that its pausing mechanism has high evolutionary conservation.

WD40 protein Wuho controls germline homeostasis via TRIM-NHL tumor suppressor Mei-p26 in Drosophila.

WD40 proteins control many cellular processes via protein interactions. Drosophila Wuho (Wh, a WD40 protein) controls fertility, although the involved mechanisms are unclear. Here, we show that Wh promotion of Mei-p26 (a human TRIM32 ortholog) function maintains ovarian germ cell homeostasis. Wh and Mei-p26 are epistatically linked, with wh and mei-p26 mutants showing nearly identical phenotypes, including germline stem cell (GSC) loss, stem-cyst formation due to incomplete cytokinesis between GSCs and daughter cells, and overproliferation of GSC progeny. Mechanistically, Wh interacts with Mei-p26 in different cellular contexts to induce cell type-specific effects. In GSCs, Wh and Mei-p26 promote BMP stemness signaling for proper GSC division and maintenance. In GSC progeny, Wh and Mei-p26 silence nanos translation, downregulate a subset of microRNAs involved in germ cell differentiation and suppress ribosomal biogenesis via dMyc to limit germ cell mitosis. We also found that the human ortholog of Wh (WDR4) interacts with TRIM32 in human cells. Our results show that Wh is a regulator of Mei-p26 in Drosophila germ cells and suggest that the WD40-TRIM interaction may also control tissue homeostasis in other stem cell systems.

Diet regulates membrane extension and survival of niche escort cells for germline homeostasis via insulin signaling.

Diet is an important regulator of stem cell homeostasis; however, the underlying mechanisms of this regulation are not fully known. Here, we report that insulin signaling mediates dietary maintenance of Drosophila ovarian germline stem cells (GSCs) by promoting the extension of niche escort cell (EC) membranes to wrap around GSCs. This wrapping may facilitate the delivery of bone morphogenetic protein stemness factors from ECs in the niche to GSCs. In addition to the effects on GSCs, insulin signaling-mediated regulation of EC number and protrusions controls the division and growth of GSC progeny. The effects of insulin signaling on EC membrane extension are, at least in part, driven by enhanced translation of Failed axon connections (Fax) via Ribosomal protein S6 kinase. Fax is a membrane protein that may participate in Abelson tyrosine kinase-regulated cytoskeletal dynamics and is known to be involved in axon bundle formation. Therefore, we conclude that dietary cues stimulate insulin signaling in the niche to regulate EC cellular structure, probably via Fax-dependent cytoskeleton remodeling. This mechanism enhances intercellular contact and facilitates homeostatic interactions between somatic and germline cells in response to diet.

Molecular control of the female germline stem cell niche size in Drosophila.

Adult stem cells have a unique capacity to renew themselves and generate differentiated cells that are needed in the body. These cells are recruited and maintained by the surrounding microenvironment, known as the stem cell niche, during organ development. Thus, the stem cell niche is required for proper tissue homeostasis, and its dysregulation is associated with tumorigenesis and tissue degeneration. The identification of niche components and the mechanisms that regulate niche establishment and maintenance, however, are just beginning to be uncovered. Germline stem cells (GSCs) of the Drosophila ovary provide an excellent model for studying the stem cell niche in vivo because of their well-characterized cell biology and the availability of genetic tools. In this review, we introduce the ovarian GSC niche, and the key signaling pathways for niche precursor segregation, niche specification, and niche extracellular environment establishment and niche maintenance that are involved in regulating niche size during development and adulthood.

Snail controls proliferation of Drosophila ovarian epithelial follicle stem cells, independently of E-cadherin.

