The synchronization of peripheral circadian clocks by simulated body temperature cycles and feeding rhythms

(1)

Thesis

Reference

The synchronization of peripheral circadian clocks by simulated body temperature cycles and feeding rhythms

SAINI, Camille

Abstract

Des oscillateurs moléculaires générant des rythmes circadiens sont présents dans chaque cellule et contrôlent de nombreux aspects de la physiologie. Ces oscillateurs restent synchronisés grâce à une horloge centrale, dans l'hypothalamus, qui règle la phase des oscillateurs périphériques dans le reste du corps. En utilisant des reporteurs circadiens bioluminescents, nous avons montré que des cycles de température peuvent synchroniser les oscillateurs des fibroblastes en culture et que le facteur de transcription HSF1 est requis durant ce processus. Ceci suggère que le rythme de température corporelle, lui-même régulé par l'horloge centrale, pourrait participer à la synchronisation des oscillateurs périphériques.

Nous avons établi un système permettant de visualiser l'expression circadienne de reporteurs bioluminescents dans le foie de souris. Ceci nous a permis de montrer que l'horloge centrale est nécessaire pour coordonner la phase des oscillateurs individuels hépatiques et pour empêcher que cette phase ne change abruptement suite à un rythme alimentaire contradictoire.

SAINI, Camille. The synchronization of peripheral circadian clocks by simulated body temperature cycles and feeding rhythms. Thèse de doctorat : Univ. Genève, 2011, no. Sc.

4310

URN : urn:nbn:ch:unige-161692

DOI : 10.13097/archive-ouverte/unige:16169

Available at:

http://archive-ouverte.unige.ch/unige:16169

Disclaimer: layout of this document may differ from the published version.

(2)

UNIVERSITE DE GENEVE FACULTE DES SCIENCES Département de Biologie Moléculaire Professeur Ueli Schibler

The Synchronization of Peripheral Circadian Clocks by Simulated Body Temperature Cycles and Feeding Rhythms

THESE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologie

par

Camille Saini de Genève (GE)

Thèse N° 4310

Genève

Atelier de reprographie Repromail 2011

(3)

Remerciements

Je remercie vivement Ueli Schibler de m’avoir donné l’opportunité de travailler dans son laboratoire, pour la confiance et l’attention qu’il m’a accordées, ainsi que pour son apport intellectuel constant et son enthousiasme débordant durant toute ma thèse.

Je remercie aussi tous les membres du groupe Schibler pour leur amitié, leur disponibilité et leurs précieux conseils durant tout mon séjour au laboratoire. L’athmosphère agréable qu’ils ont généré a grandement contribué à ma motivation de travailler au laboratoire.

Je suis particulièrement reconnaissante envers André Liani pour sa motivation et son inventivité sans limites, ayant permis la construction de nouveaux systèmes de mesure de bioluminescence pour mes expériences sur les cellules en culture, ainsi que sur les souris.

Son apport intellectuel et technique ont été déterminants pour l’accomplissement de la plupart des résultats décrits dans ce manuscrit.

J’aimerais aussi remercier particulièrement Pascal Gos pour sa contribution et ses précieuses suggestions concernant le travail avec les souris.

Je remercie tous mes collaborateurs - Jean-Pierre Wolf, Luigi Bonacina, Florian Kreppel, Paul Franken, Thomas Curie, and Yann Emmenegger - pour leur contribution essentielle à la réalisation de mes projets de recherche.

Je remercie les professeurs David Shore et Steven Brown d’avoir accepté d’être les jurés de mon travail de thèse.

Je remercie aussi Nicolas Roggli pour la réalisation d’un grand nombre de figures présentées dans ce manuscrit, tous les membres du secrétariat, de la cuisine, de la plateforme de bioimagerie, de l’animalerie et bien sûr de l’atelier pour leur gentillesse et leur contribution essentielle à la réalisation de mes projets de recherche.

Finalement, j’aimerais remercier ma famille et mes amis pour leurs encouragements, et

(4)

Durant l’évolution, les mammifères, comme la plupart des autres organismes vivant sur la terre, se sont adaptés aux variations journalières et saisonnière de luminosité et de température, résultant de la rotation de la terre sur son axe et autour du soleil. Des oscillateurs moléculaires capables de générer des rythmes d’environ 24 heures ont pris place au cœur de quasiment chaque cellule et contrôlent de nombreux aspects de la physiologie et du comportement chez les mammifères. Ces horloges moléculaires permettent à l’organisme d’anticiper les changements journaliers, afin de régler et d’optimiser sa physiologie en accord avec ce rythme géophysique. Ces oscillateurs restent synchronisés au sein du corps grâce à un groupe d’oscillateurs situés dans l’hypothalamus, appelé horloge centrale, qui elle-même est synchronisée chaque jour par la lumière. L’horloge centrale emploie ensuite divers moyens pour régler la phase des oscillateurs périphériques, tel que la sécrétion d’hormone, l’envoi d’influx nerveux, ou plus indirectement le contrôle du cycle veille/sommeil et du cycle alimentaire en résultant, ainsi que probablement la régulation journalière de la température corporelle.

Afin de mieux comprendre ces mécanismes, notamment l’influence de la température dans le processus de synchronisation, nous avons dans un premier temps examiné les effets de différents stimuli de température sur les oscillateurs moléculaires opérant dans les fibroblastes de souris en culture. Pour ce faire, nous avons utilisé des reporteurs bioluminescents reflétant l’expression de divers gènes circadiens, tel que Bmal1, Dbp ou Per2. Nous avons ainsi pu montrer que des cycles de température, similaires aux fluctuations de température corporelle, sont capables de synchroniser les horloges moléculaires présentes dans ces cellules. De plus, nous avons pu démontrer que le facteur de transcription, heat shock factor 1 (HSF1), joue un rôle durant ce processus. Ces résultats suggèrent donc que la fluctuation journalière de la

(7)

périphériques au sein de l’animal entier, et que ceci nécessiterait la participation du facteur de transcription HSF1.

Dans un deuxième temps, pour pouvoir analyser les différentes voies de synchronisation entre l’horloge centrale et les horloges des tissus périphériques dans un animal entier, nous avons mis en place un système permettant la visualisation de l’expression de gènes circadiens dans le foie d’un animal intact et libre de mouvements. Nous avons introduit des transgènes bioluminescents reflétant l’expression de gènes circadiens dans le foie des souris par l’utilisation de vecteurs adénoviraux. La mesure en temps réel de la bioluminescence émanent de l’animal a pu être effectuée à l’aide de détecteurs de photons extrêmement sensibles. Cette approche nous a permis de visualiser l’expression circadienne des gènes Bmal1 ou Per2 durant plus d’une semaine au sein d’un même animal. De plus, grâce à cette méthode, nous avons pu examiner les influences respectives des signaux émis par l’horloge centrale et des signaux induits par la prise nourriture, sur les oscillateurs du foie.

