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The Drosophila circadian pacemaker circuit: Pas De Deux or Tarantella? - PubMed

Review

The Drosophila circadian pacemaker circuit: Pas De Deux or Tarantella?

Vasu Sheeba et al. Crit Rev Biochem Mol Biol. 2008 Jan-Feb.

Abstract

Molecular genetic analysis of the fruit fly Drosophila melanogaster has revolutionized our understanding of the transcription/translation loop mechanisms underlying the circadian molecular oscillator. More recently, Drosophila has been used to understand how different neuronal groups within the circadian pacemaker circuit interact to regulate the overall behavior of the fly in response to daily cyclic environmental cues as well as seasonal changes. Our present understanding of circadian timekeeping at the molecular and circuit level is discussed with a critical evaluation of the strengths and weaknesses of present models. Two models for circadian neural circuits are compared: one that posits that two anatomically distinct oscillators control the synchronization to the two major daily morning and evening transitions, versus a distributed network model that posits that many cell-autonomous oscillators are coordinated in a complex fashion and respond via plastic mechanisms to changes in environmental cues.

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Figures

FIGURE 1
FIGURE 1

FIGURE 1A The Drosophila molecular circadian clock in pacemaker cells: the essence. The rhythm in activity levels of adult flies shows two peaks, one around dawn and the other around dusk. The pattern of activity rest rhythms is greatly influenced by level and sub cellular localization of PERIOD protein in a group of ventral lateral pacemaker neurons (small LNv and similarly in other pacemaker neurons). Highest levels of PER is seen around dawn (defined as Zeitgeber Time 0, ZT0) and peak nuclear localization occurs shortly before dawn (around ZT 22, Shafer et al., 2002; red arrow). The levels of per mRNA follow a similar pattern with an approximately 6-hour phase advance. Oscillations in mRNA, protein levels and post-translationally modified states of several other genes have been implicated in the generation of rhythmic behavioral and metabolic processes, the intricacies of which are described in Figure1B. FIGURE 1B The Drosophila molecular circadian clock in pacemaker cells: the gory details. The Drosophila circadian clock consists of interlocked sets of transcription/translation feedback loops (TTFL), which have mRNA and protein components that cycle in abundance and subcellular localization with a near 24-hour period. Dotted or dashed arrows indicate pathways that are not completely resolved or not considered essential for the self-sustained biochemical oscillator function. Lines ending in arrows indicate activation while those ending in a hatched bar indicate repression or inhibition. Degraded proteins are indicated by ovals with dotted walls. Light ordinarily recalibrates the clock at the onset of each day. During the previous night, PER and TIM proteins have accumulated at their highest levels in the nucleus, acting as transcriptional repressors of their own mRNA expression by binding to the transcriptional activators (only per repression is shown for simplicity) CLK-CYC, thus forming the negative limb of one of the TTFLs (loop1). Starting at dawn, light degrades TIM via a CRYPTOCHROME (CRY) mediated pathway, subsequently monomeric PER is phosphorylated and degraded after SLIMB an F-Box protein marks it for proteosomal degradation. By noon, PER and TIM’s degradation releases transcriptional repression and the transcription of period (per) and timeless (tim) genes is activated by CLK-CYC which bind to E-box sequences in per and tim promoters forming the positive limb of loop1 and attaining peak levels of per and tim transcripts at dusk. In the cytoplasm, DOUBLE-TIME (DBT) kinase complexes with and destabilizes PER by phosphorylating and thus facilitating its subsequent degradation. PER and TIM are each also phosphorylated by Casein kinase 2 (CK2) and SHAGGY (SGG), respectively. Since TIM is light sensitive, TIM levels can begin to rise in the cytoplasm only after dusk, after which it complexes with the DBT-PER heteromers. Levels of PER and TIM are maximal by mid-night. The entry of DBT-PER/TIM heteromultimer into the nucleus around midnight is controversial, it is possible that they dissociate such that PER (along with DBT) enters the nucleus at least 3 hours before TIM does. Cytoplasmic PER is stabilized by Protein Phosphatase 2A (PP2A), which dephosphorylates PER while Protein Phosphatase1 (PP1) dephosphorylates and stabilizes TIM thus promoting PER accumulation and hetero-dimerization. Total CLK levels remain constant with circadian oscillation in its phosphorylation state due to the action of the multifunctional DBT and perhaps other kinases and phosphatases including PP2A. CLK heterodimerizes with the constitutively present CYC and this CLK-CYC complex in addition to activation of per and tim transcription also binds to E boxes of at least two other genes vrille (vri) and par domain protein-1ε(pdp-1ε) to activate their transcription. PDP-1 in turn activates transcription of Clk, while VRI represses it by competitively binding to regulatory sequences called VRI/PDP1ε- boxes (V/P-boxes, shown by dotted and hashed line), upstream of Clk, forming a second feedback loop that interlocks with the first via CLK/CYC. Recent studies indicate that the second loop may not be an essential component of the circadian pacemaking machinery as Clk cycling is nonessential for clock function. Instead, the cycling in phosphorylation state of CLK is thought to contribute towards maintaining a robust period. In addition to core-clock genes, CLK regulates mRNA levels of several output genes (and cry via VRI and possibly PDP1ε- not shown). PER-TIM complex is also thought to repress CLK/CYC transcription of vri and pdp-1ε(not shown). PDP1εis now believed to function as an oscillator output component rather than a central oscillator component. The amplitude of the circadian biochemical oscillator may be regulated by Clk-mediated transcriptional activation of various genes including clockwork orange (cwo) which is proposed to contribute to robustness of the amplitude of mRNA oscillations of vri, pdp-1ε, tim, and per. For a depiction of how the amounts and location of these clock molecules vary over time (see Yu and Hardin 2006).

