Article Figures & Data
Figures
- Figure 1.
Early life, sex-dependent PKA activity in Mushroom Body circuit regions. A, Schematic of MB lobes and defined MBON fields (dashed outlines) shown in three layers: α’/β’ (left), α/β (middle), and γ lobes (right). B, C, Representative images of MB lobes with OK107-Gal4 driving UAS-PKA-SPARK at 0 dpe (B) and 7 dpe (C). The MBON fields (dashed circles) and arrows delineate the α’1 and β’1 quantified regions. D, E, PKA-SPARK::GFP puncta number in both regions, including α’1 (D) and β’1 (E). Scatter plots show all data points and mean ± SEM. F, G, MB lobes with OK107-Gal4 driving UAS-PKA-SPARK in female (F) and male (G) at 7 dpe. H, I, Quantification of PKA-SPARK puncta in both α’1 (H) and β’1 (I). Sample size >15 fields in all conditions. Statistics show two-tailed t tests with Welch’s correction (H) or Mann–Whitney tests (D, E, I). Significance: ***p < 0.001.
- Figure 2.
PKA-SPARK::GFP reporter levels constant across sex and age groups. A, Representative Western blotting comparing PKA-SPARK::GFP protein levels (anti-GFP) from 0 and 7 dpe time points, in both females and males with OK107-Gal4 driving UAS-PKA-SPARK::GFP. The protein loading control is α-Tubulin (α-tub). Probed proteins are indicated on the left and molecular weights on the right. B, PKA-SPARK::GFP protein levels normalized to the α-tub loading control. Scatter plots show all data points and mean ± SEM. Statistics show Brown–Forsythe and Welch ANOVA tests. Sample size: 9, all conditions. Significance: not significant (n.s.; p > 0.05).
- Figure 3.
Localized MB lobe PKA activity signaling enabled by FMRP and Rugose. A, Representative images of MB lobes with OK107-Gal4 driving UAS-PKA-SPARK at 7 dpe in control (left) and dfmr150M null (right). Arrows point to α’1 and β’1 (Fig. 1A). PKA-SPARK::GFP puncta number in α’1 (B) and β’1 (C) regions. D, Similar confocal imaging comparison at 7 dpe in control (left) and rgFDD null (right). E, F, Quantification of PKA-SPARK::GFP puncta number in α’1 (E) and β’1 (F) regions. Note that this comparison is done in males only owing to the X chromosome location of the rugose gene. Scatter plots show all data points and mean ± SEM. Sample size >12 fields in all conditions. Statistics show two-tailed t tests with Welch’s correction (B, C, F) or Mann–Whitney tests (E). Significance: ***p < 0.001.
- Figure 4.
Localized circuit PKA activity increased with Meng-Po OE. A–F, Representative images of MB lobes with OK107-Gal4 driving UAS-PKA-SPARK at 0 and 7 dpe in control (A) and with targeted OE of UAS-dFMRP (B), UAS-hFMRP (C), UAS-ΔRGG-hFMRP (D), UAS-Rugose (Rg, E), and UAS-Meng-Po (MP, F). Arrows indicate the α’1 and β’1 regions (Fig. 1A) of normally heightened PKA activity. Arrowhead (F) points to the expanded PKA activity within the γ3 region. G, H, Quantification of PKA-SPARK::GFP puncta in α’1 (G) and β’1 (H) regions. Scatter plots show all data points and mean ± SEM. Sample size: >7 fields in every genotype and at every time point. Statistics show two-tailed t tests with Welch’s correction and Mann–Whitney tests (see statistical table; Table 1). Significance: ***p < 0.001.
- Figure 5.
Localized MB lobe PKA activity signaling requires the Meng-Po kinase. A, Representative MB lobe images with OK107-Gal4 driving UAS-PKA-SPARK at both 0 dpe (left) and 7 dpe (right) in the genetic control background. Quantified MBON fields (dashed circles, left) and arrows indicate α’1 and β’1 regions. B, Representative MB lobes images with meng-po RNAi under the same conditions as in A. Scale bar: 10 μm. C, D, Quantification of PKA-SPARK::GFP puncta in α’1 (C) and β’1 (D) MBON fields. Scatter plots show all data points and mean ± SEM. Statistics show Kruskal–Wallis tests. Sample size: >9, all conditions. Significance: ***p < 0.001, not significant (n.s.; p > 0.05).