Epithelial stem cells undergo constant self-renewal and differentiation to maintain the homeostasis of epithelial tissues that undergo rapid turnover. Recent studies have shown that the epithelial-mesenchymal transition (EMT), which is primarily mediated by Snail via the suppression of E-cadherin, is able to generate cells with stem cell properties. However, the role of Snail in epithelial stem cells remains unclear. Here, we report that Snail directly controls proliferation of follicle stem cells (FSCs) in Drosophila females. Disruption of Snail expression in FSCs compromises their proliferation, but not their maintenance. Conversely, FSCs with excessive Snail expression display increased proliferation and lifespan, which is accompanied by a moderate decrease in the expression of E-cadherin (required for adhesion of FSCs to their niche) at the junction between their adjacent cells, indicating a conserved role of Snail in E-cadherin inhibition, which promote epithelial cell proliferation. Interestingly, a decrease in E-cadherin in snail-knock down FSCs does not restore the decreased proliferation of snail-knock down FSCs, suggesting that adhesion strength of FSCs to their niche is dispensable for Snail-mediated FSC division. Our results demonstrate that Snail controls epithelial stem cell division independently of its known role in the EMT, which contributes to induction of cancer stem cells.

Notch signaling mediates the age-associated decrease in adhesion of germline stem cells to the niche.

Stem cells have an innate ability to occupy their stem cell niche, which in turn, is optimized to house stem cells. Organ aging is associated with reduced stem cell occupancy in the niche, but the mechanisms involved are poorly understood. Here, we report that Notch signaling is increased with age in Drosophila female germline stem cells (GSCs), and this results in their removal from the niche. Clonal analysis revealed that GSCs with low levels of Notch signaling exhibit increased adhesiveness to the niche, thereby out-competing their neighbors with higher levels of Notch; adhesiveness is altered through regulation of E-cadherin expression. Experimental enhancement of Notch signaling in GSCs hastens their age-dependent loss from the niche, and such loss is at least partially mediated by Sex lethal. However, disruption of Notch signaling in GSCs does not delay GSC loss during aging, and nor does it affect BMP signaling, which promotes self-renewal of GSCs. Finally, we show that in contrast to GSCs, Notch activation in the niche (which maintains niche integrity, and thus mediates GSC retention) is reduced with age, indicating that Notch signaling regulates GSC niche occupancy both intrinsically and extrinsically. Our findings expose a novel role of Notch signaling in controlling GSC-niche adhesion in response to aging, and are also of relevance to metastatic cancer cells, in which Notch signaling suppresses cell adhesion.

Retinoic Acid Protects and Rescues the Development of Zebrafish Embryonic Retinal Photoreceptor Cells from Exposure to Paclobutrazol.

Paclobutrazol (PBZ) is a widely used fungicide that shows toxicity to aquatic embryos, probably through rain-wash. Here, we specifically focus on its toxic effect on eye development in zebrafish, as well as the role of retinoic acid (RA), a metabolite of vitamin A that controls proliferation and differentiation of retinal photoreceptor cells, in this toxicity. Embryos were exposed to PBZ with or without RA from 2 to 72 h post-fertilization (hpf), and PBZ-treated embryos (2-72 hpf) were exposed to RA for additional hours until 120 hpf. Eye size and histology were examined. Expression levels of gnat1 (rod photoreceptor marker), gnat2 (cone photoreceptor marker), aldehyde dehydrogenases (encoding key enzymes for RA synthesis), and phospho-histone H3 (an M-phase marker) in the eyes of control and treated embryos were examined. PBZ exposure dramatically reduces photoreceptor proliferation, thus resulting in a thinning of the photoreceptor cell layer and leading to a small eye. Co-treatment of PBZ with RA, or post-treatment of PBZ-treated embryos with RA, partially rescues photoreceptor cells, revealed by expression levels of marker proteins and by retinal cell proliferation. PBZ has strong embryonic toxicity to retinal photoreceptors, probably via suppressing the production of RA, with effects including impaired retinal cell division.

Tumor Suppressor Lzap Suppresses Wnt/ß-Catenin Signaling to Promote Zebrafish Embryonic Ventral Cell Fates via the Suppression of Inhibitory Phosphorylation of Glycogen Synthase Kinase 3.