Nous avons ainsi pu démontrer que l’horloge centrale joue un rôle essentiel dans la synchronisation des oscillateurs individuels du foie dans l’animal. Cette synchronisation est perdue an absence de l’horloge centrale, mais peut être restaurée par un rythme alimentaire imposé. Cette méthode ouvre donc la possibilité d’étudier une multitude de processus au sein d’un animal vivant, et dans le domaine circadien, notamment de mieux caractériser les diverses voies de synchronisation entre l’horloge centrale et les oscillateurs périphériques.

1.2 Abstract

During evolution, most of the organisms living on earth, including mammals, adapted to the daily and seasonal variations of luminosity and temperature resulting from the rotation of earth around its own axis and around the sun. Molecular oscillators able to generate rhythms of approximately 24 hours are present in virtually all cells. These molecular clocks are involved in the control of many aspects of physiology and behavior in mammals. They allow

(8)

the organism to anticipate daily changes in order to tune its physiology to geophysical time.

These oscillators keep synchrony within the body owing to a specific group of oscillators located in the hypothalamus, called the central clock, which is itself synchronized every day by light. The central clock uses then diverse means to reset the phase of peripheral oscillators;

these include hormones secretion, neuronal signals, or more indirectly the control of rest/activity cycle and the resulting fasting/feeding cycle, as well as probably the daily regulation of body temperature.

In order to better understand these mechanisms, notably the influence of temperature in the synchronization process, we first examined the effects of different temperature stimuli on the molecular oscillators operative in cultured mouse fibroblasts. To this end, we used bioluminescent reporters reflecting the expression of diverse circadian genes, such as Bmal1, Dbp or Per2. We were thus able to show that temperature cycles mimicking natural body temperature fluctuations can synchronize the molecular clocks present in these cells. In addition, we demonstrated that the transcription factor, heat shock factor 1 (HSF1) plays a role during this process. These results suggest therefore that daily fluctuations of body temperature may indeed participate in the synchronization of peripheral oscillators in the whole animal, and that this would necessitate the participation of HSF1.

Secondly, in order to analyze the different synchronization pathways between the central clock and the clocks in peripheral tissues in the context of a whole animal, we established a system allowing the visualization of circadian gene expression in the liver of an intact and freely moving mouse. We introduced bioluminescent transgenes - reflecting the expression of circadian genes - into mice liver by using adenoviral vectors. Real time monitoring of the bioluminescence emanating from the animal was possible using ultra sensitive photons detectors. This approach allowed us to visualize the circadian expression of Bmal1 or Per2 genes during more than one week within an individual animal. Moreover, using this method, we could examine the respective influences of signals emanating from the

(9)

central clock and food-induced signals on liver oscillators. We thus demonstrated that the central clock plays an essential role in the synchronization of individual oscillators within the liver in the animal. This synchronization is lost in the absence of the central clock, but can be restored by restricted feeding regimens. This method opens thus the possibility to study many processes within a living animal, and in the circadian field, will notably allow to better characterize the diverse synchronization pathways operative between the central clock and peripheral oscillators.

2. Introduction

2.1 The circadian clock

During evolution, all living organisms must adapt to variations in environmental conditions.

The most striking changes on earth are the alternation between day and night, the seasonal variations of their relative durations, and the temperature fluctuations associated with them.

These changes are the result of the cyclic geophysical properties of our planet, that is, it rotates around the sun in 365 days and rotates around its own axis in 24 hours with an angle of 23°27’.

Most eukaryotes and some prokaryotes are not only able to respond to these changes, but have developed an internal timing system allowing them to measure time, anticipate the environmental daily changes, and tune their physiology and behavior to these alterations. This system is based on molecular cyclic events running with a period length (τ or tau) of approximately 24 hours and is named the circadian clock (circadian been derived from the Latin words circa : about and diem : day). Because the circadian period produced by this auto regulatory mechanism is not exactly 24 hours, this timekeeper needs to be reset regularly.

This is achieved every day by the photoperiod, which also allows an adaptation to seasonal

(10)

changes. In turn, the circadian clock drives the different biological processes occurring in the organism in order to synchronize them with geophysical time in the most favorable way.

Light is the most important Zeitgeber (German for “time giver”). Nevertheless various other external stimuli such as temperature, nutrients availability, social interactions, etc. are able to reset the phase of the circadian clock. In nocturnal animals such as mice, light pulses given during the night are able to phase shifts the circadian system, while if they are given during the day, they have no effect. Moreover, if they are applied during the fist half of the night, they provoke phase delays, while if applied during the second half of the night, they engender phase advances (Daan and Pittendrigh, 1976). In a similar manner, temperature changes can phase-shift the circadian rhythm of locomotor activity and of eclosion in Drosophila (Zimmerman et al., 1968).

However, while temperature variations can reset the phase of the circadian clock, long-term high or low temperatures have nearly no influence on the period length of the oscillator. This astonishing property, called temperature compensation, allows the oscillator to continue to run with a relatively stable period length in a wide range of temperatures (Izumo et al., 2003; Tsuchiya et al., 2003). This is in sharp contrast with the strong temperature dependence observed for most biochemical reactions. This feature is very relevant for time keeping in cold-blooded organisms whose body temperature can be subject to important daily or seasonal changes.

The circadian clock is self-sustained, that is, it continues to run in constant conditions. This was first shown in 1729 by the French astronomer Jean-Jacques d'Ortous De Mairan. He demonstrated that the sunlight was not necessary for the movements of mimosa leaves by placing the plant in total darkness. Thus, even under these nearly constant conditions, the leaves opened during the subjective day and folded during the subjective night (DeMairan, 1729) (Figure 1). The existence of an internal clock was then shown to be present also in animals, including humans. In 1965, a study of Jurgen Aschoff and Rutger Wever revealed

(11)

the persistence of a close to 24 hours rhythmicity of many aspects of behavior and physiology in voluntary subjects retreated into a bunker devoid of environmental cues (Aschoff, 1965).

The presence of circadian rhythms in some prokaryotes, such as Synecocchus elongatus cyanobacteria, was the first evidence that a molecular oscillator can reside within a single cell (Golden et al., 1997). In the context of multicellular organisms, each cell represents an autonomous oscillator that does not require intercellular communication to create the oscillation. However interactions between cells can play an important role in the

synchronization of individual clocks at the level of a tissue. Indeed, autocrine or paracrine secretion can synchronize and/or reinforce cell autonomous oscillations (Harmar et al., 2002;

Liu et al., 2007; Peng et al., 2003). In addition, the coordination of multiple oscillators throughout a whole organism can be achieved owing to the establishment of a hierarchical organization of these clocks. In mammals for example, specialized groups of neurons that constitute the suprachiasmatic nuclei (SCN) in the anterior hypothalamus coordinate the oscillators present in the cells of adjacent brain regions and of the different peripheral organs Figure 1. A representation of de Mairan’s original experiment.