- Figure 6.
Conditional shibirets neurotransmission block increases PKA activity. A–D, Representative images of MB lobes with OK107-Gal4 driving UAS-shibirets at the 0 and 7 dpe time points at the indicated adult rearing temperatures. Dotted outlines define additional MBON regions of elevated PKA activity detected with the PKA-SPARK reporter, and arrowheads indicate expanded α/α’ neuropils. E–H, Quantified number of PKA-SPARK:GFP puncta in defined MB lobe regions. Scatter plots show all data points and the mean ± SEM. Sample size >12 fields in every temperature condition. Statistics show Brown–Forsythe and Welch ANOVA tests (E, G) and Kruskal–Wallis tests (F, H). Significance: *p < 0.05, ***p < 0.001 and not significant (n.s.; p > 0.05).
- Figure 7.
MB circuit PKA activity dramatically expanded by synaptic output block. A, B, MB lobes with OK107-Gal4 driving UAS-tetanus toxin light chain (TNT) at both 0 dpe (A) and 7 dpe (B). Arrows indicate expanded PKA activity regions detected with the PKA-SPARK reporter and dotted outlines indicate newly-recruited MBON regions. C–I, Quantification of PKA-SPARK::GFP puncta number in each defined MBON region, including α’1 (C), β’1 (D), γ1 (E), α’3 (F), α3 (G), β’2m (H), and β2 (I). Scatter plots show all data points and the mean ± SEM. Sample size >19 fields in every paired comparison. Statistics show Mann–Whitney tests. Significance: ***p < 0.001.
Tables
Circuit
locationComparison Data
structureTest Sample size
(# KC fields or
Western lysates)Mean Statistics p value α’1 0 dpe; 7 dpe Not normal Mann–Whitney test 38; 34 0.1; 32.5 U = 0 p < 0.0001 β’1 0 dpe; 7 dpe Not normal Mann–Whitney test 37; 38 0.1; 66.8 U = 0 p < 0.0001 α’1 Female 7 dpe; male 7 dpe normal Unpaired two-tailed t test 18; 16 50.1; 26.9 t = 6.51 df = 26.96 p < 0.0001 β’1 Female 7 dpe; male 7 dpe Not normal Mann–Whitney test 18; 16 83.2; 64.5 U = 31 p < 0.0001 N/A Female 0 dpe; male 0 dpe Normal Brown–Forsythe, Welch ANOVA test 9; 9 1.0; 1.104 t = 2.028 DF = 15.75 p = 0.2846 N/A Female 0 dpe; female 7 dpe Normal Brown–Forsythe, Welch ANOVA test 9; 9 1.0; 1.088 t = 0.8219 DF = 10.31 p = 0.9482 N/A Female 0 dpe; male 7 dpe Normal Brown–Forsythe, Welch ANOVA test 9; 9 1.0; 0.9298 t = 0.7746 DF = 11.32 p = 0.9608 N/A Male 0 dpe; female 7 dpe Normal Brown–Forsythe, Welch ANOVA test 9; 9 1.104; 1.088 t = 0.1493 DF = 9.809 p > 0.9999 N/A Male 0 dpe; male 7 dpe Normal Brown–Forsythe, Welch ANOVA test 9; 9 1.104; 0.9298 t = 1.956 DF = 10.63 p = 0.3383 N/A Female 7 dpe; male 7 dpe Normal Brown–Forsythe, Welch ANOVA test 9; 9 1.088; 0.9298 t = 1.223 DF = 15.43 p = 0.7724 α’1 Control 7dpe; dfmr1 7dpe Normal Unpaired two-tailed t test 34; 21 43.5; 26.1 t = 3.77 df = 41.34 p = 0.0005 β’1 Control 