Till Roenneberg Martha Merrow

Review article

Circadian clocks - from genes to complex behaviour

Institute for Medical Psychology, Ludwig Maximilians University, Goethestr. 31-33, 80336 Munich, Germany

(Received 4 February 1999; accepted 16 April 1999)

Abstract - Circadian clocks control temporal structure in practically all organisms and on all levels of biology, from gene expression to complex behaviour and cognition. Over the last decades, research has begun to unravel the physiological and, more recently, molecular mechanisms that underlie this endogenous temporal programme. The generation of circadian rhythms can be explained, at the molecular level, by a model based upon a set of genes and their products which form an autoregulating negative feedback loop. The elements contributing to this transcriptional feedback appear to be conserved from insects to mammals. Here, we summarize the process of the genetic and molecular research that led to ’closing the molecular loop’. Now that the reductionist approach has led to the description of a detailed clock model at the molecular level, further insights into the circadian system can be provided by combining the extensive knowledge gained from decades of physiological research with molecular tools, thereby reconstructing the clock within the organism and its environment. We describe experiments combining old and new tools and show that they constitute a powerful approach to understanding the mechanisms that lead to temporal structure in complex behaviour. © Inra/Elsevier, Paris circadian rhythm / transcription / entrainment / clock gene / autoregulating negative feedback

Résumé ― Les horloges circadiennes - depuis les gènes jusqu’aux comportements les plus complexes. Les horloges circadiennes contrôlent l’organisation du temps chez pratiquement tous les organismes et à tous les niveaux de la biologie, depuis l’expression des gènes jusqu’aux comportements les plus complexes et à la cognition. Au cours des dernières décennies, la recherche a com- mencé à dévoiler les mécanismes physiologiques et moléculaires sous-jacents à ce programme tem- porel endogène. L’origine des rythmes circadiens peut être expliquée, au niveau moléculaire, par une série de gènes et leurs produits, qui forment une boucle de rétroaction négative autorégulée. Les éléments qui contribuent à cette rétroaction transcriptionnelle apparaissent conservés depuis les insectes jusqu’aux mammifères. Nous avons résumé ici les résultats les plus récents des recherches génétiques et moléculaires qui conduisent à « refermer la bouche moléculaire ». Alors que l’approche réductionniste a conduit à la description d’un modèle détaillé d’horloge au niveau moléculaire, une vision plus perspicace des systèmes circadiens peut être effectuée en combinant les connaissances très étendues obtenues depuis plusieurs décades de recherche en physiologie, avec les outils molécu-

* Correspondence and reprints E-mail: [email protected] laires, ce qui permet une reconstruction de l’horloge à l’intérieur de l’organisme placé dans son environnement. Nous décrivons des expériences qui combinent des outils anciens et nouveaux et qui montrent qu’ils constituent une approche puissante afin de comprendre les mécanismes qui conduisent à une organisation temporelle des comportements les plus complexes. © Inra/Elsevier, Paris rythme / transcription / entraînement / gène clock / boucle d’autorégulation