When exposed to sunlight during the day (upper left), the leaves of the plant were open, and during the night (upper right) the leaves were folded. De Mairan showed that sunlight was not necessary for the leaves movements by placing the plant in total darkness: even under these constant conditions, the leaves opened during the day (lower left) and folded during the night (lower right). This figure is taken from (Moore-Ede et al., 1982).

(12)

via neural and/or humoral pathways (Reppert and Weaver, 2001). This “central clock” is located just above the optic chiasm and is synchronized by visual input from the photoperiod via direct and indirect pathways from the retina. In turn, it generates coordinated circadian outputs that regulate overt rhythms.

The ubiquitous presence of the circadian mechanism in organisms from cyanobacteria to green plants and humans implies that it fulfills functions that provide selective advantages to the organism. In all examined organisms circadian clocks are dispensable under laboratory conditions. However, under natural conditions, which are much more hostile, circadian clock must increase survival fitness. It should be emphasized that features providing only a small increase in survival per generation will eventually be selected after many generations. An increase in fitness provided by circadian clocks has been beautifully exemplified in the laboratory for cyanobacteria (Synecocchus elongatus) and the plant Arabidopsis Thaliana by competition experiments. In cyclic environmental conditions, a Synecocchus strain with a functioning clock rapidly outcompetes a clock-defective strain. However this selective advantage disappears in constant environment. In addition, competition experiments between strains with different period lengths revealed that a strain competes more effectively if its period length resonates with that of an imposed light-dark cycle (T-cycle) (Woelfle et al., 2004). Similarly, Dodd and co-workers have shown in Arabidopsis Thaliana that plants with a period length that matches that of the environmental rhythm contain more chlorophyll in their leaves, fix more carbon, grow faster, and survive better than plants with circadian periods differing those of environmental cycles (Dodd et al., 2005).

2.2 Molecular basis of the circadian oscillator

The circadian oscillator is based on molecular events that are performed in approximately one day. Once a loop is completed, the system is back to its starting point, and a new cycle can start again. The identification of the molecular components of circadian clocks in

(13)

cyanobacteria, fungi, flies, mammals and other organisms indicates that the basic clockwork circuitry in each system may be composed of only a few central components. Although the clock genes are different in different phyla, their expression shares common features and is based on negative feedback loop mechanisms. Thus, the core clock is composed of clock genes encoding positive regulatory proteins, while others encode negative regulatory proteins.

Positive factors activate the transcription of negative factors. The protein levels of the negative factors increases, and at a certain threshold these factors start to attenuate their own expression by interfering with the positive factors acting on their own promoter. As a consequence, the concentration of the negative components decreases until the autorepression can no more be performed. This brings the system back to its starting point. In parallel, the positive factors activate different target genes, whose expression is thus also dependent on the cyclic regulation exerted by the negative factors. These genes are called clock-controlled genes (CCGs). They are not part of the oscillator itself and constitute the basis of the oscillator’s output. The CCGs then influence physiology and behavior of the organism, either directly or indirectly by controlling additional downstream genes (Figure 2).

Inputs into the circadian system are responsible for resetting the phase of the oscillator. Under natural conditions, light and temperature are the dominant Zeitgebers.

However in the laboratory, it was shown that various stimuli could also reset the clock, mostly by a rapid and transient alteration of the expression of the negative core clock genes.

Such prompt responses allow classifying the negative factors in the family of immediate early genes (IEGs) (Albrecht et al., 1997; Balsalobre et al., 2000a; Balsalobre et al., 1998;

Balsalobre et al., 2000b; Shearman et al., 1997; Shigeyoshi et al., 1997).

The scheme in Figure 2 represents a simplified model of the circadian system, which in reality, can be made of one or more interconnected feedback loops. Furthermore, the situation can be even more complex since clock outputs can feed back on the clock itself. And in other cases, the oscillator might also regulate components of input pathways.

(14)

Figure 2. Molecular basis of the circadian oscillatory loop. The phase of the cycle is reset by input stimuli. The interplay between positive and negative core clock regulators generates the 24 hours cyclic expression pattern. The same positive and negative elements also drive the expression of clock- controlled genes that are not part of the oscillator itself. In turn, clock-controlled genes drive diverse biological processes making up the clock’s output.

While sharing a similar molecular basis, each organism obviously possesses different clock genes and diverse regulatory mechanisms to generate circadian rhythmicity. The circadian clock has been studied at the biochemical and genetic level in different model organisms, such as the cyanobacterium Synechococcus elongatus, the filamentous fungus Neurospora crassa, the fruit fly Drosophila melanogaster, the mouse Mus musculus, the microalgae Ostreococcus tauri, and the plant Arabidopsis thaliana. The identified positive elements include KaiA (kai means cycle in Japanese) in S.elongatus, WC-1 and WC-2 (white collar 1 and 2) in N.crassa, dCLK (clock) and CYC (cycle) in Drosophila, mCLK and mBMAL1 (brain and muscle Arnt-like protein 1) in mice, and APRR1 (pseudo-response regulator 1, also known as TOC1) in O.tauri and A.thaliana. In these six systems, the negative elements are KaiC in S.elongatus, FRQ (frequency) in N.crassa, dPER (period) and dTIM (timeless) in Drosophila, mPER1, mPER2, mPER3, and mCRY1, mCRY2 (cryptochrome 1 and 2) in mice, and CCA1 (circadian and clock associated 1) in O.tauri accompanied by LHY (late elongated hypocotyl) in A.thaliana (Corellou et al., 2009; Hardin,

(15)

transcriptional feedback loops associated with each of this model organisms are described in more details in sections 2.3 and 2.4.

Although rhythmic transcription seems to be required for rhythm generation by the core oscillator, it is becoming clear that post-transcriptional mechanisms also have important roles in producing the appropriate circadian expression profiles of some core clock components in most organisms (Dibner et al., 2009; Harmer et al., 2001; Kojima et al., 2011;

Mehra et al., 2009; O'Neill et al., 2011; Tomita et al., 2005). Post-transcriptional regulation can take place at the level of the mRNA; for example the transcription of Drosophila period gene (dper) is less rhythmic than the accumulation of its mRNAs. This suggests that a regulation at the level of mRNAs stability (modulated by differential splicing) contributes to the observed circadian accumulation (Majercak et al., 2004; Suri et al., 1999). Translational control can provide another stage of control; in Neurospora, for example, alternative initiation of translation and daytime-specific phosphorylation of the essential clock protein frequency (FRQ) give rise to two forms of FRQ proteins and drive their turnover; these controls of the ratio and abundance of FRQ isoforms turned out to be required for precise clock operation (Garceau et al., 1997). Changes in the subcellular localization of mammalian period proteins (mPERs) were also revealed to be important parameters for progression of the clockwork cycle. This subcellular gating was shown to involve post-translational modifications as well as protein-protein interactions (Vielhaber et al., 2001). Finally, protein stability also represents an important regulatory step in maintaining the robustness of the circadian clock.