7dpe; dfmr1 7dpe Normal Unpaired two-tailed t test 34; 21 75.5; 43.9 t = 3.88 df = 34.73 p = 0.0004 α’1 Control 7dpe; rg 7 dpe Not normal Mann–Whitney test 24; 18 27.4; 15.2 U = 70.50 p = 0.0001 β’1 Control 7dpe; rg 7 dpe Normal Unpaired two-tailed t test 24; 18 58.9; 20.8 t = 8.40 df = 31.59 p < 0.0001 α’1 Control 0 dpe; control 7 dpe Not normal Mann–Whitney test 14; 12 0.1; 35.7 U = 0 p < 0.0001 α’1 dFMRP OE 0 dpe; dFMRP OE 7 dpe Not normal Mann–Whitney test 16; 18 5.2; 31.6 U = 11.50 p < 0.0001 α’1 hFMRP OE 0 dpe; hFMRP OE 7 dpe Not normal Mann–Whitney test 18; 11 0.2; 39.8 U = 0 p < 0.0001 α’1 ΔRGG-hFMRP OE 0 dpe; ΔRGG-hFMRP OE 7 dpe Not normal Mann–Whitney test 8; 18 0.1; 5.3 U = 9.50 p = 0.0001 α’1 Rg OE 0 dpe; Rg OE 7 dpe Normal Unpaired two-tailed t test 14; 16 0.3; 31.6 t = 8.94 df = 15.04 p < 0.0001 α’1 Meng-Po OE 0 dpe; Meng-Po OE 7dpe Normal Unpaired two-tailed t test 16; 16 67.1; 196.4 t = 7.88 df = 16.78 p < 0.0001 α’1 Control 0 dpe; dFMRP OE 0 dpe Not normal Kruskal–Wallis test 14; 16 0.1; 5.2 Mean rank diff = −22.9 p = 0.0272 α’1 Control 0 dpe; hFMRP OE 0 dpe Not normal Kruskal–Wallis test 14; 18 0.1; 0.2 Mean rank diff = −0.7143 p > 0.9999 α’1 Control 0 dpe; ΔRGG-hFMRP OE 0 dpe Not normal Kruskal–Wallis test 14; 8 0.1; 0.1 Mean rank diff = 0.5357 p > 0.9999 α’1 Control 0 dpe; Rg OE 0 dpe Not normal Kruskal–Wallis test 14; 14 0.1; 0.3 Mean rank diff = −4.286 p > 0.9999 α’1 Control 0 dpe; Meng-Po OE 0 dpe Not normal Kruskal–Wallis test 14; 16 0.1; 67.1 Mean rank diff = −49.21 p < 0.0001 α’1 Control 7 dpe; dFMRP OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 18 35.7; 31.6 t = 0.74 DF = 23.56 p = 0.9505 α’1 Control 7 dpe; hFMRP OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 11 35.7; 39.8 t = 0.62 DF = 20.17 p = 0.9751 α’1 Control 7 dpe; ΔRGG-hFMRP OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 18 35.7; 5.3 t = 6.81 DF = 12.48 p < 0.0001 α’1 Control 7 dpe; Rg OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 16 35.7; 31.6 t = 0.74 DF = 22.91 p = 0.9508 α’1 Control 7 dpe; Meng-Po OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 16 35.7;196.4 t = 9.74 DF = 17.16 p < 0.0001 β’1 Control 0 dpe; control 7 dpe Not normal Mann–Whitney test 14; 12 0.1; 53.6 U = 0 p < 0.0001 β’1 dFMRP OE 0 dpe; dFMRP OE 7 dpe Not normal Mann–Whitney test 16; 18 3.4; 19.6 U = 48.50 p = 0.0005 β’1 hFMRP OE 0 dpe; hFMRP OE 7 dpe Not normal Mann–Whitney test 18; 11 1.0; 60.5 U = 1 p < 0.0001 β’1 ΔRGG-hFMRP OE 0 dpe; ΔRGG-hFMRP OE 7 dpe Not normal Mann–Whitney test 8; 18 0.1; 7.1 U = 0 p < 0.0001 β’1 Rg OE 0 dpe; Rg OE 7 dpe Normal Unpaired two-tailed t test 14; 16 0.4; 25.7 t = 9.12 df = 15.12 p < 0.0001 β’1 Meng-Po OE 0 dpe; Meng-Po OE 7dpe Normal Unpaired two-tailed t test 