FIGURE 1
FIGURE 1

FIGURE 1A The Drosophila molecular circadian clock in pacemaker cells: the essence. The rhythm in activity levels of adult flies shows two peaks, one around dawn and the other around dusk. The pattern of activity rest rhythms is greatly influenced by level and sub cellular localization of PERIOD protein in a group of ventral lateral pacemaker neurons (small LNv and similarly in other pacemaker neurons). Highest levels of PER is seen around dawn (defined as Zeitgeber Time 0, ZT0) and peak nuclear localization occurs shortly before dawn (around ZT 22, Shafer et al., 2002; red arrow). The levels of per mRNA follow a similar pattern with an approximately 6-hour phase advance. Oscillations in mRNA, protein levels and post-translationally modified states of several other genes have been implicated in the generation of rhythmic behavioral and metabolic processes, the intricacies of which are described in Figure1B. FIGURE 1B The Drosophila molecular circadian clock in pacemaker cells: the gory details. The Drosophila circadian clock consists of interlocked sets of transcription/translation feedback loops (TTFL), which have mRNA and protein components that cycle in abundance and subcellular localization with a near 24-hour period. Dotted or dashed arrows indicate pathways that are not completely resolved or not considered essential for the self-sustained biochemical oscillator function. Lines ending in arrows indicate activation while those ending in a hatched bar indicate repression or inhibition. Degraded proteins are indicated by ovals with dotted walls. Light ordinarily recalibrates the clock at the onset of each day. During the previous night, PER and TIM proteins have accumulated at their highest levels in the nucleus, acting as transcriptional repressors of their own mRNA expression by binding to the transcriptional activators (only per repression is shown for simplicity) CLK-CYC, thus forming the negative limb of one of the TTFLs (loop1). Starting at dawn, light degrades TIM via a CRYPTOCHROME (CRY) mediated pathway, subsequently monomeric PER is phosphorylated and degraded after SLIMB an F-Box protein marks it for proteosomal degradation. By noon, PER and TIM’s degradation releases transcriptional repression and the transcription of period (per) and timeless (tim) genes is activated by CLK-CYC which bind to E-box sequences in per and tim promoters forming the positive limb of loop1 and attaining peak levels of per and tim transcripts at dusk. In the cytoplasm, DOUBLE-TIME (DBT) kinase complexes with and destabilizes PER by phosphorylating and thus facilitating its subsequent degradation. PER and TIM are each also phosphorylated by Casein kinase 2 (CK2) and SHAGGY (SGG), respectively. Since TIM is light sensitive, TIM levels can begin to rise in the cytoplasm only after dusk, after which it complexes with the DBT-PER heteromers. Levels of PER and TIM are maximal by mid-night. The entry of DBT-PER/TIM heteromultimer into the nucleus around midnight is controversial, it is possible that they dissociate such that PER (along with DBT) enters the nucleus at least 3 hours before TIM does. Cytoplasmic PER is stabilized by Protein Phosphatase 2A (PP2A), which dephosphorylates PER while Protein Phosphatase1 (PP1) dephosphorylates and stabilizes TIM thus promoting PER accumulation and hetero-dimerization. Total CLK levels remain constant with circadian oscillation in its phosphorylation state due to the action of the multifunctional DBT and perhaps other kinases and phosphatases including PP2A. CLK heterodimerizes with the constitutively present CYC and this CLK-CYC complex in addition to activation of per and tim transcription also binds to E boxes of at least two other genes vrille (vri) and par domain protein-1ε(pdp-1ε) to activate their transcription. PDP-1 in turn activates transcription of Clk, while VRI represses it by competitively binding to regulatory sequences called VRI/PDP1ε- boxes (V/P-boxes, shown by dotted and hashed line), upstream of Clk, forming a second feedback loop that interlocks with the first via CLK/CYC. Recent studies indicate that the second loop may not be an essential component of the circadian pacemaking machinery as Clk cycling is nonessential for clock function. Instead, the cycling in phosphorylation state of CLK is thought to contribute towards maintaining a robust period. In addition to core-clock genes, CLK regulates mRNA levels of several output genes (and cry via VRI and possibly PDP1ε- not shown). PER-TIM complex is also thought to repress CLK/CYC transcription of vri and pdp-1ε(not shown). PDP1εis now believed to function as an oscillator output component rather than a central oscillator component. The amplitude of the circadian biochemical oscillator may be regulated by Clk-mediated transcriptional activation of various genes including clockwork orange (cwo) which is proposed to contribute to robustness of the amplitude of mRNA oscillations of vri, pdp-1ε, tim, and per. For a depiction of how the amounts and location of these clock molecules vary over time (see Yu and Hardin 2006).