Mammalian period 2 protein (mPER2) acetylation and Bmal1 protein (mBMAL1) phosphorylation have been shown to promote their stabilization and degradation, respectively (Asher et al., 2008; Sahar et al., 2010). So these multiple levels of post-transcriptional controls are in some cases essential for clock function, presumably to delay the steps between transcription and protein activity and thereby to prevent that the system collapses to equilibrium. In others cases, they are built into this system to maintain robust amplitude of

(16)

cycling, to buffer the clock mechanism against abrupt changes or to provide mechanisms by which the clock can be reset by environmental input.

2.3 Circadian systems of diverse organisms: variation around a theme

As mentioned above, delayed negative feedback loops are at the heart of oscillatory processes, and they are found implicated in all circadian clocks investigated to date. While central clock mechanisms are conserved, actual clock components do not seem to be conserved between kingdoms. Moreover, and somewhat paradoxically, clock components that are conserved between species can be used in diverse ways.

In three decades, the study of circadian rhythms in different model organisms has gone from observations of phenomena to the development of sophisticated biochemical, biophysical, genetic, and genomic tools with the aim to decipher the underlying mechanisms.

Genetic screens have proved useful in retrieving mutants with long-period, short-period, and arrhythmic phenotypes. In the following paragraphs, I will give a brief overview over the molecular aspects of circadian oscillators amongst representative organisms.

2.3.1 The prokaryotic Synechoccus elongatus circadian clock

Cyanobacteria are photoautotrophic prokaryotes that exhibit an exceptional variety of forms, faculties, and functions. They are found in nearly every habitat accessible to sunlight. In addition to their common photosynthetic characteristics, they represent the simplest organisms known to exhibit circadian rhythms. Physiological studies regarding the temporal separation of nitrogen fixation and oxygenic photosynthesis were among the first to suggest that cyanobacteria have endogenous timing mechanisms (Golden et al., 1997). Indeed, the cyanobacterial clock regulates many physiological processes, such as photosynthesis, nitrogenase activity, amino acid uptake, cell division or carbohydrate synthesis (for review, see (Golden, 2007). Figure 3 shows examples for the visualization of S.elongatus endogenous

(17)

Figure 3. Gene expression reflected by bioluminescence from a Ф(kaiB-luc+) reporter (left) and chromosome compaction rhythms (right) in S.elongatus (red, autofluorescence from S. elongatus;

green, DAPI-stained DNA). Cells are sampled at the indicated times during the light/dark cycle (upper) and the second free-running cycle (lower). These Figures are issued from (Smith and Williams, 2006).

rhythmicity.

The generation of a strain with an insertion of the luciferase coding sequence downstream the promoter of the circadian psbA1 gene, which encode a key component of the photosynthetic apparatus, allowed isolating a variety of clock mutants with bioluminescence as a readout. The genes corresponding to the mutants were shown to be comprised in a gene cluster composed of three genes, kaiA, kaiB and kaiC. Transcription of these three genes is rhythmic. Overexpression of kaiA increases the expression kaiBC, which share a single promoter and are co-transcribed. In contrast, overexpression of kaiC suppresses the expression of kaiBC (Ishiura et al., 1998). KaiA acts thus as a positive element that enhances KaiBC expression. KaiC, in turn, represses its own transcription as well as that of kaiB (Figure 4). KaiA protein levels do not oscillate, whereas KaiB and KaiC protein levels cycle.

This transcription/translation-based model had to be reconsidered when Tomita and co-workers observed that KaiC is phosphorylated in a circadian manner, even in absence of transcription and translation (Tomita et al., 2005). In the same perspective, an in vitro experiment assembling recombinant KaiA, KaiB and KaiC in the presence of ATP, revealed robust oscillations in KaiC phosphorylation with a period close to 24 hours for a few days

(18)

(Nakajima et al., 2005). KaiC possesses auto-kinase and auto-phosphatase activities that are regulated by KaiA and KaiB. KaiA dimers transiently bind KaiC hexamer units, and this promotes KaiC phosphorylation. Once phosphorylated, KaiC associates with KaiB, which inactivates KaiA and induces the auto-phosphatase activity of KaiC (Figure 5). These findings place KaiC phosphorylation at the heart of the S.elongatus clock. However, a recent study by the same group revealed that KaiA-overexpressing cyanobacteria, which have constitutively phosphorylated KaiC, show circadian gene expression (Kitayama et al., 2008). In fact, the KaiC phosphorylation cycle and the transcription/translation cycle appear to compensate each other, depending on the conditions, in order to maintain robust and precise circadian rhythm.

KaiC also interacts with a histidine protein kinase SasA (Synechococcus adaptive sensor A).

In turn, SasA mediates the activation of RpaA (regulator of phycobilisome-associated A), a DNA-binding protein that is believed to be necessary for coordinating genome-wide circadian gene expression with proper phase relationships and period lengths and for maintaining the robust oscillation of KaiC phosphorylation (Figure 5) (Takai et al., 2006).

Another study suggests that a cyclic change in genome topology (see Figure 3) could explain the universal rhythmicity of promoter activity in S. elongatus and the residual rhythmicity observed in sasA and rpaA mutants (Smith and Williams, 2006). Thus, the clock could include output pathways from the oscillator to gene expression using transcriptional modulation via SasA and RpaA and through whole scale remodeling of the chromosome.

Cyanobacterial circadian rhythms can be synchronized by light, nutrients and Figure 4. KaiC negatively regulates its own (kaiBC) expression to generate a molecular feedback loop. In contrast, KaiA activates kaiBC expression as a positive element to make the loop oscillate. This model implies circadian fluctuation in the levels of the Kai proteins.

This Figure is issued from (Harmer et al., 2001)

(19)

Figure 5. The oscillation in KaiC phosphorylation results from the opposing actions of KaiA, which stimulates KaiC autokinase activity, and KaiB, which abolishes the positive effect of KaiA. Temporal information is transduced from the Kai oscillator to SasA through the stimulation of SasA autophosphorylation by KaiC. SasR (RpaA) is activated by SasA via transfer of a phosphoryl group.

