A visual screen for diet-regulated proteins in the Drosophila ovary using GFP protein trap lines
Introduction
Reproduction demands high levels of energy and resources to support oocyte growth and/or embryonic development. In addition, adequate food availability maximizes the chance of offspring survival. Millions of years of evolution have therefore ensured the tight coupling of nutrient availability and reproductive processes, including oogenesis (Ables et al., 2012). In women, for example, malnutrition due to low food availability or eating disorders is associated with decreased fertility, whereas obesity is also linked to infertility and other reproductive issues (Fontana and Della Torre, 2016). Much remains to be learned, however, about the cellular and molecular underpinnings of this connection.
A well-described example of how the ovary responds to diet is seen in Drosophila melanogaster, a model organism highly amenable to genetic, molecular and cell biological analyses (Ables et al., 2012, Hudson and Cooley, 2014). The ovary is composed of 15–20 ovarioles, each of which contains an anterior germarium followed by developing egg chambers, or follicles (Fig. 1A). The germarium houses germline stem cells (GSCs) and follicle stem cells (FSCs) that continuously produce germ cells and follicle cells, respectively (Fig. 1B). Two to three GSCs reside in a well-defined niche composed of cap cells, terminal filament cells and a subset of escort cells. Each GSC division yields a GSC and a cystoblast that undergoes four rounds of incomplete division to produce two-, four-, eight- and 16-cell cysts. Early germ cells have a unique membranous structure, called the fusome, which undergoes stereotypical morphological changes: in GSCs, the predominantly round fusome always abuts the interface with cap cells; as germline cyst divisions progress, the fusome becomes gradually more branched as it interconnects all of the cells in the cyst. In the 16-cell cyst, one of the cells is determined as the oocyte and undergoes meiosis, while the remaining 15 become supportive polyploid nurse cells. FSC-derived follicle cells envelop the 16-cell cyst to form a follicle that leaves the germarium and progresses through fourteen developmental stages. Follicle cells divide mitotically until stage 7, after which they undergo endoreplication. During stage 8, oocytes initiate yolk uptake, or vitellogenesis, and at stage 14, mature eggs are ready to be ovulated, fertilized, and laid (Hudson and Cooley, 2014, Spradling, 1993).
Drosophila oogenesis is highly regulated by diet at multiple steps, resulting in up to ∼60-fold differences in rates of egg laying on protein-rich versus -poor diets. On a protein-rich diet, GSCs and FSCs have relatively high division rates, and their progeny proliferate and grow robustly. On a protein-poor diet, proliferation and growth slow down two-to four-fold, GSC loss increases, early germline cysts die more frequently within the germarium, follicles entering vitellogenesis degenerate at high rates, and ovulation is partially blocked (Drummond-Barbosa and Spradling, 2001). In addition, developing previtellogenic follicles undergo a rearrangement of the microtubule cytoskeleton and accumulation of ribonucleoproteins in large processing bodies under starvation (Burn et al., 2015, Shimada et al., 2011). This dietary response is fast and largely reversible; for example, upon switching from poor to rich diets, changes in proliferation and growth rates occur within less than 18 hours, while from rich to poor diets these rates change within less than 24 hours (Drummond-Barbosa and Spradling, 2001).
Multiple diet-dependent factors mediate the Drosophila ovarian response to diet. Insulin-like peptides directly modulate GSC proliferation (Hsu et al., 2008, LaFever and Drummond-Barbosa, 2005), and act on cap cells to control GSC maintenance (Hsu and Drummond-Barbosa, 2009, Hsu and Drummond-Barbosa, 2011, Yang et al., 2013). In addition, insulin signaling cell autonomously controls the growth of germline cysts and vitellogenesis (Hsu et al., 2008, LaFever and Drummond-Barbosa, 2005), and act through follicle cells to modulate the microtubule cytoskeleton and processing body formation in underlying germline cysts (Burn et al., 2015). The nutrient sensor Target of rapamycin (TOR) has multiple roles in the GSC and follicle stem cell lineages (LaFever et al., 2010, Sun et al., 2010), and its appropriate downregulation on a poor diet is important for survival of previtellogenic egg chambers (Wei et al., 2014). Ecdysone stimulates GSC responsiveness to niche signals, controls germline differentiation, and stimulates oocyte lipid accumulation (Ables et al., 2015, Ables and Drummond-Barbosa, 2010, Konig et al., 2011, Morris and Spradling, 2012, Sieber and Spradling, 2015). Adiponectin signaling (which in mammals is stimulated by the adipocyte proteohormone adiponectin) and AMP-activated protein kinase are also required for GSC maintenance (Laws et al., 2015, Laws and Drummond-Barbosa, 2016), and amino acid sensing by adult adipocytes controls GSC numbers and ovulation (Armstrong et al., 2014). Here, to identify additional factors potentially involved in the ovarian response to diet, we screened 887 green fluorescent protein (GFP) protein trap lines. In these lines, an artificial GFP exon is inserted in frame into endogenous loci, and the resulting GFP fusion proteins therefore exhibit levels and subcellular localization patterns that reflect those of their native counterparts (Buszczak et al., 2007, Kelso et al., 2004, Morin et al., 2001, Quinones-Coello et al., 2007). For each line, we compared the ovarian GFP pattern of females kept on a rich diet to that of females switched to a poor diet for 12–16 hours, with the goal of capturing early changes in the response to diet. Of the lines we screened, 61 (corresponding to 53 proteins) exhibited distinct GFP fusion expression levels or subcellular localization patterns in the ovary on different diets. These lines identify candidates to be investigated in the future for potential regulatory or early effector roles in the ovarian response to diet.
Section snippets
Drosophila GFP protein trap lines and culture conditions
Drosophila GFP protein trap lines from Yale University and Carnegie Institution for Science were maintained at room temperature on standard medium, consisting of cornmeal, yeast, molasses and agar. For the screen, ten pairs of two-to three-day-old flies from each GFP protein trap line were first cultured in plastic vials containing standard medium supplemented with wet yeast paste (yeast-rich diet) for three days. Half of the flies were maintained on a rich diet, whereas the other half was
Fifty-three GFP trap lines respond to diet within 12–16 h
To uncover additional factors involved in the ovarian response to diet, we searched for GFP fusion proteins that undergo rapid changes in expression levels or subcellular localization upon switching females from yeast-rich to -poor diets. We screened 887 publically available GFP trap lines by initially placing females on a rich diet for three days and then switching them to a poor diet for 12–16 hours. We then compared the GFP pattern of dissected and fixed ovaries to those of control females
Acknowledgments
We thank L. Cooley and A. Spradling for providing GFP protein trap lines. This work was supported by American Cancer Society RSG-07-182-01 and National Institutes of Health R01 GM069875.
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Present address: Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei 115, Taiwan.