Wnt/ß-catenin signaling controls various cell fates in metazoan development, and its dysregulation is often associated with cancer formation. However, regulations of this signaling pathway are not completely understood. Here, we report that Lzap, a tumor suppressor, controls nuclear translocation of ß-catenin. In zebrafish embryos disruption of lzap increases the expression of chordin (chd), which encodes a bone morphogenetic protein (BMP) antagonist that is localized in prospective dorsal cells and promotes dorsal fates. Consistently, lzap-deficient embryos with attenuated BMP signaling are dorsalized, which can be rescued by overexpression of zebrafish lzap or bmp2b or human LZAP. The expansion of chd expression in embryos lacking lzap is due to the accumulation of nuclear ß-catenin in ventral cells, in which ß-catenin is usually degraded. Furthermore, the activity of GSK3, a master regulator of ß-catenin degradation, is suppressed in lzap-deficient embryos via inhibitory phosphorylation. Finally, we also report that a similar regulatory axis is also likely to be present in a human tongue carcinoma cell line, SAS. Our results reveal that Lzap is a novel regulator of GSK3 for the maintenance of ventral cell properties and may prevent carcinogenesis via the regulation of ß-catenin degradation.

FOXO/Fringe is necessary for maintenance of the germline stem cell niche in response to insulin insufficiency.

The stem cell niche houses and regulates stem cells by providing both physical contact and local factors that regulate stem cell identity. The stem cell niche also plays a role in integrating niche-local and systemic signals, thereby ensuring that the balance of stem cells meets the needs of the organism. However, it is not clear how these signals are merged within the niche. Nutrient-sensing insulin/FOXO signaling has been previously shown to directly control Notch activation in the Drosophila female germline stem cell (GSC) niche, which maintains the niche and GSC identity. Here, we demonstrate that FOXO directly activates transcription of fringe, a gene encoding a glycosyltransferase that modulates Notch glycosylation. Fringe facilitates Notch inactivation in the GSC niche when insulin signaling is low. We also show that the Notch ligand predominantly involved is GSC niche-derived Delta. These results reveal that FOXO-mediated regulation of fringe links the insulin and Notch signaling pathways in the GSC niche in response to nutrition, and emphasize that stem cells are regulated by complex interactions between niche-local and systemic signals.

Specific roles of Target of rapamycin in the control of stem cells and their progeny in the Drosophila ovary.

Stem cells depend on intrinsic and local factors to maintain their identity and activity, but they also sense and respond to changing external conditions. We previously showed that germline stem cells (GSCs) and follicle stem cells (FSCs) in the Drosophila ovary respond to diet via insulin signals. Insulin signals directly modulate the GSC cell cycle at the G2 phase, but additional unknown dietary mediators control both G1 and G2. Target of rapamycin, or TOR, is part of a highly conserved nutrient-sensing pathway affecting growth, proliferation, survival and fertility. Here, we show that optimal TOR activity maintains GSCs but does not play a major role in FSC maintenance, suggesting differential regulation of GSCs versus FSCs. TOR promotes GSC proliferation via G2 but independently of insulin signaling, and TOR is required for the proliferation, growth and survival of differentiating germ cells. We also report that TOR controls the proliferation of FSCs but not of their differentiating progeny. Instead, TOR controls follicle cell number by promoting survival, independently of either the apoptotic or autophagic pathways. These results uncover specific TOR functions in the control of stem cells versus their differentiating progeny, and reveal parallels between Drosophila and mammalian follicle growth.

Inhibition of acetyl-CoA carboxylase impaired tubulin palmitoylation and induced spindle abnormalities.