RpaA is necessary for overt rhythmicity of gene expression. Additionally, the Kai oscillator regulates the compaction rhythm of the cyanobacterial nucleoid (double-stranded loop), which would control accessibility of transcriptional machinery to promoter regions. An input to the oscillator is provided by CikA, which influences the state of KaiC phorphorylation in response to light variations. This Figure is issued from (Ditty et al., 2003)

period) have been identified as factors needed to transmit indirectly light cues to the circadian clock. CikA senses not light but rather the redox state of the plastoquinone pool, which, in photosynthetic organisms varies as a function of the light environment. Furthermore, CikA associates with the Kai proteins of the circadian oscillator, and it influences the phosphorylation state of KaiC (Ivleva et al., 2006). LdpA protein is also sensitive to the redox state of the cell, and this has been suggested to affect not only CikA, but also KaiA (Ivleva et al., 2005). This suggest that in S.elongatus, the clock receives at least some information about light not directly from a photoreceptor, but indirectly from a signal that reflects the redox state of the cell, which in photosynthetic organisms greatly depends on light intensity.

Important evidence emanating from the studies on the cyanobacterial clock is the

(20)

observation that circadian rhythm can be detected also in cells that are dividing faster than circadian frequency. This implies that DNA replication and cell division do not interfere with circadian oscillations, and that the oscillators pass time to daughter cells (Kondo et al., 1997).

2.3.2 The picoeukaryotic Ostreococcus tauri circadian clock

Picoeukaryote microalgaes represent an important, yet poorly characterized aspect of marine phytoplankton that have a worldwide distribution and are important contributors to biogeochemical cycles. Genome-wide analysis of gene expression in Ostreococcus tauri cells exposed to light/dark cycles revealed that nearly all expressed genes displayed rhythmic patterns of expression. Clusters of genes were associated with the main biological processes such as transcription in the nucleus and the organelles, photosynthesis, DNA replication and mitosis (Monnier et al., 2010; Moulager et al., 2007) (Figure 6). Recent results strongly suggest that its circadian clock is a simplified version of Arabidopsis thaliana clock (see below). Indeed, two putative homologs of higher-plant core clock genes, namely TOC1- and CCA1-like, referred to as TOC1 and CCA1, have been identified. Analysis of stably transformed lines expressing luciferase reporter genes to monitor TOC1 and CCA1 transcriptional and translational activity, in combination with overexpression or knockdown of either of these two components, show that TOC1 is an activator of CCA1, while CCA1 might repress the expression of TOC1, similar to what has been found in higher plants

Figure 6. The smallest free-living eukaryote, Ostrecoccus tauri, exhibits circadian cycles of cell division in continuous dim light (center) and gene expression of full TOC1 and CCA1 genes fused to luciferase upon release from LD: 12/12 into LL (right). These Figures are issued from (Moulager et

(21)

(Corellou et al., 2009). However, several lines of evidence, such as the robustness of the clock to CCA1 repression in LL, suggest the existence of additional unidentified components.

Measuring oxidation of peroxiredoxin (PRX) proteins as a rhythmic biomarker, a recent report shows that non-transcriptional mechanisms are sufficient to sustain circadian timekeeping in O.tauri (O'Neill et al., 2011). Although in the context of a living cell, transcription is ultimately required for any biological process, the natural state of a eukaryotic cellular clock might revolve around reciprocal interplays between post-translational oscillations and established transcriptional feedback loops.

2.3.3 The Neurospora crassa circadian clock

The filamentous fungus Neurospora crassa represents another model organism for the analysis of the cellular and molecular bases of circadian rhythms in eukaryotes, because of its relative simplicity and its easily assayed circadian rhythmicity. N.crassa is a haploid fungus that can grow and propagate asexually (production of conidia) or via a sexual cycle (formation of ascospores), both of these developments being regulated in a circadian fashion (Figure 7) (Bobrowicz et al., 2002; Dunlap and Loros, 2004). Classical genetic screens allowed the characterization and cloning of core clock and clock controlled genes (Aronson et al., 1994a; Loros et al., 1989). The N.crassa circadian system is based on interlocked negative and positive feedback loops. The positive core components include the transcription factors WC-1 and WC-2 that heterodimerize and drive the transcription of the negative element, frq, and of other clock-controlled genes (CCGs). WC-1 and WC-2 are able to bind DNA via their zinc finger domains and are held together via the interaction of protein dimerization domains called PAS domains (from PER-ARNT-SIM that were the first proteins shown to have this domain) (Aronson et al., 1994b; Crosthwaite et al., 1997; Froehlich et al., 2003). The presence of PAS domains in positive regulatory proteins was shown to be a characteristic

(22)

Figure 7. In N.crassa, the endogenous clock controls conidia formation. Correlation between circadian conidial bands formation (left, upper track) and bioluminescence expression from frq core clock promoter fused to luciferase (frq-luc-l) under free running condition in DD. Rhythms are most intense at the growth front, although there is a clear, but dampening, rhythm in older conidial bands (left, lower track). The trace on the right represents quantification of the luminescence of the entire race tube. These Figures are issued from (Hastings et al., 2008) and (Gooch et al., 2008).

of other eukaryotic circadian systems, such as Drosophila (Allada et al., 1998; Rutila et al., 1998) and mouse (King et al., 1997). Wc-1 and wc-2 transcription are not rhythmic. WC-2 proteins level is high and constant throughout the circadian period, while WC-1 proteins display an abundance rhythm almost antiphasic to frq. The FRQ proteins participate in separate actions during the clock cycle, each being essential to the daily rhythm. First, the FRQ proteins homodimerize and interfere with WC complex (WCC) by modulating the phosphorylation status of both subunits WC-1 and WC-2 (Schafmeier et al., 2005). FRQ thus feeds back on its own synthesis and regulates at the same time other genes controlled by WCC. This feedback regulation results in high amplitude circadian rhythms of frq RNA and protein levels. A second role of FRQ occurs in the cytoplasm, where FRQ is phosphorylated by several kinases and becomes an activator. In this state, it may promote the translation of wc-1 RNA, generating the cyclic accumulation of WC-1 proteins and facilitating the formation of WCC on a post-translational level. Finally, hyperphosphorylated FRQ is targeted to degradation. WCC is thus progressively dephosphorylated and becomes again able to activate frq transcription, thereby initiating a new circadian cycle (Figure 8) (Lee et al., 2000;

Schafmeier et al., 2006), for review, see (Brunner and Schafmeier, 2006; Dunlap and Loros,

(23)

2004).

