Neuron-specific knockouts indicate the importance of network communication to Drosophila rhythmicity

Animal circadian rhythms persist in constant darkness and are driven by intracellular transcription-translation feedback loops. Although these cellular oscillators communicate, isolated mammalian cellular clocks continue to tick away in darkness without intercellular communication. To investigate these issues in Drosophila, we assayed behavior as well as molecular rhythms within individual brain clock neurons while blocking communication within the ca. 150 neuron clock network. We also generated CRISPR-mediated neuron-specific circadian clock knockouts. The results point to two key clock neuron groups: loss of the clock within both regions but neither one alone has a strong behavioral phenotype in darkness; communication between these regions also contributes to circadian period determination. Under these dark conditions, the clock within one region persists without network communication. The clock within the famous PDF-expressing s-LNv neurons however was strongly dependent on network communication, likely because clock gene expression within these vulnerable sLNvs depends on neuronal firing or light.


Several studies have investigated interactions between different clock neurons. Artificially
How neuronal communication influences the fly core feedback loop is not well understood. 75 The latter consists of several interlocked transcriptional-translational feedback loops, which 76 probably underlie rhythms in behavior and physiology (Hardin, 2011). A simplified version of ). In addition, neuronal activation is able to mimic a light pulse and phase shift the clock 93 due to firing-mediated TIM degradation . 94 To investigate more general features of clock neuron interactions on the circadian machinery, 95 we silenced the majority of the fly brain clock neurons and investigated behavior and clock 96 protein cycling within the circadian network in a standard light-dark cycle (LD) as well as in 97 constant darkness (DD). Silencing abolished rhythmic behavior but had no effect on clock 98 protein cycling in LD, indicating that the silencing affects circadian output but not oscillator was not affected by neuronal silencing in DD, the sLNvs dampened almost immediately. 102 Interestingly, this differential effect is under transcriptional control, suggesting that some 103 Drosophila clock neurons experience activity-regulated clock gene transcription. Cell-specific 104 CRISPR/Cas9 knockouts of the core clock protein PER further suggests that network properties 105 are critical to maintain wild-type activity-rest rhythms. Our data taken together show that clock 106 neuron communication and firing-mediated clock gene transcription are essential for high 107 amplitude and synchronized molecular rhythms as well as rhythmic physiology.

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Results: 110 To investigate the effects of clock network communication on fly behavior, we silenced most 111 adult brain clock neurons using UAS-Kir (Johns et al., 1999). To this end, we used the clk856-112 GAL4 driver, which is expressed in most clock neurons (Gummadova et al., 2009) and first 113 addressed locomotor activity behavior in a 12:12 LD cycle.

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To address possible developmental defects, we added tub-GAL80ts as an additional 120 transgene to silence the clock network in an adult-specific manner. In this system, GAL80 is 121 active at low temperatures (18ºC) and inhibits GAL4 expression. By increasing the temperature 122 to 30ºC, GAL80 is inactivated, GAL4 is then functional and the clk856 network silenced  At the low temperature, the controls and experimental lines show a typical wild-type 125 bimodal activity pattern, which disappeared in experimental flies after switching to the high 126 temperature (Suppl. Fig. 1). This shows that the clk856>Kir phenotype is not caused by defects 127 during development. 128 We next compared the behavior to flies with silenced PDF neurons. Adult-specific 129 silencing of the PDF neurons using the gene-switch system reduced M anticipation and 130 significantly advanced the timing of the E peak ( Fig. 1E and 1F Fig. 1G-1L). This indicates that network silencing has no detectable effect on clock protein 140 timing or cycling amplitude in LD. These data further suggest that either the different neuron 141 clocks are self-sustained, comparable to the mammalian liver, or that light can drive rhythmic 142 gene expression even in absence of neuronal communication.

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To distinguish between these possibilities, we assayed behavior and molecular cycling in 144 constant darkness (DD). Only 17% of the silenced flies were rhythmic, indicating that network 145 silencing causes high levels of DD arrhythmicity ( Fig. 2A). To rule out developmental effects, 146 we applied the tub-GAL80ts system as described above: 80 percent of the experimental flies were 147 8 rhythmic at 18ºC, but they were profoundly arrhythmic at 30ºC with only two rhythmic flies 148 (Suppl. Fig. 2). In contrast, adult-specific silencing of only the PDF neurons more weakly 149 reduced rhythmicity ( Fig. 2A) and also caused a short period (Fig. 2B), phenotypes that are 150 essentially indistinguishable from those of the classical pdf 01 mutant (Renn et al., 1999).

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To address why network silencing has such a profound effect, we assayed PER and PDP1 152 protein cycling after five days in constant darkness (DD5). As expected, all assayed clock 153 neurons from control strains maintain robust and coordinated cycling in DD (Fig. 2C-H); the 154 sLNvs, LNds and DN1s peak slightly sooner than in LD, consistent with the slightly less than 24 155 hr circadian period in DD (Fig. 2B).

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In striking contrast, silencing the clock network causes clock protein cycling within the 157 individual neuronal subgroups to differ strongly from each other, in amplitude and in phase.