Tubulin s-palmitoylation involves the thioesterification of a cysteine residue in tubulin with palmitate. The palmitate moiety is produced by the fatty acid synthesis pathway, which is rate-limited by acetyl-CoA carboxylase (ACC). While it is known that ACC is phosphorylated at serine 79 (pSer79) by AMPK and accumulates at the spindle pole (SP) during mitosis, a functional role for tubulin palmitoylation during mitosis has not been identified. In this study, we found that modulating pSer79-ACC level at the SP using AMPK agonist and inhibitor induced spindle defects. Loss of ACC function induced spindle abnormalities in cell lines and in germ cells of the Drosophila germarium, and palmitic acid (PA) rescued the spindle defects in the cell line treated transiently with the ACC inhibitor, TOFA. Furthermore, inhibition of protein palmitoylating or depalmitoylating enzymes also induced spindle defects. Together, these data suggested that precisely regulated cellular palmitate level and protein palmitoylation may be required for accurate spindle assembly. We then showed that tubulin was largely palmitoylated in interphase cells but less palmitoylated in mitotic cells. TOFA treatment diminished tubulin palmitoylation at doses that disrupt microtubule (MT) instability and cause spindle defects. Moreover, spindle MTs comprised of α-tubulins mutated at the reported palmitoylation site exhibited disrupted dynamic instability. We also found that TOFA enhanced the MT-targeting drug-induced spindle abnormalities and cytotoxicity. Thus, our study reveals that precise regulation of ACC during mitosis impacts tubulin palmitoylation to delicately control MT dynamic instability and spindle assembly, thereby safeguarding nuclear and cell division.

Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands.

Adult stem cells reside in specialized microenvironments, or niches, that are essential for their function in vivo. Stem cells are physically attached to the niche, which provides secreted factors that promote their self-renewal and proliferation. Despite intense research on the role of the niche in regulating stem cell function, much less is known about how the niche itself is controlled. We previously showed that insulin signals directly stimulate germline stem cell (GSC) division and indirectly promote GSC maintenance via the niche in Drosophila. Insulin-like peptides are required for maintenance of cap cells (a major component of the niche) via modulation of Notch signaling, and they also control attachment of GSCs to cap cells and E-cadherin levels at the cap cell-GSC junction. Here, we further dissect the molecular and cellular mechanisms underlying these processes. We show that insulin and Notch ligands directly stimulate cap cells to maintain their numbers and indirectly promote GSC maintenance. We also report that insulin signaling, via phosphoinositide 3-kinase and FOXO, intrinsically controls the competence of cap cells to respond to Notch ligands and thereby be maintained. Contrary to a previous report, we also find that Notch ligands originated in GSCs are not required either for Notch activation in the GSC niche, or for cap cell or GSC maintenance. Instead, the niche itself produces ligands that activate Notch signaling within cap cells, promoting stability of the GSC niche. Finally, insulin signals control cap cell-GSC attachment independently of their role in Notch signaling. These results are potentially relevant to many systems in which Notch signaling modulates stem cells and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function.

Pregnenolone stabilizes microtubules and promotes zebrafish embryonic cell movement.

Embryonic cell movement is essential for morphogenesis and the establishment of body shapes, but little is known about its mechanism. Here we report that pregnenolone, which is produced from cholesterol by the steroidogenic enzyme Cyp11a1 (cholesterol side-chain cleavage enzyme, P450scc), functions in promoting cell migration during epiboly. Epiboly is a process in which embryonic cells spread from the animal pole to cover the underlying yolk. During epiboly, cyp11a1 is expressed in an extra-embryonic yolk syncytial layer. Reducing cyp11a1 expression in zebrafish using antisense morpholino oligonucleotides did not perturb cell fates, but caused epibolic delay. This epibolic defect was partially rescued by the injection of cyp11a1 RNA or the supplementation of pregnenolone. We show that the epibolic delay is accompanied by a decrease in the level of polymerized microtubules, and that pregnenolone can rescue this microtubule defect. Our results indicate that pregnenolone preserves microtubule abundance and promotes cell movement during epiboly.

Insulin levels control female germline stem cell maintenance via the niche in Drosophila.

Stem cell maintenance depends on local signals provided by specialized microenvironments, or niches, in which they reside. The potential role of systemic factors in stem cell maintenance, however, has remained largely unexplored. Here, we show that insulin signaling integrates the effects of diet and age on germline stem cell (GSC) maintenance through the dual regulation of cap cell number (via Notch signaling) and cap cell-GSC interaction (via E-cadherin) and that the normal process of GSC and niche cell loss that occurs with age can be suppressed by increased levels of insulin-like peptides. These results underscore the importance of systemic factors for the regulation of stem cell niches and, thereby, of stem cell numbers.