FIGURE 2
FIGURE 2

Neuronal network that regulates circadian rhythmicity in the adult Drosophila brain. Locations and putative arborization patterns of Drosophila clock neurons as originally illustrated by Helfrich-Forster et al. (2007). Each neuronal cluster is depicted in distinct color for clarification of neuronal morphology in a frontal view of a brain: Large LNv, brown; PDF-positive small LNv, red; PDF-negative fifth small LNv, dark violet; LNd, orange; DN1a,p, lilac; DN1, blue; DN2, light blue; DN3, navy; LPN cell bodies, green; photoreceptors including H-B eyelet, yellow. Bedsides PDF-positive LNv, fifth small LNv (dark violet arrowhead ), DN1a,p (lilac arrowhead ), LNd (orange arrowhead ), and DN3 (navy arrowhead) invade the AMe ipsilaterally. Large LNv (brown arrow ) and LNd (orange arrow ) send contralateral projections to the AMe. Brown arrowhead points to the ventral elongation of the AMe which receives innervations from large LNv. (Reprinted with permission of Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.)

FIGURE 3
FIGURE 3

LNv regulation of morning and evening locomotor activity. Representative activity profiles of three genotypes under 12:12 hour light/dark cycles, blue shaded areas represent darkness, and the yellow shaded area represents light. All three genotypes show enhanced activity around dawn and dusk. The controls (solid black curve) show an increase in activity both in anticipation of dawn and dusk as indicated by the black asterisk (morning anticipation) and arrow (evening anticipation). When LNv neurons are electrically silenced by targeted expression of the dORKΔ-C1 channel (red dotted curve) evening anticipation is phase advanced as indicated by the red arrow and occurs around 3 hour before lights OFF while morning anticipation is not significantly altered (red asterisk). A more severe phenotype is obtained with expression of Kir2.1 channel with both a loss of morning anticipation and a shift in evening anticipation (data not shown). The null mutant pdf01 (green dashed curve) shows complete loss of morning anticipation as well as a large phase advance in the evening anticipation. These results indicate a role of the LNv cells in mediating both peaks of the activity rest cycle in Drosophila.

