1. Wnt Signaling Is Required for Long-Term Memory Formation
Ying Tan, Dinghui Yu, Germain U. Busto, Curtis Wilson, Ronald L. Davis 
Cell Reports 
Volume 4, Issue 6, Pages (September 2013)
DOI: /j.celrep
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2. Cell Reports 2013 4, 1082-1089DOI: (10.1016/j.celrep.2013.08.007)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1 Schematic of the Canonical Wnt Signaling Pathway
(A) In the absence of Wnt protein, newly synthesized β-catenin (Arm in Drosophila) is sequestered by a “destruction complex” comprised of Adenomatous polyposis coli (Apc), Axin, Gsk-3β, and CkI. The phosphorylation of β-catenin protein by Gsk-3β and CkI targets it for degradation by the proteasome. This keeps β-catenin at a low level in the cytoplasm. The transcription of Wnt target genes is inhibited through the action of the Tcf/Lef complex.
(B) In the presence of Wnt (Wg is the homolog for Wnt-1 in Drosophila) protein, Wnt binds to the seven-transmembrane-domain protein Fz and its coreceptor, Lrp (Arr in Drosophila). This triggers the activation of Dishevelled (Dvl) and phosphorylation of the cytoplasmic tail of Lrp. The interaction among activated Dvl, the destruction complex, and the docking of Axin to phosphorylated Lrp promotes the disassembly of the destruction complex, allowing β-catenin to accumulate in the cytoplasm. β-catenin then translocates into the nucleus, where it activates the transcription of Wnt target genes by binding to the Tcf/Lef complex. β-catenin also binds to the cytoplasmic domain of N-cadherin to regulate the cadherin complex, controlling cell-cell adhesion.
Cell Reports 2013 4, DOI: ( /j.celrep )
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4. Figure 2 Impaired Olfactory LTM with arm Knockdown in Adult MBs
(A) Performance of flies expressing armRNAi in adult MBs. Flies carrying one copy each of P{MB-GeneSwitch}12-1, uas-dcr2, and uas-armRNAi were fed with RU486 to silence arm expression in the adult MBs. These flies exhibited impairment in 24 hr memory after 5X spaced forward conditioning compared with the no-RU control (n = 12, ∗p < 0.05). Performance at 24 hr after 5X massed conditioning or at 3 min after 1X conditioning was indistinguishable between RU-fed and no-RU flies (n = 6 for each group). N.S., no significant difference.
(B) Performance of uas-armRNAi/+; P{MB-GeneSwitch}12-1, uas-dcr2/+ flies kept on regular food for 9 days (no-RU group), on RU486-containing food for 5 days and then on regular food for 4 days (RU to no-RU group), or on RU486-containing food for 9 days (RU group). The RU to no-RU group performed significantly better than the RU group, whereas there was no significant difference between the no-RU group and the RU to no-RU group (n = 8 for all groups, ∗p < 0.05).
(C) Performance of flies expressing armRNAi in adult MBs compared with those coexpressing armRNAi with a uas-arm transgene. These flies also carried one copy of P{MB-GeneSwitch}12-1 and one copy of uas-dcr2. Flies expressing only armRNAi exhibited a significant decrement in performance compared with flies of the same genotype that remained uninduced (n = 12 for each group, ∗p < 0.05). In addition, the performance levels of flies expressing both armRNAi and uas-arm in adult MBs were indistinguishable from those observed in flies in which transgene expression remained uninduced.
(D) Effects of overexpression of uas-arm on LTM. The performance of flies with induced expression of uas-arm in adult MBs is shown. The P{MB-GeneSwitch}12-1/uas-arm flies were fed with RU486 for 5 days to induce the expression of arm in adult MBs. The 24 hr performance index after 5X spaced conditioning of the RU-fed flies was indistinguishable from that of the no-RU control (n = 6 for all groups). Error bars are the SEM.
See also Figures S1–S4.
Cell Reports 2013 4, DOI: ( /j.celrep )
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5. Figure 3 Expression of armRNAi Blocks the Formation of an LTM Trace
The flies used to obtain data for this figure were uas-armRNAi/+; P{MB-GeneSwitch}12-1,uas-dcr2/MB-GCamP and were trained using 5X spaced forward conditioning.
(A) Performance of flies with and without RU486 feeding at 24 hr after conditioning using benzaldehyde (Ben) as the CS+ and 3-octanol (Oct) as the CS−. The RU-fed group showed a significantly reduced performance index compared with the unfed group of the same genotype (n = 8, ∗p < 0.05).
(B–D) A small fraction of the behaviorally trained flies were removed prior to testing and used for functional imaging.
