The role of PDF neurons in setting the preferred temperature before dawn in Drosophila
- Cincinnati Children's Hospital Medical Center, United States
- University College London, United Kingdom
- Queen Mary University of London, United Kingdom
- Hokkaido University, Japan
- PRESTO, Japan Science and Technology Agency, Japan
- University of Cincinnati, United States
Figures
Figure 1
PDF neurons are required for preferred temperature before dawn.
(A) TPR of Pdf-Gal4/UAS-Kir2.1 (red), Pdf-Gal4/+ (dark gray) and UAS-Kir2.1/+ (light gray) flies over 24 hr. Numbers represent the number of assays. Pdf-Gal4 drives sLNvs and lLNvs. (B) TPR of Pdf-Ga…
Figure 2 with 1 supplement
sLNvs contact DN2s, and the number of contacts peaks before dawn.
(A1) GRASP between DN2s and sLNvs. The terminal area of sLNvs in dorsal lateral brain was magnified. The reconstituted GFP signals (green) were only detected in the dorsal protocerebrum, but not in …
Figure 2—figure supplement 1
GRASP between DN2s and sLNvs.
Clk9M-Gal4::UAS-CD4:spGFP1-10; Pdf-Gal80 and Pdf-LexA::LexAop-CD4:spGFP11 flies were crossed. The reconstituted GFP signals (green, arrowheads) were only detected in the dorsal protocerebrum, but …
Figure 3 with 2 supplements
sLNvs activate DN2s, and the loss of DN2 activation results in a lower temperature preference.
(A1) A representative graph of sLNv activation via P2X2 expression in sLNvs. Both P2X2 and GCaMP3.0 were expressed in sLNvs by using Clk9M-Gal4::UAS-GCaMP3.0 / Pdf-LexA:: LexAop-P2X2 flies. The …
Figure 3—figure supplement 1
Lack of P2 × 2 expression in sLNvs leads to no responses to ATP application in both sLNvs and DN2s.
(A) Representative graphs of GCaMP fluorescence in sLNv (A1) and DN2 (A2) using Clk9M-Gal4::UAS-GCaMP3.0 / LexAop-P2X2 flies. GCaMP3.0 were expressed both in sLNvs and DN2s. Without Pdf-LexA to …
Figure 3—figure supplement 2
The DN2 driver is only expressed in one of the two sets of DN2s.
(A) Clk9M-Gal4;Pdf-Gal80 is expressed in one of the two sets of DN2s. While less than 20% flies had both sets of DN2s labeled with GFP (red), more than 80% flies had only one of the sets of DN2s …
Figure 4 with 2 supplements
TrpA1SH-Gal4 and Pdf-Gal4 expressing neurons contact and are involved in regulating temperature preference before dawn.
(A) GRASP between ACs and sLNvs. TrpA1SH-Gal4::UAS-CD4:spGFP1-10 and Pdf-LexA::LexAop-CD4:spGFP11 flies were used to express split-GFP1-10 in ACs and split-GFP11 in LNvs, respectively. When these …
Figure 4—figure supplement 1
TRPA1 knockdown in ACs causes lower temperature preference before dawn.
(A–B) NP0002-Gal4 is expressed in AC neurons (A). Expression of NP0002-Gal4/ UAS-mCD8-RFP (green) in the fly brain (B). NP0002-Gal4/ UAS-mCD8-RFP expression (green) was overlapped in AC neurons …
Figure 4—figure supplement 2
TrpA1SH-Gal4 expressing cells do not overlap with clock neurons in the brain.
(A) TrpA1SH-Gal4::UAS-mCD:GFP flies were stained with anti-GFP (green) and anti-Tim (red) (A1). AC neurons (arrow head) were labeled by GFP (A2) but not by the Tim antibody (A3). (B, C) TrpA1SH-Gal4 …
Figure 5 with 2 supplements
AC neuron silencing using the inwardly rectifying K+ channel Kir2.1 and TRPA1 knock down do not interfere with temperature entrainment.
Locomotor behavior of male flies of the genotypes indicated above each plot were analyzed in LD 20°C, followed by two 20°C: 25°C temperature cycles (TC) in DD, which were delayed by 6 hr compared to …
Figure 5—figure supplement 1
Blocking synaptic transmission of AC neurons using tetanus toxin light chain (TNT) does not interfere with temperature entrainment.
Locomotor behavior of male flies of the genotypes indicated above each plot (UAS-IMP-TNT: inactive toxin, UAS-TNT: active toxin) were analyzed in LD 20°C, followed by two 20°C: 25°C TC in DD, that …
Figure 5—figure supplement 2
Silencing AC neurons does not interfere with synchronization to high temperature cycles (25°C: 29°C) (A) and down regulation of the serotonin receptor 5HT1B in Pdf neurons does not interfere with temperature entrainment (B).
(A) Locomotor behavior of male flies of the genotypes indicated above each plot were analyzed in LD 25°C, followed by two 25°C: 29°C TC in DD, that were delayed by 6 hr compared to the previous …
Figure 6
A model depicting the roles of AC, sLNv and DN2 neurons.