The circadian oscillator regulates many biological processes, including the well studied conidia formation, but also CO2 production, a number of enzymatic activities, lipid and diacylglycerol metabolism, and growth rate. Attempts to identify CCGs in N.crassa have led to the estimation that approximately 10% of its transcripts are cycling. Some of them contain a C box identical to the one found in the frq promoter and bound by the WCC, thus suggesting a direct regulation by WCC. Some harbor ACEs (activating circadian elements) and/or LREs (light responsive elements) within their promoters. Still others have neither element, suggesting additional clock-controlled cis-acting elements that are perhaps the targets of transcriptional regulation by yet unknown CCGs. Interestingly, some genes were discovered to continue to cycle in a frq-null strain, albeit with a somewhat altered

Figure 8. Model for interconnected negative feedback loop and positive loop of N.crassa circadian system. (Morning) Newly synthesized FRQ is active as a repressor by promoting phosphorylation and inactivation of WCC. (Evening) High levels of hyperphosphorylated cytosolic FRQ are required to support WC-1 accumulation.

Cytosolic FRQ may also act on the level of WCC assembly. Newly assembled WCC accumulates in the nucleus and is still inactivated via FRQ-dependent phosphorylation. frq transcription remains repressed. (Night) FRQ is progressively phosphorylated and degraded. As the FRQ concentration decreases, WCC is progressively dephosphorylated (Schafmeier et al., 2005). Dephosphorylated WCC binds to the frq promoter and activates transcription. This Figure is taken from (Schafmeier et al., 2006)

(24)

phase (Correa et al., 2003). This is in accordance with the fact that frq-null strain have long been known to occasionally express a rhythm in conidial banding, although one that has lost most typical circadian features (Aronson et al., 1994a). Thus, these residual oscillations appeared to be irregular, damping, not resettable by input pathways, and, not temperature compensated. Nonetheless, they were observed under certain conditions, and their period length was dependent on temperature (for conidiation) and nutrition (for nitrate reductase (NR) activity) period length (Christensen et al., 2004; Merrow et al., 1999). Such non- circadian oscillators, which can exist in the absence of the FRQ/WC loop, were named FRQ- less oscillators (FLOs). These are possible slave oscillators that can be coupled to the FRQ/WCC circadian feedback loop, but can run on their own in a non-circadian manner in absence of the FRQ/WCC loop (Figure 9).

In a rhythmic environment, the phase of the N.crassa clock is adjusted to light and temperature cycles. The light response is maximal at blue light and acts through WCC to reset the clock by a rapid frq induction. WC-1 is associated with a flavin adenine dinucleotide (FAD) chromophore via its flavin-binding LOV domain (He et al., 2002). Light perceived by this LOV domain may induce a conformational change in WCC, thereby promoting its binding to LREs within the frq promoter. At the same time, light exposure renders WCC susceptible to phosphorylation, which then leads, with a kinetic delay, to the inactivation of WCC by reducing its affinity for LREs (He and Liu, 2005). The VVD protein allows the

Figure 9. A model for oscillators and networks controlling and integrating light and temperature influences in the N.crassa circadian system. Solid loops, lines, and arrows represent known feedback loops or regulatory relationships, and dotted lines represent those predicted to exist.

This Figure is issued from (Dunlap and Loros, 2004)

(25)

circadian clock to modulate its sensitivity to light resetting cues, as a function of daytime. In response to light, VVD mediates a photoadaptation process that modulates the organism’s response to subsequent light signals. Vvd is an immediate early light-induced gene controlled by WCC (Heintzen et al., 2001). It appears to be a negative regulator of light responses, since vvd mutants exhibit prolonged transcription of immediate early light-induced genes and altered light-induced phase-shifting properties. VVD proteins are present 30 minutes after light exposure and may participate in the alteration of WCC activity (Brunner and Schafmeier, 2006). VVD may help to entrain the N.crassa circadian clock properly with natural photoperiods. After completion of the Neurospora genome project,two putative red- light photoreceptors (N. crassa phytochrome orthologs phy-1 and phy-2) and one additional blue-light photoreceptor (N. crassa cryptochrome orthologue cry) were identified. Cry transcript and protein levels are strongly induced by blue light in a wc-1 dependent manner.

However, due to the lack of a detectable phenotype and atypical light responses in the respective knockout strains, the biological functions of PHY-1, PHY-2 and CRY remain to be discovered (Chen and Loros, 2009) (Figure 9).

Resetting of the clock by temperature is mediated via the levels of FRQ. As mentioned previously (section 2.2, last paragraph), N.crassa frq ORF encodes two versions of FRQ proteins, a large (l-FRQ) and a small (s-FRQ) isoform. Thermosensitive splicing and translation regulate the ratio and abundance of these isoforms, respectively; which are crucial for high-amplitude, self-sustained rhythmicity and for temperature compensation of the circadian clock (Garceau et al., 1997; Liu et al., 1997). At low temperature, frq precursor mRNA is preferentially spliced to give rise to s-FRQ. There is thus more s-FRQ at low temperature and more l-FRQ at high temperature. In parallel, the global abundance of both isoforms is regulated at the level of translation initiation. In the high-temperature range, ribosomes are scanning through nonconsensus AUGs of the upstream ORFs (uORFs) present in the 5′-UTR of frq, but mostly initiate translation at the main frq ORF, leading to efficient

(26)

expression of FRQ. In contrast, at low temperatures, scanning ribosomes initiate translation more efficiently at the uORFs, leading to a reduced translation of the downstream frq ORF.

Accordingly, the levels of both isoforms are elevated at high temperature and diminishe while temperature is decreasing (Diernfellner et al., 2005). So after a temperature change, the relation among frq mRNAs and FRQ proteins are instantaneously changed allowing a rapid response accordingly to the new situation. In this way, temperature changes reset the circadian clock directly within the core loop, and it has been shown that physiologically natural temperature differences can be dominant over light/dark transitions (Liu et al., 1998).

One of the goals of studying the circadian clocks in multiple model organisms is to determine if the properties of the clock in one organism are conserved in other organisms. It is interesting to note that another member of the fungi kingdom, Saccharomyces cerevisiae, which represents the most powerful genetic experimental system, still remains absent from the repertoire of circadian model organisms. It seems indeed that yeast cells do not harbor a circadian clock and that they have no homologs of known clock genes. They have however developed mechanisms allowing them to partition time allocated to diverse aspects of their metabolism (Chen et al., 2007; Robertson et al., 2008; Tu et al., 2007). Interestingly, a recent study has shown that the application of temperature cycle protocols reveals a circadian timing mechanism in S.cerevisiae, using pH as readout. The metabolism in yeast shows systematic circadian entrainment, responding to temperature cycle length and Zeitgeber (stimulus) strength, and rapidly damps in free running constant conditions. The ammonium permease, MEP2, and the general amino acid permease, GAP1, are expressed several hours ahead of the acidification of the media under temperature entrainment (Eelderink-Chen et al., 2010).