(B) Average peak calcium responses in the α branch of the α/β MB neurons to the CS+ (Ben) and CS− (Oct) at 24 hr after conditioning with benzaldehyde as the CS+. There was a significant difference in response to the CS+ between the RU-fed and no-RU groups (∗p < 0.05). There was no significant difference in response to the CS− between the RU-fed and no-RU groups (n = 9 and 10 for no-RU and RU-fed groups, respectively).
(C) Group time course for calcium responses in the α branch of the α/β MB neurons during presentation of the CS+ (Ben) at 24 hr after conditioning with Ben as the CS+ (n = 9 and 10 for no-RU and RU-fed groups, respectively). The graph was made using the data from the same flies used for the bar graph in (B).
(D) Group time course for calcium responses in the α branch of the α/β MB neurons across time during presentation of the CS− (Oct) at 24 hr after conditioning with Ben as the CS+ (n = 9 and 10 for no-RU and RU-fed groups, respectively). The graph was made using the data from the same flies used for the bar graph in (B).
(E) Performance of flies with and without RU486 feeding at 24 hr after conditioning using Oct as the CS+ and Ben as the CS−. The RU-fed group showed a significantly reduced performance index compared with the no-RU group of the same genotype (n = 8, ∗p < 0.05).
(F–H) A small fraction of the behaviorally trained flies were removed prior to testing and used for functional imaging.
(F) Average peak calcium responses in the α branch of the α/β MB neurons to the CS+ (Oct) and CS− (Ben) at 24 hr after conditioning with octanol as the CS+. There was a significant difference in response to the CS+ between the RU-fed and no-RU groups (∗p < 0.05). There was no significant difference in response to the CS− between the RU-fed and no-RU groups (n = 9 and 8 for no-RU and RU-fed groups, respectively).
(G) Group time course for calcium responses in the α branch of the α/β MB neurons across time during the presentation of the CS+ (Oct) at 24 hr after conditioning with octanol as the CS+ (n = 9 and 8 for no-RU and RU-fed groups, respectively). The graph was made using the data from the same flies used for the bar graph in (F).
(H) Group time course for calcium responses in the α branch of the α/β MB neurons across time during the presentation of the CS− (Ben) at 24 hr after conditioning with octanol as the CS+ (n = 9 and 8 for no-RU and RU-fed groups, respectively). The graph was made using the data from the same flies used for the bar graph in (F). Error bars are the SEM.
Cell Reports 2013 4, DOI: ( /j.celrep )
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6. Figure 4 Impaired Olfactory LTM with arr or wg Knockdown in Adult MBs
(A) Performance of flies expressing arrRNAi6707 in adult MBs. P{MB-GeneSwitch}12-1, uas-dcr2/uas-arrRNAi6707 flies were fed RU-containing food or no-RU food. The RU-fed group exhibited a significant lower performance score at 24 hr after 5X spaced conditioning (n = 12, ∗p < 0.05). There was no significant difference (N.S.) in performance scores at 24 hr after 5X massed conditioning or at 3 min after 1X conditioning between the RU-fed and no-RU groups (n = 6 for each group).
(B) Performance of flies expressing arrRNAi6708 in adult MBs. The RU-fed group exhibited a significantly lower performance score at 24 hr after 5X spaced conditioning (n = 12, ∗p < 0.05). There was no significant difference in performance scores at 24 hr after 5X massed conditioning or at 3 min after 1X conditioning between the RU-fed and no-RU groups (n = 6).
(C) Performance of flies expressing wgRNAi in the adult MBs. The RU-fed P{MB-GeneSwitch}12-1, uas-dcr2/uas-wgRNAi flies showed a significantly lower performance score at 24 hr after 5X spaced conditioning compared with the no-RU control (n = 12, ∗p < 0.05). There was no significant difference (N.S.) in performance scores at 24 hr after 5X massed conditioning or at 3 min after 1X conditioning between the RU-fed and no-RU groups (n = 6). Error bars are the SEM. See also Figures S3 and S4.
Cell Reports 2013 4, DOI: ( /j.celrep )
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7. Figure 5
Increased Expression of wg mRNA after Spaced Forward Conditioning
w(CS10) flies were trained using 5X spaced forward, 5X spaced backward, 5X massed forward, and 1X forward conditioning protocols. Total RNA samples were isolated from the heads of these flies at various time points after conditioning and assayed by qRT-PCR relative to an untrained control group.
(A) Relative wg mRNA level in the heads of trained flies at 15, 30, and 45 min after 5X spaced forward conditioning. The expression of wg mRNA was significantly higher in heads at both 30 and 45 min compared with the untrained control (∗p < 0.05). There was no significant difference in wg mRNA expression between the control and trained flies collected at 15 min after conditioning (n = 5 for all groups).
(B) In a separate experiment, the relative wg mRNA level in the heads of trained flies at 30, 60, and 90 min after 5X spaced forward conditioning was determined. There was a significant increase in wg expression at 30 min after 5X spaced conditioning compared with the untrained control group (∗p < 0.05), replicating the results from (A). There were no significant differences in wg expression between the control and trained flies collected at 60 or 90 min after conditioning (n = 5 for all groups).