1. INTRODUCTION isms, at the cellular level. Even at this level

- for in unicellular One of the most fascinating questions in example organisms - circadian have to with a tem- biology concerns the mechanisms by which systems cope organisms direct metabolism quasi inde- poral environment influencing metabolism. Not does the external environment have pendently of astronomical time. These tem- only an but so do states, such poral programmes are controlled by bio- impact, endogenous logical clocks that reflect the four temporal as energy charge or nutrient stores, which are modified over time. Like uni- ‘spaces’ governing life on our planet (tides, regularly day, lunar cycle and year). Among these, cells, individual cells of multicellular organ- the circadian system has been investigated isms (e.g. pacemaker neurons) may also contain all the elements of a circadian most intensively, and we are beginning to system oscillator understand how a circa-24-h rhythmicity is (input ! ! output): recep- generated. tors, intracellular signal transduction, a machinery that generates the rhythm and In his classic that in the paper appeared outputs (e.g. rhythmic electric activity). proceedings of the first dedicated interna- These cell clocks are entrained by a tempo- tional conference on clocks in biological rally structured micro-environment, involv- Cold Harbor, Colin Spring Pittendrigh transmitters, hormones or other chemi- defined circadian their ing systems by unique cal [12]. Some of these cells in These have been signals (e.g. properties [76]. properties the retina and the pineal) respond directly a guideline ever since to describe, dissect to exogenous such as and to model circadian in signals (zeitgeber) systems organ- light [15, 75]. isms of all phyla. The change in models over the decades reflects both fresh past insights The descriptions established in the pre- and the of tools. development experimental molecular era provided us with the basic Methods new the open possibilities, shape concepts that are unique to intact circadian to the and approach overlying questions, systems [9] and have served as the basis for thus, have an impact on how explanations all subsequent genetic and molecular are found. In the 1960s and bio- 1970s, research. They, for example, help us to dis- chemical methods allowed the dissection of tinguish between rhythms that are merely cellular metabolism, and cybernetics pro- driven by environmental changes and those vided formal tools for the description of that respond actively through the robustness the models complex systems. Accordingly, of an ongoing endogenous circadian oscil- the mechanisms cir- describing underlying lator. They, however, describe the contents cadian involved a rhythmicity, cybernetic of a Pandora’s box: i) a feedback loop pro- view of cellular metabolism [25]. With the ducing rhythmicity (independent of its fre- advent of molecular tools, circadian mod- quency); ii) the circadian range of the period; els centred around gene regulation. iii) an amplitude sufficiently robust to drive The mechanisms underlying circadian output rhythms; iv) the fact that the rhyth- rhythmicity are implemented, in all organ- micity is sufficiently self-sustained to con- tinue unabated; v) temperature compensa- system. A future task is to determine the tion; and vi) entrainability [90]. Now, that function of these clock elements and their the box is being opened by molecular cir- ’location’ within the system. Figure 7 sum- cadian biology, the generalized features of a marizes schematically the circadian pheno- clock producing the known circadian phe- types that can result from mutations of clock notype may turn out to be a sum of qualities, genes and indicates their possible location each implemented by different cellular func- within the circadian system. tions. Neither rhythmicity nor autoregulating feedback are exclusive to elements of the 2. DISCOVERING THE COGS OF THE CIRCADIAN CLOCK oscillator but may also be inherent in inputs and The of outputs [26, 91]. discovery genes 2.1. The search for circadian that affect circadian properties when mutated genes is currently progressing with breathtaking First attempts to identify circadian clock All of these can be as speed. regarded impor- used the unicellular tant elements within the circadian genes alga Chlamy- complex domonas [ 14], the fungus Neurospora [27] and the fruit fly Drosophila [50] as classic model organisms for genetic research. They resulted in the identification of several mutant strains - mainly with altered circadian periods in constant conditions. Among these, the period gene (per) in Drosophila and the frequency gene (frq) in Neurospora were the first to be cloned [13, 66, 83]. While the search for circadian mutants in long period mutants were obtained, as well Chlamydomonas, Neurospora and Droso- as one with reduced amplitude [69, 106]. phila was based on mutagenesis and subse- Bioluminescent reporter constructs were quent screening for stable and heritable later also used for Drosophila (see below). period changes, the first step into mam- a mutation malian circadian genetics was fortuitous. A The identification of genetic normal hamster shipment contained an indi- resulting in altered circadian qualities, (e.g. vidual male (the tau mutant) that displayed period length), is by itself not enough to a significantly shorter period than any ham- conclude that this gene is centrally involved ster previously recorded [82]. Subsequent in circadian rhythmicity (see also figure 1 ). mutagenesis and screening were initiated in Many cellular functions must