Fluorescence circadian imaging reveals a PDF-dependent transcriptional regulation of the Drosophila molecular clock

2019

Preprint

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Section: Methodsmentioning

confidence: 99%

Nitric Oxide Mediates Neuro-Glial Interaction that Shapes Drosophila Circadian Behavior

Kozlov

2019

Preprint

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“…To examine the intrinsic properties of Drosophila clock neurons, we recently developed two fluorescent reporters of the molecular clockwork that permit single-cell recording of the transcriptional and post-transcriptional machineries (Sabado et al, 2017 ). The transcriptional reporter 3 × 69-VNP expresses the short-lived yellow fluorescent protein (VNP; VENUS-NLS-PEST) under the control of three tandem repeats of the E-box-containing 69-bp clock regulatory sequence (CRS) of per (Hao et al, 1997 , 1999 ).…”

Section: Introductionmentioning

confidence: 99%

Evaluating the Autonomy of the Drosophila Circadian Clock in Dissociated Neuronal Culture

Sabado

Vienne

2017

Front. Cell. Neurosci.

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Circadian behavioral rhythms offer an excellent model to study intricate interactions between the molecular and neuronal mechanisms of behavior. In mammals, pacemaker neurons in the suprachiasmatic nucleus (SCN) generate rhythms cell-autonomously, which are synchronized by the network interactions within the circadian circuit to drive behavioral rhythms. However, whether this principle is universal to circadian systems in animals remains unanswered. Here, we examined the autonomy of the Drosophila circadian clock by monitoring transcriptional and post-transcriptional rhythms of individual clock neurons in dispersed culture with time-lapse microscopy. Expression patterns of the transcriptional reporter show that CLOCK/CYCLE (CLK/CYC)-mediated transcription is constantly active in dissociated clock neurons. In contrast, the expression profile of the post-transcriptional reporter indicates that PERIOD (PER) protein levels fluctuate and ~10% of cells display rhythms in PER levels with periods in the circadian range. Nevertheless, PER and TIM are enriched in the cytoplasm and no periodic PER nuclear accumulation was observed. These results suggest that repression of CLK/CYC-mediated transcription by nuclear PER is impaired, and thus the negative feedback loop of the molecular clock is incomplete in isolated clock neurons. We further demonstrate that, by pharmacological assays using the non-amidated form of neuropeptide pigment-dispersing factor (PDF), which could be specifically secreted from larval LNvs and adult s-LNvs, downstream events of the PDF signaling are partly impaired in dissociated larval clock neurons. Although non-amidated PDF is likely to be less active than the amidated one, these results point out the possibility that alteration in PDF downstream signaling may play a role in dampening of molecular rhythms in isolated clock neurons. Taken together, our results suggest that Drosophila clocks are weak oscillators that need to be in the intact circadian circuit to generate robust 24-h rhythms.

“…This means that some important features of Ca 2+ activity may not have been detected. Therefore, studies using complementary approaches, such as short-term in vivo Ca 2+ imaging [56], recording of bioluminescence Ca 2+reporter in freely moving flies [57], live-imaging of molecular clockwork in cultured brains using bioluminescent [45] or fluorescent clock gene reporters [58][59][60], are warranted to fill the knowledge gaps. PDF/PDFR signaling triggers multiple downstream processes, which are cell-typedependent, temporally gated and occur at different time scales.…”

mentioning

confidence: 99%

“…It affects membrane electrical status, calcium levels and molecular clocks. One pathway is through the increase in cAMP levels and protein kinase A (PKA) activity, leading to the increase in the stability of PER and TIM [53, 54,58]. It was also shown that PDF signaling upregulates per transcription in ex vivo brain culture [58].…”

mentioning

confidence: 99%

“…One pathway is through the increase in cAMP levels and protein kinase A (PKA) activity, leading to the increase in the stability of PER and TIM [53, 54,58]. It was also shown that PDF signaling upregulates per transcription in ex vivo brain culture [58]. The mechanism of the third downstream effect, inhibition of Ca 2+ levels, is currently unknown and should entail more complex processes than hyperpolarization.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Decoding <em>Drosophila</em> Circadian Pacemaker Circuit

Kozlov

2019

Preprint

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Drosophila circadian circuit is one of the best described neural circuits but is complex enough to obscure our understanding of how it actually works. Animals’ rhythmic behavior, the seemingly simple outcome of their internal clocks, relies on the interaction of heterogeneous clock neurons that are spread across the brain. Direct observations of their coordinated network interactions can bring us forward in understanding the circuit. The current challenge is to observe activity of each of these neurons over a long span of time –hours to days– in live animals. Here we review the progress in circadian circuit interrogation powered by in vivo calcium imaging.