1. Volume 28, Issue 5, Pages 779-787.e3 (March 2018)
Feeding-State-Dependent Modulation of Temperature Preference Requires Insulin Signaling in Drosophila Warm-Sensing Neurons 
Yujiro Umezaki, Sean E. Hayley, Michelle L. Chu, Hanna W. Seo, Prasun Shah, Fumika N. Hamada 
Current Biology 
Volume 28, Issue 5, Pages e3 (March 2018)
DOI: /j.cub
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2. Current Biology 2018 28, 779-787.e3DOI: (10.1016/j.cub.2018.01.060)
Copyright © 2018 Elsevier Ltd Terms and Conditions

3. Figure 1 Starvation Causes a Lower Tp
(A) A schematic diagram of the experimental conditions. The flies were reared under normal food conditions (fed, blue-colored bars) and then transferred to starved conditions (water-soaked paper) for either 18–21 hr (overnight [Starved O/N]) or 42–45 hr (Starved 2 O/N, orange-colored bars) and tested in behavioral assays for temperature preference. The flies starved for 24 hr were refed overnight (refed O/N). The details are provided in the experimental procedures.
(B) Comparison of Tp among fed (white box), starved overnight (gray box), starved 2 O/N (green box), and refed (purple box) conditions in w1118 (control) flies. The behavioral experiments were performed at zeitgeber time (ZT) 4–7. The behavioral experiments performed at ZT 1–3, 4–6, 7–9, and 10–12 are shown in Figure S1B. Whiskers indicate the minimum-maximum data range of Tp, and boxes indicate the 25th–75th percentile data range of Tp. A horizontal line and a cross in each box represent the median and mean, respectively. Italicized numbers indicate the number of trials. One-way ANOVA followed by a post hoc Dunnett’s test was performed to compare each Tp to the control (fed condition). ∗∗∗∗p < NS indicates no significance.
(C) Comparison of Tp among flies that were normally fed (white box), starved overnight (gray box), refed with normal food (red box), and refed with the following sugar solutions: glucose, 1% glucose (orange box); sucrose, 1% sucrose (yellow box); fructose, 1% fructose (green box); and lactose, 1% lactose (blue box). The behavioral experiments were performed at ZT 1–3. The plotting pattern is the same as in Figure 1B. One-way ANOVA and post hoc Dunnett’s test were performed to compare Tp under each condition to starved conditions (gray box). ∗p < ∗∗p < ∗∗∗p < NS indicates no significance.
See also Figure S1 and Table S2.
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4. Figure 2
Ilp6 in the Fat Body Is Necessary and Sufficient for Regulation of the Starvation-Induced Reduction in Tp
Comparison of Tp between fed (F: fed, white box) and starved overnight (S: starved O/N, gray box) conditions in the groups of flies indicated below.
(A) yw (control), ilp6 loss-of-function (LOF) (ilp6 LOF), and ilp6 LOF rescue (ilp6 LOF; Cg-Gal4/uas-ilp6) flies, in which ilp6 is expressed in the fat body using Cg-Gal4 in the ilp6 LOF mutant background. Whiskers show the minimum-maximum data range of Tp, and boxes show the 25th–75th percentile data range of Tp. A horizontal line and a cross in each box represent the median and mean, respectively. Italicized numbers indicate the number of trials. A t test was performed to compare Tp under fed and starved conditions in each genotype. The behavioral experiments performed at ZT 1–3, 4–6, 7–9, and 10–12 are shown in Figure S2B.
(B) yw (control), ilp6 null, and ilp6 null rescue (ilp6 null; Cg-Gal4/uas-ilp6) flies, in which ilp6 is expressed in the fat body using Cg-Gal4 in the ilp6 null mutant background. In the fed condition, the Tp of both ilp6 LOF and ilp6 null flies was lower than that of yw flies; however, it did not differ from that of the rescued flies (Table S1).
(C) RNAi knockdown of ilp6 in the fat body: CgG4/+ (Cg-Gal4/+); ilp6-RNAi/+ (uas-ilp6-RNAi/+); and CgG4 > ilp6-RNAi (Cg-Gal4/uas-ilp6-RNAi) flies.
(D) RNAi knockdown of ilp6 in the fat body: 3.1Lsp2G4/+ (3.1Lsp2-Gal4/+); ilp6-RNAi/+; and 3.1Lsp2G4>ilp6-RNAi (uas-ilp6-RNAi/+; 3.1Lsp2-Gal4/+) flies.
∗p < 0.05. ∗∗p < NS indicates no significance. See also Figures S2 and S3 and Tables S1 and S2.
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5. Figure 3
InR and Its Downstream Pathways in ACs Are Important for the Starvation-Induced Reduction in Tp
Comparison of Tp between the fed (F: fed, white box) and starved overnight (S: starved O/N, gray box) conditions in the groups of flies indicated below.
(A) Inhibition of ACs by Kir2.1 expression: TrpA1G4/+; Kir/+ (UAS-Kir/+); and TrpA1G4>Kir (TrpA1SH-Gal4/+; UAS-Kir/+) flies. AC-inhibited flies still avoided warm temperatures and preferred a temperature of ∼25°C in fed conditions (white box). These data suggest that ACs may not be the only neurons that regulate the warm avoidance behavior and that other redundant temperature-sensing mechanisms could contribute to this warm avoidance phenotype under the conditions applied in the present study. Given that ACs are responsible for the starvation-induced reduction in Tp, ACs represent the critical thermosensor for setting a new temperature set point in starved conditions but might not be sole regulator of the warm avoidance phenotype.
