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. 2021 Jul 12;10:e67753. doi: 10.7554/eLife.67753

Figure 6. E2F/Dp in fat body exerts systemic effects modulated by sugar supplement.

(A–B) Number of viable adults (relative to pupa) fed on control diet (7.9% carbohydrate, 0.08% fat, and 1.9% protein) and increasing levels of sugar in food (12, 16% and 24% carbohydrate). Data are represented as mean ± SD, n = 6 repeats per condition. (A) Left panel: cg>mCherry RNAi and cg>DpRNAi, right panel: r4>mCherryRNAi and r4>DpRNAi. (B) Mef2>mCherry-RNAi and Mef2>Dp-RNAi. (C) Diagram of trehalose synthesis pathway. The enzymes that are significantly downregulated in Dp-deficient fat body are indicated in blue, and upregulated in magenta, based on proteome data. (D–E) Circulating trehalose levels measured in third instar larval hemolymph. (D) cg>mCherry-RNAi and cg>Dp-RNAi, and (E) Mef2>mCherry-RNAi and Mef2>Dp-RNAi larvae fed on control diet (7.9% carbohydrate) and high sugar diet (16% carbohydrate). Data are represented as mean ± SD, two-way ANOVA followed by Tukey’s multiple comparisons test, three independent experiments were done, one representative experiment is shown. (D) n = 6 per group and **p = 0.0005. (E) n = 3–6 per group and p = 0.5. (F) Top panel: Confocal single plane images of third instar larval fat bodies stained with 4,6-diamidino-2-phenylindole (DAPI) and BODIPY red. The cg>mCherry-RNAi and cg>Dp-RNAi animals were fed on control diet (7.9% carbohydrate) and supplemented with sugar (16% carbohydrate, high sugar diet). Scale: 40 μm. Bottom panel: Measurement of lipid droplet size in fat body. Data are represented as box and whiskers, min to max showing all points, n = 10–17 fat bodies per genotype, two-way ANOVA followed by Tukey’s multiple comparisons test, *p = 0.02, ***p < 0.0001, three independent experiments, one representative experiment is shown. (G–H) Triglycerides content measured in third instar larva and normalized to total protein content. (G) cg>mCherry-RNAi and cg>DpRNAi, and (H) Mef2>mCherry RNAi and Mef2>DpRNAi larvae fed on control diet (7.9% carbohydrate) and high sugar diet (16% carbohydrate). Data are represented as mean ± SEM, two-way ANOVA followed by Tukey’s multiple comparisons test, one representative experiment is shown, (G) n = 5–6 per group and *p = 0.004. Three independent experiments were done. (H) n = 5–6 per group and p = 0.2, two independent experiments were done. Full genotypes are (A) cg-GAL4,UAS-mCherry-RNAi, cg-GAL4/UAS-Dp[GD4444]-RNAi, r4-GAL4/UAS-mCherry-RNAi, UAS-Dp[GD4444]-RNAi/r4-GAL4 (B, E, H) Mef2-GAL4/UAS-mCherry-RNAi, and UAS-Dp[GD4444]-RNAi,Mef2-GAL4 (D, F, G) cg-GAL4,UAS-mCherry-RNAi and cg-GAL4/UAS-Dp[GD4444]-RNAi.

Figure 6—source data 1. Measurements of circulating trehaloselevels and the statistical analysis.
Figure 6—source data 2. Measurements of triglycerides levelsnormalized to protein content and the statistical analysis.
Figure 6—source data 3. Average values of the lipid droplet size measured for eachimage and the statistical analysis.
Figure 6—source data 4. MacroScript used to analyze particle size or LipidDroplets in ImageJ.

Figure 6.

Figure 6—figure supplement 1. Supplementing food modifies viability of animals with E2F/Dp-deficient fat bodies.

Figure 6—figure supplement 1.

(A–B) Number of viable adults (relative to pupa) fed on different food conditions. Left panel: Increasing levels of protein in food (0.9, 1.96% and 6.3% protein). Right panel: Increasing levels of fat in food (0.04, 0.08, 1.04, 2.5 and 5% lecithin). Data are represented as mean ± SEM, n = 2–4 samples per condition. Experiment was repeated at least three times. (A) cg>mCherry-RNAi and cg>Dp-RNAi, (B) Mef2>mCherry-RNAi and Mef2>Dp-RNAi. (C) Representative images of cg>mCherry-RNAi and cg>Dp-RNAi larvae fed on increasing levels of protein in food (0.9, 1.96% and 6.3% protein). Black arrowheads point to melanotic masses. Scale: 5 mm. (D) Quantification of number of larvae fed showing melanotic masses relative to total number of larvae harvested as in C. Data are represented as mean ± SEM, n=2–5. Three independent experiments were done. (E) Top panel: Confocal single plane images of third instar larval fat bodies stained with 4,6-diamidino-2-phenylindole (DAPI) and BODIPY. The Mef2>mCherry-RNAi and Mef2>Dp-RNAi animals were fed on control diet (7.9% carbohydrate) and supplemented with sugar (16% carbohydrate, high sugar diet). Scale: 40 μm. Bottom panel: Measurement of lipid droplet size in fat body. Data are represented as box and whiskers, min to max showing all points, n = 55–60 images per group in three independent experiments, two-way ANOVA (main effects only) followed by Tukey’s multiple comparisons test, p = 0.08 for variation between genotypes. (F–G) Total whole-body glycogen content normalized to protein content. Third instar larvae (F) cg>mCherry-RNAi and cg>Dp-RNAi (G) Mef2>mCherry-RNAi and Mef2>Dp-RNAi fed on control diet (7.9% carbohydrate, left panel) and high sugar diet (16% carbohydrate, right panel). Data are represented as mean ± SEM, n = 6 per genotype, three independent experiments were done, two-way ANOVA followed by Tukey’s multiple comparisons test, (F) p = 0.5 and (G) p = 0.9 for variation between genotypes. One representative experiment is shown. (H–I) Quantification of the percentage of binucleated cells in third instar larval fat bodies. (H) cg>mCherry-RNAi and cg>Dp-RNAi (I) Mef2>mCherry-RNAi and Mef2>Dp-RNAi were fed on control diet (7.9% carbohydrate) and high sugar diet (16% carbohydrate). Data are represented as mean ± SD, n>200 cells for Mef2>Dp-RNAi, and cg>Dp-RNAi, and n=200 cells for Mef2>RFP and cg>RFP, which did not show binucleated cells. Full genotypes are (A, C–D, F) cg-GAL4,UAS-mCherry-RNAi, cg-GAL4/UAS-Dp[GD4444]-RNAi (B, E) Mef2-GAL4/UAS-mCherry-RNAi, and UAS-Dp[GD4444]-RNAi,Mef2-GAL4 (H) cg-GAL4/UAS-RFP,Dp[GFP], cg-GAL4/Dp[GFP],UAS-Dp[GD4444]-RNAi, and (I) UAS-RFP,Dp[GFP];Mef2-GAL4, and Dp[GFP],UAS-Dp[GD4444]-RNAi,Mef2-GAL4.