Although the authors suggest that the temperature cycle could entrain a circadian system that actively regulates the timing of the observed oscillations, more experiments are required to investigate circadian behavior and to identify the underlying genes. Nevertheless, the preliminary observations on the putative yeast clock could open new approaches to study

(27)

circadian timing system in eukaryotes, since yeast is easily cultured in the laboratory, its genome is sequenced, and null-strains are available for every non-essential gene.

2.3.4 The Drosophila circadian system

Drosophila, similar to N.crassa, represents one of the most successful systems in advancing the molecular dissection of circadian oscillators. In Drosophila, the clock controls a number of rhythmic outputs, including adult emergence (eclosion), locomotor activity, and olfactory physiology (Figure 10). Thus, the circadian clock gates the emergence of adults from their pupal state to dawn. Indeed in the wild, at this time of the day, the atmosphere is cool and moist. This minimizes the risk of desiccation as the emerging fly expands its folded wings and hardens its cuticle. Once emerged, adult flies restrict flying, foraging and mating activities to the day, while they tend to rest during the night. In contrast, their olfactory responses are highest at night.

The Drosophila circadian timing system serves as a good model for clocks in other animals, given that many of the components have been conserved. The molecular-genetic

Figure 10. Drosophila activity/rest cycle and per::luciferase in tissue explant. Circadian cycle of locomotor activity (monitored by infra-red beams) of a Drosophila melanogaster kept in continuous darkness (left). Activity occurs during anticipated day (grey bar), around dawn and dusk. This fly’s intrinsic period is slightly less than 24 hours and so activity drifts to the left. In period::luciferase transgenic flies light-sensitive, autonomous local pacemakers can be monitored as cycles of bioluminescence (right), here recorded from an isolated wing, initially synchronized to LD cycles, and then in continuous darkness. This Figure is issued from (Hastings et al., 2008).

(28)

study of circadian rhythms was initiated with the identification of the period (dper) mutant gene, based on its perturbation of the timing of fly eclosion and activity rhythms (Konopka and Benzer, 1971). Since then, several additional clock genes have been isolated and characterized (Williams and Sehgal, 2001).

The Drosophila circadian oscillator is based on two feedback loops in gene expression: a per/tim loop and a dClk loop. Transcriptional and post-transcriptional events are required for the connection and function of both loops (Figure 11). The first clues for the per/tim feedback loop came from the work of Hardin and co-workers in which mutations in per were shown to alter the period and phase, not only of the fly’s daily behavior, but also of per mRNA accumulation. Introduction of an active per gene rescued cycling of mutated per RNA as well as rhythmic locomotor activity, suggesting the implication of the active PER protein in an autoregulatory feedback loop mechanism (Hardin et al., 1990). This finding occurred before the discovery of the tim mutation, which was isolated in 1994 and shown to produce also arrhythmia of eclosion and locomotor activity (Sehgal et al., 1994). It is now known that to initiate the per/tim feedback loop, dCLK–CYC heterodimers bind E-box regulatory elements with a CACGTG consensus sequence on per and tim promoters, thereby activating the transcription of these genes (Taylor and Hardin, 2008). Mutations in either dClk or Cyc reduce levels of per and tim RNA as well as protein and also render flies arrhythmic for rest/activity and eclosion (Allada et al., 1998; Rutila et al., 1998). dCLK and CYC heterodimerize via helix-loop-helix motives and PAS domains. The latter domains also contribute to the dimerization of PER and the N.crassa proteins WC-1 and WC-2 (see above).

However, while WC-1 and WC-2 use Zinc fingers to bind DNA, dCLK and CYC bind DNA via bHLH (basic helix-loop-helix) domains (Darlington et al., 1998). The levels of per and tim transcripts and proteins are highly rhythmic, however, there is a lag in protein accumulation of ~5h with regard to mRNA accumulation. This delay is the result of

(29)

phosphorylation-dependent destabilization of PER by DBT (doubletime, the homolog of mammalian casein kinase 1ε), and possibly also CK2, followed by stabilization of phosphorylated PER by TIM binding (Lin et al., 2002; Nawathean and Rosbash, 2004). Loss- of-function mutations in the DBT kinase slow or stop the oscillator because of delayed PER degradation and hypophosphorylated PER accumulation (Price et al., 1998). The nuclear translocation of the PER/TIM heterodimer is regulated by the phorphorylation of TIM by SGG/GSK-3 (shaggy, glycogen synthase kinase 3 ortolog). Reducing SGG/GSK-3 activity lengthens the period, because of a delayed nuclear entry of PER/TIM (Martinek et al., 2001).

DBT remains bound to PER to form DBT/PER/TIM complexes that can enter the nucleus. In the nucleus, PER is thought to repress dCLK/CYC dependent transcription by binding to dCLK and inhibiting the DNA binding activity of CLK/CYC dimers (Chang and Reppert, 2003; Lee et al., 1999). Recent analysis also suggests that DBT-dependent phosphorylation destabilizes dCLK (Yu et al., 2009). Thus PER and TIM inhibit their own transcription as well as the transcription of other dCLK/CYC-dependent clock controlled genes, and the approximately 24h period is in part due to the lag in PER and TIM protein accumulation prior

Figure 11. A scheme illustrating current knowledge about the molecular machinery of the Drosophila circadian clock. See text for details. This Figure is taken from (Tomioka and Matsumoto, 2010).

(30)

to nuclear entry (Figure 11).

The second loop drives the circadian oscillation of dClk expression. Besides per and tim, dCLK/CYC activates the transcription of vrille (vri) and PAR domain protein 1ε (Pdp1ε).

The vri mRNA is translated into its product protein VRI, which enters the nucleus and inhibits transcription of dClk by binding to a promoter region, V/P-box. The translation of Pdp1ε occurs in a rather delayed manner. PDP1ε is thought to bind to V/P-box competitively with VRI and activate the transcription of dClk (Cyran et al., 2003). While Cyc is constantly expressed, dClk expression is thus highly rhythmic, with an accumulation profile that is antiphasic to that of per and tim. The dClk feedback loop contributes to sustaining a high- amplitude circadian oscillation (Figure 11).

In addition, the transcriptional repressor CWO (clockwork orange) was recently shown to inhibit not only its own transcription, but also the one of other clock genes, such as per and tim, through E-box elements. CWO belongs to the bHLH ORANGE family (proteins that contain bHLH and Orange domains) and is rhythmically expressed under the regulation by CLK/CYC (Matsumoto et al., 2007). CWO protein is a structural ortholog of DEC1 and DEC2 (deleted in esophageal cancer 1 and 2) proteins in mammals. It might be part of a third feedback loop participating in the generation of high-amplitude oscillations (Figure 11).