(C) Relative wg mRNA level in the heads of trained flies at 45 min after 5X spaced backward, 5X massed forward, and 1X forward conditioning. No significant differences in wg mRNA level were observed in the heads of trained flies at 45 min after conditioning compared with the untrained control group (n = 5; N.S., no significant difference). Error bars are the SEM.
Cell Reports 2013 4, DOI: ( /j.celrep )
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8. Figure S1 Arm Expression in the Adult Fly Brain, Related to Figure 2
(A1–B2) Maximum projection images of the brain immunostained with anti-Arm (A1 and A2) and anti-Drk (B1 and B2) antibodies.
(C1 and C2) Merged images. Panels A1/B1/C1 show an anterior section and A2/B2/C2 a posterior one. The anti-Drk antibody was used as a marker and stains all lobes (MBL) and calyces (C) of the MBs, antennal lobe glomeruli (AL), and the protocerebral bridge (PB). A low level of Armadillo protein expression was observed throughout the brain, including in the antennal lobes, mushroom body lobes, calyces and protocerebral bridge. Scale bar in all panels: 50 μm.
(D1 and D2) Reporter gene expression driven by arm-GAL4. The arm-GAL4 was used to drive a uas-mCD8-gfp transgene. The expression pattern of mCD8-GFP was highlighted with an anti-GFP antibody. Strong reporter expression was detected in the cell bodies (MBC), calyces (C), and all lobes (α, α’, β, β’, γ) of the MBs, the AL and and PB.
Cell Reports 2013 4, DOI: ( /j.celrep )
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9. Figure S2
Effects of Induced Expression of uas-dcr2 on Memory Performance, Related to Figure 2
Performance of flies with and without induced uas-dcr2 expression in the adult MBs. One group of P{MB-GeneSwitch}12-1, uas-dcr2/+ flies were fed with RU486 for five days to induce the expression of dcr-2 in the adult MBs. This group exhibited normal 24 hr memory after 5X spaced forward conditioning or 5X massed forward conditioning, and normal 3 min memory after 1X forward conditioning compared to the unfed control (n = 6 for all groups). Error bars are the SEM. N. S., no significant difference.
Cell Reports 2013 4, DOI: ( /j.celrep )
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10. Figure S3
Effects of Induced Knockdown of Wnt Signaling Components in Adult MBs on Shock and Odor Avoidance, Related to Figures 2 and 4
Flies carrying one copy each of P{MB-GeneSwitch}12-1, uas-dcr2, and uas-RNAi were fed with RU486 for five days to knock down the expression of various Wnt signaling components in the adult MBs. These flies were then challenged with a 90V or 30V shock versus no shock choice, or an odor (benzaldehyde or octanol) at the concentration used for conditioning or a 10-fold dilution versus a stream of fresh air and required to make a binary choice. Error bars are the SEM.
(A) Shock and odor avoidance of flies with induced arm knock down in the adult MBs. No significant difference was detected between RU-fed group and the No-RU control group (n = 12 for each group).
(B and C) Shock and odor avoidance of flies with induced expression of uas-arrRNAi6707 (B) or uas-arrRNAi6708 (C) in the adult MBs. Neither uas-arrRNAi line had significant effects on shock or odor avoidance (n = 12 for each group).
(D) Shock and odor avoidance of flies with induced wg knock down in the adult MBs. The avoidance indexes of RU-fed group were indistinguishable from the No-RU control group (n = 12 for each group).
Cell Reports 2013 4, DOI: ( /j.celrep )
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11. Figure S4
Knockdown Efficiency of RNAi Lines of the Wnt Signaling Pathway, Related to Figures 2 and 4
The c155-gal4 line was used to drive expression of RNAi lines against various Wnt signaling components. The relative level of arm, arr, or wg mRNA in the heads of each genotype was measured by quantitative RT-PCR and normalized to the 18S rRNA internal control. Error bars are the SEM.
(A) Knockdown efficiency of the armRNAi line. The expression of uas-armRNAi with c155-gal4 significantly decreased arm mRNA levels compared to the c155-gal4 or uas-armRNAi parental controls (n = 6, ∗p < 0.05).
(B) Knockdown efficiency of the two arrRNAi lines. The expression of both uas-arr RNAi transgenes with c155-gal4 significantly decreased arr mRNA levels compared to the parental controls (n = 6, ∗p < 0.05).
(C) Knockdown efficiency of the wgRNAi line. The expression of uas-wgRNAi with c155-gal4 significantly decreased wg mRNA levels compared to the parental controls (n = 6, ∗p < 0.05).
Cell Reports 2013 4, DOI: ( /j.celrep )
Copyright © 2013 The Authors Terms and Conditions