16; 22 138.9; 251.9 t = 7.49 df = 26.44 p < 0.0001 β’1 Control 0 dpe; dFMRP OE 0 dpe Not normal Kruskal–Wallis test 14; 16 0.1; 3.4 Mean rank diff = −18.48 p = 0.1195 β’1 Control 0 dpe; hFMRP OE 0 dpe Not normal Kruskal–Wallis test 14; 18 0.1; 1.0 Mean rank diff = −6.127 p > 0.9999 β’1 Control 0 dpe; ΔRGG-hFMRP OE 0 dpe Not normal Kruskal–Wallis test 14; 8 0.1; 0.1 Mean rank diff = −1.634 p > 0.9999 β’1 Control 0 dpe; Rg OE 0 dpe Not normal Kruskal–Wallis test 14; 14 0.1; 0.4 Mean rank diff = −9.179 p > 0.9999 β’1 Control 0 dpe; Meng-Po OE 0 dpe Not normal Kruskal–Wallis test 14; 16 0.1; 138.9 Mean rank diff = −50.82 p < 0.0001 β’1 Control 7 dpe; dFMRP OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 18 53.6; 19.6 t = 5.13 DF = 20.78 p = 0.0002 β’1 Control 7 dpe; hFMRP OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 11 53.6; 60.5 t = 0.76 DF = 19.17 p = 0.9426 β’1 Control 7 dpe; ΔRGG-hFMRP OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 18 53.6; 7.1 t = 8.31 DF = 12.07 p < 0.0001 β’1 Control 7 dpe; Rg OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 16 53.6; 25.7 t = 4.55 DF = 16.56 p = 0.0014 β’1 Control 7 dpe; Meng-Po OE 7 dpe Normal Brown–Forsythe, Welch ANOVA test 12; 22 53.6; 251.9 t = 13.07 DF = 26.60 p <0.0001 γ3 Control 0 dpe; Meng-Po OE 0 dpe Not normal Mann–Whitney test 14; 16 0.0; 99.3 U = 14 p <0.0001 γ3 Control 7 dpe; Meng-Po OE 7 dpe Not normal Mann–Whitney test 12; 16 0.4; 291.8 U = 0 p <0.0001 α’1 Control 0 dpe; meng-po RNAi 0 dpe Not normal Kruskal–Wallis test 10; 13 1.0; 0.5 Mean rank diff = 2.212 p > 0.9999 α’1 Control 0 dpe; control 7 dpe Not normal Kruskal–Wallis test 10; 17 1.0; 31.7 Mean rank diff = −24.75 p = 0.0004 α’1 Control 0 dpe; meng-po RNAi 7 dpe Not normal Kruskal–Wallis test 10 ; 16 1.0; 0.3 Mean rank diff = 6.125 p > 0.9999 α’1 meng-po RNAi 0 dpe; control 7 dpe Not normal Kruskal–Wallis test 13; 17 0.5; 31.7 Mean rank diff = −26.96 p < 0.0001 α’1 meng-po RNAi 0 dpe; meng-po RNAi 7 dpe Not normal Kruskal–Wallis test 13; 16 0.5; 0.3 Mean rank diff = 3.913 p > 0.9999 α’1 Control 7 dpe; meng-po RNAi 7 dpe Not normal Kruskal–Wallis test 17; 16 31.7; 0.3 Mean rank diff = 30.88 p < 0.0001 β’1 Control 0 dpe; meng-po RNAi 0 dpe Not normal Kruskal–Wallis test 10; 13 0.8; 0.3 Mean rank diff = 7.138 p > 0.9999 β’1 Control 0 dpe; control 7 dpe Not normal Kruskal–Wallis test 10; 17 0.8; 41.7 Mean rank diff = −23.90 p = 0.0007 β’1 Control 0 dpe; meng-po RNAi 7 dpe Not normal Kruskal–Wallis test 10 ; 16 0.8; 0.6 Mean rank diff = 4.194 p > 0.9999 β’1 meng-po RNAi 0 dpe; control 7 dpe Not normal Kruskal–Wallis test 13; 17 0.3; 41.7 Mean rank diff = −31.04 p < 0.0001 β’1 meng-po RNAi 0 dpe; meng-po RNAi 7 dpe Not normal Kruskal–Wallis test 13; 16 