In Drosophila, the circadian system is divided into a central clock, which resides in the brain and is known to control rhythms in locomotor activity, and peripheral clocks, which comprise all other clock tissues and which are thought to control other rhythmic outputs. In the brain, about 150 neurons express clock genes. These neurons are divided into seven groups distributed around the central brain; three groups (DN1, DN2, and DN3) are located in the dorsal region, and the remaining four (LNd, l-LNv, s-LNv, and LPN) are located laterally.

l-LNv and s-LNv (for large- and small-lateral-ventral neurons) groups are close to the optic lobes and are suggested to represent the Drosophila counterpart of mammalian

(31)

suprachiasmatic nucleus (SCN) (Figure 12). Their importance for overt activity rhythm generation was revealed by various surgical and electrophysiological experiments (Tomioka and Matsumoto, 2010). Similar to the SCN in mammals, LNvs receive light input from retinal photoreceptors in the compound eyes and extra-retinal photoreceptors within the brain;

however, they can also be entrained directly by light that penetrates the cuticle.

The pathways through which overt rhythms are regulated are not well understood.

Pigment-dispersing factor (PDF) is the best characterized output molecule of the Drosophila circadian clock system. It is thought to be the principal neurotransmitter implicated in the coordination of circadian behavior and metabolism throughout the fly’s body. Pdf is expressed in the l-LNv and s-LNv groups of neurons close to the optic lobes. Flies in which

Figure 12. Circadian oscillators in Drosophila heads. In Drosophila, there are seven main groups of circadian neurons (left). DN1, DN2, and DN3, dorsal neurons; LNd, lateral-dorsal-neurons; l-LNv and s-LNv, large- and small-lateral-ventral-neurons; and LPN, lateral-posterior-neurons. The s-LNv and l- LNv express PDF neuropeptide. l-LNvs have their processes in the optic lobe and send their axonal projection to the contralateral optic lobe through the posterior optic tract (POT). s-LNvs have axonal projection to the dorsomedial region of the protocerebrum. External structures containing circadian oscillators (right). A frontal view of a Drosophila head is shown. OC, ocelli; CE, compound eyes;

AN2, second antennal segment; AN3, third antennal segment; MP, maxillary palps; PR, proboscis.

These Figures are issued from (Tomioka and Matsumoto, 2010) and (Hardin, 2005) respectively.

(32)

LNv neurons have been selectively ablated are arrhythmic under constant conditions. The same phenotype is observed in pdf mutant animals (Renn et al., 1999). However, in both case, some aspect of rhythmicity remains, revealing the presence of additional timekeeping mechanisms in the other groups of clock neurons and/or in peripheral tissues. dClk and vri independently contribute to pdf regulation, with vri affecting a posttranscriptional stage of pdf expression. While pdf mRNA levels do not cycle, rhythmic outputs are thus likely the result of cyclic PDF protein activity (Blau and Young, 1999). Pdp1ε and takeout (to) have also been located downstream of the circadian clock in Drosophila. PDP1ε seems to drive behavioral rhythms when some specific genes, in addition to dClk, are controlled by its binding to the V/P- box (Benito et al., 2007). To mutant flies show aberrant locomotor activity and die quickly upon starvation, indicating that this clock-controlled output gene may control feeding behavior (Sarov-Blat et al., 2000). Recent work suggests that in fact, PDP1ε is a regulator of to (Benito et al., 2010). In addition, the levels of mRNAs of hundreds of other genes have been shown to oscillate (Matsumoto, 2006).

A crucial discovery was that circadian oscillators exist in almost every part of the body. Transgenic Drosophila with the luciferase expressed under the control of the per promoter have shown that per is rhythmically expressed in cells of the eyes, antennae, proboscis, wings and legs, and that this rhythm can be reset by light. This supports that every cell in the body possesses a functional oscillator and a system to transduce photic signals to this oscillator (Plautz et al., 1997). One remarkable difference between the molecular machinery of the clock in peripheral tissues and in the central brain pacemaker is the blue light photoreceptor Cryptochrome (CRY)’s function (see below). A Drosophila cry mutant, cry^b, leaves circadian oscillator function intact in the central circadian pacemaker neurons, but renders peripheral circadian oscillators largely arrhythmic (Krishnan et al., 2001). Therefore, in addition of transducing light signal in the central and peripheral clock cells, CRY also acts

(33)

as a core component necessary for cell autonomous circadian oscillator function in some peripheral tissues (Hao et al., 2008; Ivanchenko et al., 2001). Another important evidence for the functional importance of peripheral clocks comes from an experiment in which the expression of the essential clock gene per was restricted to the LNvs. Such flies exhibited a rhythmic behavior, but had lost any rhythm in electrophysiological responses of antennae to olfactory stimuli. This showed that, in contrast to the rhythm of rest-activity cycles, the circadian rhythm of olfactory responses requires functional oscillators outside the central clock (Krishnan et al., 1999). All together, this suggests that fly peripheral oscillators depend less on the central clocks than do their mammalian counterparts on the SCN (see below). How much influence the central clocks have over fly peripheral oscillators is not known, given that light penetrating through the cuticle and perceived by the blue light receptor CRY can entrain peripheral clocks. The scheme of a “master clock” required to synchronize other oscillators in the fly thus has to be reconsidered, without ruling out the possibility that there may be communication between central and peripheral oscillators.

The Drosophila clock can be entrained by environmental light and temperature. The link between light signals and circadian oscillators is mediated by both rhodopsin and the blue light photoreceptor Cryptochrome (CRY). Photic organs (compound eyes, ocelli, H-B eyelet that is a remnant of larval eye) perceive light via rhodopsin photoreceptors and then synchronize circadian neurons (Figure 12) (Ashmore and Sehgal, 2003). The exact neural pathways toward circadian neurons and the molecular mechanisms for synchronizing them are not yet elucidated. Blind flies (i.e. flies lacking signaling components of visual phototransduction system in the retina) are less sensitive to light synchronization, but their circadian response is not eliminated. This shows that although the visual system plays a role in entrainment, there must be other photoreceptive pathways that entrain circadian rhythms (Suri et al., 1998; Yang et al., 1998). Drosophila indeed uses a parallel extra-ocular pathway, by which light signals are perceived across the cuticle by CRY intracellular photoreceptor,

The synchronization of peripheral circadian clocks by simulated body temperature cycles and feeding rhythms

Thesis

Reference

The synchronization of peripheral circadian clocks by simulated body temperature cycles and feeding rhythms

The Synchronization of Peripheral Circadian Clocks by Simulated Body Temperature Cycles and Feeding Rhythms

Table of contents