Real time, in vivo measurement of neuronal and peripheral clocks in Drosophila melanogaster
- Department of Biochemistry and Molecular Biology, Dalhousie University, Canada
- Department of Pharmacology, Dalhousie University, Canada
- Institute of Neuro- and Behavioral Biology, Westfälische Wilhelms University, Germany
- You.i Labs Inc, Canada
- Department of Physics, University of Miami, United States
Figures
Figure 1 with 1 supplement
Design and activity of LABL reporter.
(A) The Locally Activatable Bioluminescence (LABL) reporter construct. In architecture, the per promoter (pPer) is fused to mCherry followed by luciferase (Luc). The mCherry gene and three stop …
Figure 1—figure supplement 1
LABL cloning strategy.
The region of per promoter between restriction sites SphI and XbaI (Bargiello et al., 1984) was cloned into pattB cloning vector. The per 5’UTR was amplified from plasmid containing the entire per …
Figure 2 with 1 supplement
Measurement, quantification, and analysis of luminescence from LABL flies.
(A) LABL activation strategy in Drosophila brain. Fly brain schematic illustrates how LABL can be activated using tissue-specific Gal4 drivers which express UAS-FLP2 to excise mCherry out of the …
Figure 2—figure supplement 1
Dip in luminescence signal in the first peak of transcription oscillation.
Luminescence signal recorded from TUG-activated LABL flies presented in Figure 2C was reproduced and the first peak enhanced for clarity (red box). The red arrow points to the dip in luminescence …
Figure 3 with 1 supplement
LABL oscillations are comparable to other luminescence reporters and respond to circadian clock manipulation.
(A) Comparison of luminescence reporters. LABL activated by tim-UAS-Gal4 (black curve) is plotted alongside per promoter fused to luciferase (plo; blue curve) and PER-BG::Luc (pink curve). (B) LABL …
Figure 3—figure supplement 1
Behavioural period analysis of wild-type, han5304 and tim01 flies.
(A) Behavioural period (left panels) is calculated in the 16-hour to 32-hour range or 48-hr to 72-hr range for wild-type (iso31), han5304 and tim01 mutant flies and plotted. Black bars represent the …
Figure 4 with 2 supplements
Luminescence oscillations of distinct clocks measured in young and aged flies.
Luminescence signal using different Gal4 drivers to activate LABL compared in young (1–3 day old) and old (39–41 day old) flies. Drivers used to activate LABL are TUG (black), Pdf-Gal4 (blue), …
Figure 4—figure supplement 1
Body drivers create ~24 hr oscillation patterns with different amplitude decay in wild-type LABL flies.
Luminescence oscillations measured from LABL flies activated by the indicated Gal4 drivers, in otherwise wild-type flies, plotted over time. Drivers used are Clk4.1-Gal4 (brown), Mai179-Gal4 …
Figure 4—figure supplement 2
Aged flies exhibit weak locomotor rhythms.
Curve represents rhythmic locomotor activity of 16 young (1–3 days old) flies (black) and 11 old (46–48 days old) flies (dark red), +/-SEM (thickness of curve). Vertical solid lines divide days. …
Figure 5
Simulations of oscillations with phase decay and measurement of distinct clocks in aged flies reveal rapidly decaying clock amplitude.
(A) Abrupt phase change simulation. Half of 100 simulated oscillations were made to phase advance at 1.5 days (arrow) by the indicated amount of time (red line). Oscillators with no phase change …
Figure 6
Different circadian Gal4 drivers activate LABL in distinct neurons and tissues.
(A) Schematic of a Drosophila brain and Gal4 driver expression patterns. Pdf-Gal4 (blue): LNvs. DvPdf-Gal4 (pink) and Mai179-Gal4 (purple): LNvs, 3 LNds and 5th s-LNv. R18H11-Gal4 (green) and …
Figure 7 with 1 supplement
Different circadian Gal4 drivers create distinct oscillation patterns that respond to loss of PDF signaling differently.
(A) Luminescence oscillations measured from LABL flies activated by the indicated Gal4 drivers, in a wild-type or PDF signaling deficient genetic background, plotted over time. Colored curves …
Figure 7—figure supplement 1
Circadian clock mutant flies perS and perL reveal distinct oscillatory properties in different parts of the brain.
Luminescence signal and locomotor activity of flies carrying the perS (A–D) or perL (E–H) mutations compared. Drivers used to activate LABL are TUG (black; A, E), Pdf-Gal4 (blue; B, F), DvPdf-Gal4 …
Figure 8 with 1 supplement
Peripheral clocks have distinct characteristics in a wild-type and PDF receptor mutant genetic background.
(A) Schematic of target expression pattern of peripheral tissue Gal4 drivers in Drosophila. elav-Gal4 (neurons; orange), mef2-Gal4 (muscle; red), esg-Gal4 and NP3084-Gal4 (intestines; blue), and …
Figure 8—figure supplement 1
Peripheral clocks exhibit ~24 hr oscillation patterns in wild-type LABL flies.
Luminescence oscillations measured from LABL flies activated by the indicated Gal4 drivers, in otherwise wild-type flies, plotted over time. Drivers used are as labelled: mef2-Gal4 (brown), esg-Gal4 …
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/77029/elife-77029-transrepform1-v2.pdf
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Supplementary file 1
Genotypes of flies used in the figures.
For LABL experiments, stocks are maintained with one parental line expressing both the Gal4 driver and LABL reporter, and the other parental line expressing UAS-FLP.
- https://cdn.elifesciences.org/articles/77029/elife-77029-supp1-v2.docx