0.3; 0.6 Mean rank diff = −2.945 p > 0.9999 β’1 Control 7 dpe; meng-po RNAi 7 dpe Not normal Kruskal–Wallis test 17; 16 41.7; 0.6 Mean rank diff = 28.09 p < 0.0001 α’1 shibirets 7 dpe 20°C; 25°C Normal Brown–Forsythe, Welch ANOVA test 15; 14 7.5; 25.5 t = 3.74 DF = 16.00 p = 0.0053 α’1 shibirets 7 dpe 20°C; 33°C Normal Brown–Forsythe, Welch ANOVA test 15; 17 7.5; 62.2 t = 11.90 DF =20.01 p < 0.0001 α’1 shibirets 7 dpe 25°C; 33°C Normal Brown–Forsythe, Welch ANOVA test 14; 17 25.5; 62.2 t = 5.844 DF = 28.31 p < 0.0001 β’1 shibirets 7 dpe 20°C; 25°C Not normal Kruskal–Wallis test 16; 14 18.8; 25.6 Mean rank diff = −3.808 p > 0.9999 β’1 shibirets 7 dpe 20°C; 33°C Not normal Kruskal–Wallis test 16; 17 18.8; 66.9 Mean rank diff = −24.08 p < 0.0001 β’1 shibirets 7 dpe 25°C; 33°C Not normal Kruskal–Wallis test 14; 17 25.6; 66.9 Mean rank diff = −20.27 p = 0.0001 α2 shibirets 7 dpe 20°C; 25°C Normal Brown–Forsythe, Welch ANOVA test 16; 13 11.1; 53.8 t = 9.33 DF = 16.03 p <0.0001 α2 shibirets 7 dpe 20°C; 33°C Normal Brown–Forsythe, Welch ANOVA test 16; 17 11.1; 9.3 t = 0.67 DF = 30.76 p = 0.8747 α2 shibirets 7 dpe 25°C; 33°C Normal Brown–Forsythe, Welch ANOVA test 13; 17 53.8; 9.3 t = 9.53 DF = 17.13 p < 0.0001 γ1 shibirets 7 dpe 20°C; 25°C Not normal Kruskal–Wallis test 16; 14 1.9; 0.9 Mean rank diff = 4.353 p > 0.9999 γ1 shibirets 7 dpe 20°C; 33°C Not normal Kruskal–Wallis test 16; 17 1.9; 101.8 Mean rank diff = −21.47 p < 0.0001 γ1 shibirets 7 dpe 25°C; 33°C Not normal Kruskal–Wallis test 14; 17 0.9; 101.8 Mean rank diff = −25.82 p < 0.0001 α2 shibirets 7 dpe 20°C; 7 dpe 20°C, 3 h 33°C Not normal Mann–Whitney test 30; 26 3.6; 26.6 U = 82.50 p <0.0001 α’1 UAS-TNT 0 dpe; UAS-TNT 7 dpe Not normal Mann–Whitney test 32; 57 1.2; 72.4 U = 0 p <0.0001 β’1 UAS-TNT 0 dpe; UAS-TNT 7 dpe Not normal Mann–Whitney test 32; 57 0.5; 75.2 U = 3.50 p < 0.0001 γ1 UAS-TNT 0 dpe; UAS-TNT 7 dpe Not normal Mann–Whitney test 31; 57 0.6; 18.5 U = 165.50 p < 0.0001 α'3 UAS-TNT 0 dpe; UAS-TNT 7 dpe Not normal Mann–Whitney test 29; 54 0.3; 30.1 U = 154 p < 0.0001 α3 UAS-TNT 0 dpe; UAS-TNT 7 dpe Not normal Mann–Whitney test 20; 49 0.2; 66.8 U = 0 p < 0.0001 β’2m UAS-TNT 0 dpe; UAS-TNT 7 dpe Not normal Mann–Whitney test 31; 57 0.6; 44.1 U = 0 p < 0.0001 β2 UAS-TNT 0 dpe; UAS-TNT 7 dpe Not normal Mann–Whitney test 32; 58 0.2; 85.1 U = 0 p < 0.0001 α’1 Control; UAS-TNT 7 dpe Not normal Mann–Whitney test 68; 57 35.9; 72.4 U = 451.50 p < 0.0001 β’1 Control; UAS-TNT 7 dpe Normal Unpaired two-tailed t test 72; 57 70.4; 75.2 t = 0.83 df = 75.77 p = 0.41 α’1 and
β’1α’1 UAS-TNT 7 dpe; β’1 UAS-TNT 7 dpe Normal Unpaired two-tailed t test 57; 57 72.4; 75.2 t =0.43 df = 101.59 p = 0.67
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