AC neurons detect ambient temperatures, and the information could be transmitted to sLNvs. At dawn (ZT22-24) sLNvs maximally activate DN2s, and the loss of DN2 activation results in flies preferring …
Tables
Table 1
AC neuron silencing does not interfere with synchronization to high temperature cycles (25°C: 29°C) but reduces viability
| Genotype | Total n | Survived until end experiment n | Entrain of survived n |
|---|---|---|---|
| Canton-S | 10 | 6 | 6* |
| TrpA1SH-gal4 > UAS-TeTxLC V1b (inactive) | 9 | 4 | 4 |
| TrpA1SH-gal4 > UAS-TeTxLC R3 (active) | 9 | 0 | |
| + > UAS-kir2.1 | 10 | 8 | 8 |
| TrpA1SH-gal4 > UAS-kir2.1 | 10 | 1 | 1* |
| TrpA1SH-gal4 > UAS-TrpA1 RNAi | 8 | 3 | 3* |
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*actograms shown in Figure 5—figure supplement 2A.
Table 2
Rhythm and temperature compensation analysis of control and TrpA1 loss-of-function mutant flies under free running conditions at different ambient temperatures.
| Genotype | Free run | n | % Rh | Period (h) ± SEM | RS ± SEM | Q10 |
|---|---|---|---|---|---|---|
| +/+ | 16°C | 9 | 64 | 24.67 ± 0.56 | 2.6 ± 0.20 | 0.97 |
| trpA11/+ | 16°C | 8 | 57 | 24.22 ± 0.57 | 2.9 ± 0.24 | 0.99 |
| +/trpA1w903* | 16°C | 12 | 75 | 23.83 ± 0.25 | 2.9 ± 0.28 | 1.01 |
| +/Df(3L)ED4415 | 16°C | 15 | 94 | 23.85 ± 0.19 | 3.2 ± 0.19 | 1.00 |
| trpA11/trpA11 | 16°C | 6 | 55 | 23.29 ± 0.38 | 2.0 ± 0.19 | 1.02 |
| trpA11/trpA1w903* | 16°C | 13 | 81 | 24.23 ± 0.17 | 3.4 ± 0.27 | 1.01 |
| trpA11/Df(3L)ED4415 | 16°C | 14 | 88 | 23.54 ± 0.25 | 2.9 ± 0.19 | 1.02 |
| +/+ | 29°C | 16 | 100 | 23.75 ± 0.06 | 4.8 ± 0.24 | |
| trpA11/+ | 29°C | 13 | 93 | 23.75 ± 0.12 | 3.9 ± 0.31 | |
| +/trpA1w903* | 29°C | 16 | 100 | 24.16 ± 0.09 | 3.5 ± 0.25 | |
| +/Df(3L)ED4415 | 29°C | 11 | 79 | 23.98 ± 0.07 | 4.1 ± 0.36 | |
| trpA11/trpA11 | 29°C | 7 | 78 | 23.57 ± 0.46 | 2.6 ± 0.26 | |
| trpA11/trpA1w903* | 29°C | 13 | 93 | 24.56 ± 0.11 | 3.3 ± 0.26 | |
| trpA11/Df(3L)ED4415 | 29°C | 14 | 88 | 24.25 ± 0.08 | 3.9 ± 0.31 |
Additional files
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Supplementary file 1
One-way ANOVA and Tukey-Kramer tests showing a comparison of each genotype of flies (Gal4/UAS, Gal4/+ or UAS/+) within the same time zone.
The preferred temperatures among Gal4/UAS, Gal4/+ and UAS/+ flies were analyzed using One-way ANOVA and Tukey-Kramer tests. In each time zone, F and p values and degrees of freedom are shown. The comparison between Gal4/UAS and Gal4/+, Gal4/UAS and UAS/+ as well as Gal4/+ and UAS/+ are shown in green, blue and red, respectively (****p<0.0001, ***p<0.001, **p<0.01 or *p<0.05). Stars are shown in Figures 1 and 4D–F and Figure 4—figure supplement 1C–D when Gal4/UAS are statistically different from both Gal4/+ (stars in green) and UAS/+ (stars in blue). These time zones are highlighted in orange.
- https://doi.org/10.7554/eLife.23206.017
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Supplementary file 2
One-way ANOVA and Tukey-Kramer tests showing a comparison of each control fly line (Pdf-Gal4/+, UAS-Kir/+, UAS-∆clock/+, UAS-5-HT1B-RNAi/+ and R6-Gal4 within the same time zone
The preferred temperatures among Pdf-Gal4/+, UAS-Kir/+, UAS-∆clock/+, UAS-5-HT1B-RNAi/+ and R6-Gal4 flies were analyzed using one-way ANOVA and Tukey-Kramer tests. In the each time zone, F and p values and degrees of freedom are shown. ***p<0.001, **p<0.01 or *p<0.05.
- https://doi.org/10.7554/eLife.23206.018
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Supplementary file 3
One-way ANOVA and Tukey-Kramer tests showing TPR comparisons of each fly line
The daytime TPR of each fly line was analyzed using one-way ANOVA. The preferred temperatures of ZT1-3 were further compared to that of each time zone (ZT 4–6, 7–9 or 10–12) by Tukey-Kramer tests. F and p values and degrees of freedom are shown. ***p<0.001, **p<0.01 or *p<0.05
- https://doi.org/10.7554/eLife.23206.019