(B) RNAi knockdown of InR in ACs: TrpA1G4/+; InR-RNAi(1)/+ (UAS- InR-RNAi(1)/+); and TrpA1G4 > InR-RNAi(1) (TrpA1SH-Gal4/UAS-InR-RNAi(1)) flies.
(C) RNAi knockdown of InR in ACs: NP0002/+ (NP0002-Gal4/+); InR-RNAi(1)/+ (UAS- InR-RNAi(1)/+); and NP0002 > InR-RNAi(1) (NP0002-Gal4/UAS-InR-RNAi(1)) flies.
(D) RNAi knockdown of InR in ACs: DCR2; TrpA1G4/+ (uas-DCR2/+; TrpA1SH-Gal4/+); InR-RNAi(2)/+ (uas-InR-RNAi(2)/+); and DCR2; TrpA1G4 > InR-RNAi(2) (uas-DCR2/+; TrpA1SH-Gal4/+; uas-InR-RNAi(2)/+) flies. InR-RNAi(1) and InR-RNAi(2) are different RNAi lines targeting different regions of the InR sequence.
(E) RNAi knockdown of chico in ACs: DCR2; TrpA1G4/+; chico-RNAi(1)/+ (uas-chico-RNAi(1)/+); and TrpA1G4 > chico-RNAi(1) (uas-DCR2/+; TrpA1SH-Gal4/+; uas-chico-RNAi(1)/+) flies.
(F) RNAi knockdown of chico in ACs: TrpA1G4/+; chico-RNAi(2)/+ (uas-chico-RNAi(2)/+); and TrpA1G4 > chico-RNAi(2) (TrpA1SH-Gal4/uas-chico-RNAi(2)) flies. chico-RNAi(1) and chico-RNAi(2) are different RNAi lines targeting different regions of the chico sequence.
(G) Expression of a dominant-negative form of p110 (p110-DN) in ACs: TrpA1G4/+; p110-DN/+ (uas-p110-DN/+); and TrpA1G4>p110-DN (TrpA1SH-Gal4/uas-p110-DN) flies. DN means a dominant-negative form of p110.
(H) Expression of a constitutive-active form of p110 (p110-CA) in ACs: TrpA1G4/+; p110-CA/+ (uas-p110-CA/+); and TrpA1G4>p110-CA (uas-p110-CA/+; TrpA1SH-Gal4/+) flies. CA means a constitutive-active form of p110.
(I) RNAi knockdown of p60 in ACs: TrpA1G4/+; p60-RNAi/+ (uas-p60-RNAi/+); and TrpA1G4 > p60-RNAi (TrpA1SH-Gal4/uas-p60-RNAi) flies.
(J) RNAi knockdown of Pten in ACs: TrpA1G4/+; Pten-RNAi (1)/+ (uas-Pten-RNAi(1)/+); and TrpA1G4 > Pten-RNAi (1) (TrpA1SH-Gal4/uas-Pten-RNAi(1)) flies.
(K) RNAi knockdown of Pten in ACs: TrpA1G4/+; Pten-RNAi(2)/+ (uas-Pten-RNAi(2)/+); and TrpA1G4 > Pten-RNAi(1) (uas-DCR2/+; TrpA1SH-Gal4/+; uas-Pten-RNAi(2)/+) flies. Pten-RNAi(1) and Pten-RNAi(2) are different RNAi lines targeting different regions of the Pten sequence.
(L) A schematic diagram of IIS.
The plotting pattern and statistical analysis are the same as in Figure 2A. ∗p < ∗∗p < ∗∗∗p < ∗∗∗∗p < NS indicates no significance. See also Figure S4 and Tables S1 and S2.
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6. Figure 4
Ilp6 Is Important for Resetting the Responding Temperature in ACs
(A–F) Representative profiles of the changes in GCaMP fluorescence in ACs under fed (A, C, and E, blue-colored line) and starved overnight (B, D, and F, orange-colored line) conditions using yw; TrpA1SH-Gal4, UAS-GCaMP3.0 (yw [control]; A and B), ilp6 LOF (C and D), and ilp6 null (E and F) flies. The temperature ramp is shown with gray dotted lines. F indicates the averaged basal fluorescence signal before warm stimulation. ΔF/F is the change in GCaMP fluorescence relative to the basal level of fluorescence.
(G–I) Mean temperature (G) and mean ΔF/F (H) at peak points and mean dT/dt (I). Blue- and orange-colored bars indicate fed and starvation conditions, respectively. Italicized F and S indicate fed and starved overnight conditions, respectively. Italicized numbers indicate the number of trials. A t test was utilized to compare the raw data between the fed and starvation conditions. ∗p < NS indicates no significance. Error bars indicate SEM.
(J) Schematic representation of the experimental conditions for wortmannin treatment. Fed flies were starved with wortmannin overnight, and GCaMP fluorescence was then examined.
(K) Representative profiles of changes in GCaMP fluorescence in the ACs of TrpA1SH-Gal4, UAS-GCaMP3.0 flies under starved plus wortmannin conditions (B, green line). The temperature ramp is shown with gray dotted lines.
(L–N) Mean temperature (L) and mean ΔF/F (M) at peak points as well as the mean dT/dt (N). The same data from starved conditions (orange-colored bars) employed in Figures 4G–4I are used. ∗p < NS indicates no significance. Error bars indicate SEM.
(O) A schematic diagram of IIS and the inhibition of PI3K activity by wortmannin. Wortmannin blocks phosphatidylinositol 3-kinase (PI3K) activity [34]. Starvation causes an increase in ilp6 expression in the fat body [21].
See also Table S2.
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