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Clock protein cycling in the LNds is least affected by neuronal silencing and with little to no 159 change in phase or amplitude, suggesting a robust and possibly self-autonomous clock in these 160 neurons; see Discussion ( Fig. 2D and 2G). The sLNvs in contrast dampen and rapidly become 161 arrhythmic, suggesting that these cells are rather weak oscillators and require network activity or 162 light for proper molecular rhythms ( Fig. 2C and 2F). The DN1s also dampen but less strongly.

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They manifest low amplitude cycling, which is phase-advanced; this intermediate situation 164 suggests a fast and somewhat network dependent clock in DN1s ( Fig. 2E and 2H). The DN2s 165 were similar to the DN1s (data not shown). A comparable set of effects were observed in adult-166 specific silencing experiments (Suppl. Fig. 3).

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To further address the molecular basis of the silencing dependence, we applied a 168 fluorescent in-situ hybridization (fish) protocol to whole-mount Drosophila brains. Because per 169 mRNA was undetectable, likely due to low expression within the clock network (data not shown robustly in both sLNvs and LNds with a peak towards the beginning of the night as expected. In 173 addition, clock network silencing had no effect on tim mRNA cycling amplitude or phase in LD, 174 which parallels the protein cycling results ( Fig. 3A and 3B). In constant darkness (DD5), the 175 controls show robust cycling in both sLNvs and LNds as expected, but silencing causes a 176 profound decrease in tim mRNA signal in the sLNvs; the LNds cycle normally ( Fig. 3C and 3D).

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These data indicate a direct correlation between neuronal activity and tim RNA levels at least in 178 the sLNvs and suggest that the silencing-mediated changes in clock protein cycling are in part 179 transcriptional in origin.

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Network silencing therefore reveals different levels of autonomy and endogenous speeds 181 among clock neuron clusters. This leads to a drifting apart of the different subgroups from their 182 usual well-synchronized and robust clock protein expression pattern. Interestingly, it appears that 183 these phase differences are too big to re-establish coordinated rhythms after one week of 184 silencing; there is no indication of rhythmic behavior upon lowering the temperature in the 185 tubGAL80ts experiment (Suppl. Fig. 4).

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The results to this point indicate that neuronal activity/communication is essential for 187 rhythmicity as well as synchronized, high amplitude clock protein cycling in DD conditions.

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However, these results do not provide a hierarchy among the different groups, nor do they 189 address a need for the circadian clock within these neurons. To distinguish between these 190 possibilities and to develop a general knock-out strategy within the adult fly brain, we   activity (E cells: 3 LNds and the 5th sLNv) with MB122B-split-GAL4 had no effect on 217 rhythmicity (Fig. 4J). However, a PERKO in both groups achieved with Mai179-GAL4, lowered 218 rhythmicity to less than 20% (Fig. 4K). Similar results were obtained with dvPDF-GAL4, which 219 expresses in similar neuron groups (data not shown).

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To address whether other neurons have similar effects, we expressed the PER guides 221 elsewhere: knockout in the retina (GMR-GAL4), glial cells (repo-GAL4) or DN1s (clk4.1M-222 GAL4 and AstC-GAL4) did not affect rhythmicity (Suppl. Fig. 6). These findings taken together 223 suggest that a clock in either of two key places, the sLNvs or the LNds, can drive rhythmic 224 behavior. 225 We also assayed the free-running DD periods of flies lacking PER in individual neuron  showing that silencing the PDF neurons had no effect on PER cycling within these neurons DD; in contrast, robust cycling was maintained in the LNds (Fig 2D and 2G). This suggests that  The second approach was a cell-specific knockout strategy, applied to the clock neuron 295 network. We generated three guides targeting the CDS of per and also expressed CAS9 in a cell-296 specific manner. The guides caused double strand breaks in the per gene, which in turn led to 297 cell-specific per mutations. This adult brain knockout strategy worked reliably and specifically, 298 in glial cells as well as neurons, with a greater than 90% efficiency and with no apparent 299 background effects (Fig. 4B-G). We have successfully used this strategy to knock out most if not 300 all Drosophila GPCRs (data not shown) and believe it will be superior to RNAi for most 301 purposes.Importantly, expression of the guides with the clk856-GAL4 driver phenocopied per 01 302 behavior ( Fig. 4C and 4H). To focus on individual clock neurons, we generated cell-specific   Fly line generation: 365 We generated a UAS-per-g line following the protocol published by (Port and Bullock, 2016). In  continued recording the behavior for 6 more days in DD at 18°C. 393 We generated actograms using ActogramJ (Schmid et al., 2011). We next generated average 394 activity profiles of at least the last 3 days of LD condition as previously described (Schlichting 395 and Helfrich-Förster, 2015). Each experiment consists of at least 2 biological repeats. DD 396 analysis was performed using chi2-analysis. Statistical analysis was performed using a student's 397 t-test or one-way ANOVA followed by post-hoc Tukey analysis.       whereas both controls (black) show high levels of rhythmicity. We observed no effect on free-552 running period at 18 degrees (C) but flies experimental flies showed the tendency towards a long 553 period at 30 degrees (D), similar to clk856>Kir (Fig. 2B).