Genetic Mosaic Analysis of Stem Cell Lineages in the Drosophila Ovary

Laws, Kaitlin M.; Drummond‐Barbosa, Daniela

doi:10.1007/978-1-4939-2851-4_4

Cited by 26 publications

(28 citation statements)

References 22 publications

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“…To quantify GSC loss, we analyzed all germaria containing GFP-negative cystoblasts and/or cysts, and calculated the percentage of germaria that no longer contained GFP-negative GSCs (i.e. “GSC loss events”), as described (Laws and Drummond-Barbosa, 2015). To measure GSC and follicle cell proliferation, flies were maintained on a rich diet for 4 days following the last heat shock, then either switched to a poor diet or maintained on a rich diet for an additional 3 days.…”

Section: Methodsmentioning

confidence: 99%

“…To measure GSC and follicle cell proliferation, flies were maintained on a rich diet for 4 days following the last heat shock, then either switched to a poor diet or maintained on a rich diet for an additional 3 days. The frequency of EdU-positive, GFP-negative GSCs or follicle cells was calculated as a percentage of the total number of GFP-negative GSCs or follicle cells, respectively, for multiple single plane images of follicle epithelia, as described (Laws and Drummond-Barbosa, 2015). …”

Section: Methodsmentioning

confidence: 99%

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AMP-activated protein kinase has diet-dependent and -independent roles in Drosophila oogenesis

Laws

Drummond‐Barbosa

2016

Developmental Biology

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Multiple aspects of organismal physiology influence the number and activity of stem cells and their progeny, including nutritional status. Previous studies demonstrated that Drosophila germline stem cells (GSCs), follicle stem cells (FSCs), and their progeny sense and respond to diet via complex mechanisms involving many systemic and local signals. AMP-activated protein kinase, or AMPK, is a highly conserved regulator of energy homeostasis known to be activated under low cellular energy conditions; however, its role in the ovarian response to diet has not been investigated. Here, we describe nutrient-dependent and -independent requirements for AMPK in Drosophila oogenesis. We found that AMPK is cell autonomously required for the slow down in GSC and follicle cell proliferation that occurs on a poor diet. Similarly, AMPK activity is necessary in the germline for the degeneration of vitellogenic stages in response to nutrient deprivation. In contrast, AMPK activity is not required within the germline to modulate its growth. Instead, AMPK acts in follicle cells to negatively regulate their growth and proliferation, thereby indirectly limiting the size of the underlying germline cyst within developing follicles. Paradoxically, AMPK is required for GSC maintenance in well-fed flies (when AMPK activity is presumably at its lowest), suggesting potentially important roles for basal AMPK activity in specific cell types. Finally, we identified a nutrient-independent, developmental role for AMPK in cyst encapsulation by follicle cells. These results uncover specific AMPK requirements in multiple cell types in the ovary and suggest that AMPK can function outside of its canonical nutrient-sensing role in specific developmental contexts.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

AMP-activated protein kinase has diet-dependent and -independent roles in Drosophila oogenesis

Laws

Drummond‐Barbosa

2016

Developmental Biology

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“…Immunofluorescence analysis in targeted RNAi knockdown ovaries can be complemented by molecular analysis of the ovary, including gene expression (RT-PCR, RNA-seq, or in situ hybridization) or protein analysis (Western blotting). All phenotypes derived from targeted RNAi should be validated using other gene loss-of-function models, including the phenotypic assessment of genetic mutants by mosaic clonal analysis [45, 48]. …”

Section: Methodsmentioning

confidence: 99%

RNAi-Based Techniques for the Analysis of Gene Function in Drosophila Germline Stem Cells

Blake

Finger

Hardy

et al. 2017

Methods in Molecular Biology

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Elucidating the full repertoire of molecular mechanisms that promote stem cell maintenance requires sophisticated techniques for identifying and characterizing gene function in stem cells in their native environment. Ovarian germline stem cells in the fruit fly, Drosophila melanogaster, are an ideal model to study the complex molecular mechanisms driving stem cell function in vivo. A variety of new genetic tools make RNAi a useful complement to traditional genetic mutants for the investigation of the molecular mechanisms guiding ovarian germline stem cell function. Here, we provide a detailed guide for using targeted RNAi knockdown for the discovery of gene function in ovarian germline stem cells and their progeny.

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“…Other genetic tools are described in FlyBase. Genetic mosaics were generated using FLP/FRT-mediated recombination in 1-3 day old females carrying a mutant allele in trans to a wild-type allele (linked to a Ubi-GFP or NLS-RFP marker) on homologous FRT arms with a hs-FLP transgene, as previously described (Hinnant et al, 2017;Laws and Drummond-Barbosa, 2015). Flies were heat shocked at 37°C twice per day 6-8 hours apart for 3 days, then incubated at 25°C on standard media supplemented first with dry yeast, then with wet yeast 3 days prior to dissection.…”

Section: Genetic Mosaic Generation and Stem Cell Analysesmentioning

confidence: 99%

“…GSCs were identified by the location of their fusomes adjacent to the cap cells (de Cuevas and Spradling, 1998). GSC loss was measured by the number of germaria that contain a GFP-negative cyst (generated from the original GFP-negative stem cell) but lack a GFP-negative GSC, compared to the total number of germaria containing a germline clone (Laws and Drummond-Barbosa, 2015). Results were analyzed by Chi-square tests using Microsoft Excel.…”

Section: Genetic Mosaic Generation and Stem Cell Analysesmentioning

confidence: 99%

Ecdysone signaling promotes expression of multifunctional RNA binding proteins essential for ovarian germline stem cell self-renewal inDrosophila

Ables

2018

Preprint

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RUNNING TITLEhnRNPs promote stem cell self-renewal SUMMARY STATEMENTEcdysone signaling promotes expression of heterogeneous ribonucleoproteins that modulate BMP-dependent germline stem cell self-renewal in the Drosophila ovary. ABSTRACTSteroid hormones promote stem cell self-renewal in many tissues; however, the molecular mechanisms by which hormone signaling is integrated with niche-derived signals are largely uncharacterized. In the Drosophila ovary, the steroid hormone ecdysone promotes germline stem cell (GSC) self-renewal. Despite strong evidence that ecdysone modulates the reception of bone morphogenetic protein (BMP) signals in GSCs, transcriptional targets of ecdysone signaling that facilitate BMP reception are unknown. Here, we report that ecdysone signaling promotes the expression of the heterogeneous nuclear ribonucleoproteins (hnRNPs) squid, hephaestus, Hrb27C, and Hrb87F in GSCs. These hnRNPs functionally interact with ecdysone signaling to control GSC number and are cell autonomously required in GSCs for their maintenance. We demonstrate that hnRNPs promote GSC self-renewal by binding to transcripts essential for proper BMP signaling, including the BMP receptors thickveins and punt. Our findings support the model that stem cells coordinate local and long-range signals at the transcriptional and post-transcriptional levels to maintain self-renewal in response to physiological demand.

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Genetic Mosaic Analysis of Stem Cell Lineages in the Drosophila Ovary

Cited by 26 publications

References 22 publications

AMP-activated protein kinase has diet-dependent and -independent roles in Drosophila oogenesis

AMP-activated protein kinase has diet-dependent and -independent roles in Drosophila oogenesis

RNAi-Based Techniques for the Analysis of Gene Function in Drosophila Germline Stem Cells

Ecdysone signaling promotes expression of multifunctional RNA binding proteins essential for ovarian germline stem cell self-renewal inDrosophila

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