Expansion Microscopy-based imaging for visualization of mitochondria in Drosophila ovarian germline stem cells.

Recent studies have shown that mitochondrial morphology can modulate organelle function and greatly affect stem cell behavior, thus affecting tissue homeostasis. As such, we previously showed that the accumulation of fragmented mitochondria in aged Drosophila ovarian germline stem cells (GSCs) contributes to age-dependent GSC loss. However, standard immunofluorescence methods to examine mitochondrial morphology yield images with insufficient resolution for rigorous analysis, while 3-dimensional electron microscopy examination of mitochondrial morphology is labor intensive and allows only limited sampling of mitochondria. To overcome these issues, we utilized the expansion microscopy technique to expand GSC samples by 4-fold in combination with mitochondrial immunofluorescence labeling. Here, we present a simple, inexpensive method for nanoscale optical imaging of mitochondria in the germline. This protocol may be beneficial for studies that require visualization of mitochondria or other fine subcellular structures in the Drosophila ovary.

Canonical Wnt Signaling Promotes Formation of Somatic Permeability Barrier for Proper Germ Cell Differentiation.

Morphogen-mediated signaling is critical for proper organ development and stem cell function, and well-characterized mechanisms spatiotemporally limit the expression of ligands, receptors, and ligand-binding cell-surface glypicans. Here, we show that in the developing Drosophila ovary, canonical Wnt signaling promotes the formation of somatic escort cells (ECs) and their protrusions, which establish a physical permeability barrier to define morphogen territories for proper germ cell differentiation. The protrusions shield germ cells from Dpp and Wingless morphogens produced by the germline stem cell (GSC) niche and normally only received by GSCs. Genetic disruption of EC protrusions allows GSC progeny to also receive Dpp and Wingless, which subsequently disrupt germ cell differentiation. Our results reveal a role for canonical Wnt signaling in specifying the ovarian somatic cells necessary for germ cell differentiation. Additionally, we demonstrate the morphogen-limiting function of this physical permeability barrier, which may be a common mechanism in other organs across species.

Zebrafish monosex population reveals female dominance in sex determination and earliest events of gonad differentiation.

The zebrafish is a popular model for genetic analysis and its sex differentiation has been the focus of attention for breeding purposes. Despite numerous efforts, very little is known about the mechanism of zebrafish sex determination. The lack of discernible sex chromosomes and the difficulty of distinguishing the sex of juvenile fish are two major obstacles that hamper the progress in such studies. To alleviate these problems, we have developed a scheme involving methyltestosterone treatment followed by natural mating to generate fish with predictable sex trait. Female F1 fish that gave rise to all-female offspring were generated. This predictable sex trait enables characterization of gonadal development in juvenile fish by histological examination and gene expression analysis. We found the first sign of zebrafish sex differentiation to be ovarian gonocyte proliferation and differentiation at 10 to 12 days post-fertilization (dpf). Somatic genes were expressed indifferently at 10 to 17 dpf, and then became sexually dimorphic at three weeks. This result indicates clear distinction of male and female gonads derived independently from primordial gonads. We classified the earliest stages of zebrafish sex determination into the initial preparation followed by female germ cell growth, oocyte differentiation, and somatic differentiation. Our genetic selection scheme matches the prediction that female-dominant genetic factors are required to determine zebrafish sex.

Regulation of adult female germline stem cells by nutrient-responsive signaling.

Insect oogenesis is greatly affected by nutrient availability. When nutrients are abundant, oocytes are rapidly generated, but the process is slowed to conserve energy under nutrient-deficient conditions. To properly allocate limited resources toward oogenesis, systemic factors coordinate the behavioral response of ovarian germline stem cells (GSCs) to nutritional inputs by acting on the GSC itself, GSC supporting cells (the niche), or the adipose tissue surrounding the ovary. In this review, we describe current knowledge of the Drosophila ovarian GSC-niche-adipocyte system and major nutrient sensing pathways (insulin/IGF signaling, TOR signaling, and GCN2-dependent amino acid sensing) that intrinsically or extrinsically regulate GSC responses to nutrient signals.