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A resetting signal between Drosophila pacemakers synchronizes morning and evening activity


The biochemical machinery that underlies circadian rhythms is conserved among animal species and drives self-sustained molecular oscillations and functions, even within individual asynchronous tissue-culture cells1,2,3. Yet the rhythm-generating neural centres of higher eukaryotes are usually composed of interconnected cellular networks, which contribute to robustness and synchrony as well as other complex features of rhythmic behaviour4,5,6,7. In mammals, little is known about how individual brain oscillators are organized to orchestrate a complex behavioural pattern. Drosophila is arguably more advanced from this point of view: we and others have recently shown that a group of adult brain clock neurons expresses the neuropeptide PDF8 and controls morning activity (small LNv cells; M-cells), whereas another group of clock neurons controls evening activity (CRY+, PDF- cells; E-cells)6,9. We have generated transgenic mosaic animals with different circadian periods in morning and evening cells. Here we show, by behavioural and molecular assays, that the six canonical groups of clock neurons10 are organized into two separate neuronal circuits. One has no apparent effect on locomotor rhythmicity in darkness, but within the second circuit the molecular and behavioural timing of the evening cells is determined by morning-cell properties. This is due to a daily resetting signal from the morning to the evening cells, which run at their genetically programmed pace between consecutive signals. This neural circuit and oscillator-coupling mechanism ensures a proper relationship between the timing of morning and evening locomotor activity.

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Figure 1: The M-cell clock influences the timing of events related to E-cells.
Figure 2: The M-cell clock dictates the period and phase of E-cell molecular oscillation, and DN2 cells control the phase of l-LN v cells.
Figure 3: The duration of subjective day is correlated with the genotype of E-cells, whereas the period is correlated with the genotype of M-cells.
Figure 4: The evening oscillator times its output according to its own intrinsic clock programme.


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We thank J. Hall, R. Allada, P. Emery and R. Stanewsky for critical comments on the manuscript; J. Agosto, J. Menet, E. Nagoshi and S. Kadener for discussions and advice; E. Dougherty for assistance in confocal microscopy; and H. Felton for administrative assistance. The work was supported in part by grants from the NIH to M.R.

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Correspondence to Michael Rosbash.

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Supplementary information

Supplementary Figure 1

Analysis of DD locomotor rhythms of flies expressing SGG in different clock-cell groups. (PDF 355 kb)

Supplementary Figure 2

Temporal variation of tim RNA expression level in each clock neuronal group during the fourth day of constant darkness (DD4) in brains of EP1576 flies. (PDF 116 kb)

Supplementary Figure 3

Representative examples of whole-mount brain in-situ hybridization results in EP1576 flies. (PDF 254 kb)

Supplementary Figure 4

Temporal variation of tim RNA expression level during DD4 in each clock neuronal group in timGAL4/UAS-SGG brains. (PDF 109 kb)

Supplementary Figure 5

Representative whole-mount brain in-situ hybridization pictures of timGAL4/UAS-SGG flies. (PDF 222 kb)

Supplementary Figure 6

Temporal variation of tim RNA expression level in each clock neuronal group in PdfGAL4/UAS-SGG brains. (PDF 123 kb)

Supplementary Figure 7

Representative whole-mount brain in-situ hybridization pictures of PdfGAL4/ EP1576 flies. (PDF 299 kb)

Supplementary Figure 8

Temporal variation of tim RNA expression level in each clock neuronal group in timGAL4/UAS-SGG/PdfGAL80 brains. (PDF 126 kb)

Supplementary Figure 9

Representative whole-mount brain in-situ hybridization pictures of timGAL4/ EP1576/PdfGAL80 flies. (PDF 433 kb)

Supplementary Figure 10

Temporal variation of tim RNA expression level of each clock neuronal group in timGAL4/UAS-SGG/cryGAL80 brains. (PDF 116 kb)

Supplementary Figure 11

Representative whole-mount brain in-situ hybridization pictures of timGAL4/ EP1576/cryGAL80 flies. (PDF 367 kb)

Supplementary Figure Legends

Legends to accompany the above Supplementary Figures. (DOC 29 kb)

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Stoleru, D., Peng, Y., Nawathean, P. et al. A resetting signal between Drosophila pacemakers synchronizes morning and evening activity. Nature 438, 238–242 (2005).

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