FIGURE 4
FIGURE 4

Schematic summary of locomotor activity level and its relationship with molecular oscillation in Drosophila brain circadian pacemaker neuronal subgroups. Horizontal bars at the bottom of each panel depict the light regime. White and black bars indicate the light and dark duration under light/dark (LD) cycles. Constant darkness (DD) is denoted by pale and dark grey bars corresponding to the light and dark durations of previous entrainment regime and similarly constant light (LL) is denoted by white and pale grey bars. Activity level is indicated by the grey filled wave above the LD bars. The neuronal subgroups are color coded in most panels except in cases where the oscillation is synchronous in all the cells assayed, in which case a double lined curve is used. (A) The adult locomotor activity shows two clear peaks in activity under LD 12:12 hr and the level of PER in all cells occur at approximately the same phase and coincides with the morning peak in activity (Bachleitner et al., 2007). Upon transfer to DD, locomotor activity of wild-type flies usually shows only one peak; very often this peak appears to be derived from the evening peak of prior entrainment. (B-C) Lin et al., (2004), showed that molecular oscillations in sLNv and LNd stay tightly synchronized in case of wild-type controls (yw) for up to 6 days in DD, and that in pdf01 flies the dampening in activity rhythm is accompanied by a dampening in molecular oscillations among the sLNv probably due to loss in intercellular communication among sLNv and a phase advance as well as dampening in oscillations among LNd, suggesting that PDF is the agent of synchrony both within and between sLNv and LNd subgroups. (D) Such a dampening and ultimate stop in molecular oscillations was also seen when LNv are electrically silenced using Kir2.1 channels along with arrhythmic locomotor behavior in DD (Nitabach et al., 2002). (E-H) mRNA levels have been used as an indicator of the state of the clock in some studies (Stoleru et al., 2004, 2005, 2007). (E) Under LD 12:12 hr, high level of per mRNA is seen soon after lights OFF and the level is lowest around early morning. (F) Stoleru et al. (2005) report that mRNA level oscillates with a single peak on the fourth day of DD in all the neuronal groups examined and the phase of oscillation remains in close synchrony among the different neuronal subgroups. (G) When SGG is expressed in LNv, the “morning” cells, activity is phase advanced and so is the phase of mRNA oscillation in all cells; with greatest advance in sLNv and DN1 cells, followed by LNd and lastly DN2. (H) Such a phase advance is seen in all the neuronal subgroups to a similar degree when SGG is expressed using the tim-GAL4 driver. (H1) When SGG expression is restricted to non-LNv cells, only DN2 cells (and lLNv, not shown) are phase advanced and other cells are similarly phased as sLNv indicating a dominance of sLNv. (H2) Alternatively when DN2 cells alone express SGG, the advance in molecular oscillation does not alter any of the other cells except lLNv (not shown) (I-J) Veleri et al. (2003) assayed molecular oscillations in control (yw) and disco mutant flies after 5 days in DD and report that oscillation in DN2 cells are now in anti-phase with the rest of the neuronal groups. disco mutants which lack almost all the LN neurons exhibit anti-phase oscillations in both DN1 and DN2 cells compared to DN3 oscillations, which have similar phase as controls. (K) Out-of-phase oscillations in DN2 were also detected in rhythmic control flies expressing dORK-NC1 channel after five days in DD (Nitabach et al., 2006). (L) In contrast, flies expressing NaChBac channel in LNv exhibit arrhythmic behavior and asynchrony in molecular oscillations among the different neuronal subgroups when assayed after 5 days in DD. (M) After 14 days in DD, control flies continue to exhibit robust rhythmic activity with a single peak in activity level. The peak in PER levels in the sLNv occurs at the trough of activity level. The oscillation in DN1 and DN2 are delayed with respect to the sLNv, and LNd shows a dampened oscillation with a clear trough just before activity onset. (N) NaChBac flies show two clear bouts of activity at day 14 DD, one bout exhibits a shorter than 24-hour free running period and the other a longer than 24-hour free running period. The sLNv show peak PER levels coinciding with the long-period activity bout. The DN1 show two peaks each coinciding with one peak of activity. DN2 cells also show higher PER levels coinciding with activity peaks, although they are not significantly different from the other two time points. Under LL, while most wild-type flies are arrhythmic, cryb mutants exhibit two periodicities. This is evident after at least 5 days of LL (Rieger et al., 2006). (O) On the first day of LL only one peak in activity is seen and at that time point, the sLNv, DN2 and a subset of LNd express low levels of PER. At the trough of activity profile, the levels of PER is high in these cells. Other cells do not exhibit significant oscillation. On the fifth day of LL, the activity pattern shows two distinct bouts. DN2, fifth sLNv and one large LNd (extra LNd, dotted orange line) appear synchronous and express high PER coinciding with the activity peak of the short-period bout, and low PER levels corresponding to the time of the long-period bout. PER levels in other four sLNv are in anti-phase with the above cells and have high PER coinciding with the long-period bout. The oscillations in other cell groups as determined by sampling at these two active phases are not statistically significant.

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