be involved the superior genetic model system, the in circadian rhythmicity: transcription, trans- mouse, and resulted in the identification of lation, protein modification, RNA and pro- a gene (clock) that lengthens the circadian tein degradation, energy metabolism, and period [114]. elements transducing zeitgeber signals to the clock. There are good examples showing The that finding prokaryotes (Syne- that altered circadian properties can be due also co-ordinated their chococcus) daily to mutants affecting other cellular functions. metabolism with the of an help endogenous The mutation of a gene encoding a neuronal the temporal programme [110] toppled cell adhesion molecule (ncam-1 ) results in a that circadian clocks were to dogma unique phenotype similar to the clock mutation in For of eukaryotes [76]. purposes isolating mice [103]. Neurospora mutants defective in clock genes, cyanobacteria offer several lipid metabolism (chol-I and cel) have The time is short advantages. generation extremely long (up to 70 h) and indi- and the number of individual periods organisms cate interactions with frq (as available for and is - complicated mutagenesis screening shown with double mutants) [51]. orders of than in Finally, by magnitude - larger any biochemical experiments in algae show that of the other model systems; recording of circadian rhythmicity can be influenced by circadian rhythmicity was made very simple as well as by peroxisomal with the of a luciferase photosynthesis, help reporter gene and nitrate metabolism [86, 91]. Although construct; and automation enables finally, these effects to be with- the of thousands of individual appear non-specific, screening out the information as to how these cells rather their colonies) genes (or concurrently. and metabolic functions affect circadian With these tools, mutants were identified our of the with decreased bimodal struc- properties, understanding system amplitudes, remains ture and altered periods in circadian rhyth- incomplete. [49]. micity In view of the numerous ways that circadian can be it is sur- For the mutant search in Synechococcus, properties affected, that few clock were the open reading frame of the bacterial prising relatively genes identified in the screens. luciferase gene was fused with a clock-con- originally genetic The of alleles isolated trolled promoter (psbAl, a photosystem majority originally II gene). This construct reports circadian in Drosophila and Neurospora mapped to rhythmicity by emitting light when the per and frq, respectively, giving rise to the are in fusion gene is expressed. Similarly, in Ara- optimism that few genes involved the bidopsis, the firefly luciferase gene was generation of circadian rhythmicity. Thir- fused with a rhythmically expressed pro- teen years after per was identified, a mutant moter involved in photosynthesis (cab2, screen in Drosophila revealed another clock chlorophyll a/b binding protein). The trans- gene, timeless (tim), conferring short or long formants were mutagenized, and short and periods as well as arhythmicity [100]. 2.2. Building a simple molecular to be experimentally addressed. Both per clock and frq participate in a negative feedback loop [7, 34, 117]. Discrete induction results Demonstration that a gene and its prod- in a stable phase shift of the overt rhythm ucts are directly responsible for the molec- [7, 24], and conversely, zeitgebers can ular generation of the circa-24-h rhythmic- induce changes in the phase of the molecu- ity has been based on a set of five criteria lar oscillation of per and frq [18, 52, 58]. which were first formulated 20 years ago Constitutive expression results in arhyth- and have been marginally modified since micity [7, 117]. additional see [7] (for references, [90]). As a result of the experiments in 1) Mutations in a clock component should Drosophila and Neurospora the first molec- affect canonical clock and null properties ular clock model was constructed, a model mutations should abolish normal rhythmic- that had been proposed several years before The amount of the ity. 2) (activity) compo- based on the fact that per mRNA was con- nent must oscillate in a self-sustained man- stitutively expressed in the per null mutant ner with an Induced appropriate periodicity. 3) [34]. The clock gene (per or frq) produces a in the amount of the com- changes (activity) protein that in turn inhibits the transcription must act to the ponent (by feedback) change of its mRNA. Due to RNA and protein amount (activity) of the component. 4) The degradation, the self-inhibitory effect is of the oscillation must be phase component’s eventually relieved and the cycle starts reset by shifts in the light/dark growth reg- again. imen, and conversely, the overt rhythm must be reset by changes in the amount (activity) of the component. 5) Prevention of the 2.3. A matter of details component’s oscillation should result in loss of the overt rhythm. In particular, there Once the basic molecular models were should be no degree of constitutive expres- constructed and the criteria fulfilled for per sion that will support rhythmicity. These and frq, several questions had to be criteria basically describe the behaviour of answered concerning the detailed progres- an element involved in an autoregulating sion of the loop. Which processes are negative feedback loop that constitutes the responsible for the long time constant in the circadian oscillator, i.e. a molecule that is circadian range? How much does de novo rhythmic with the same periodicity as the transcription and how much does turnover of observed circadian outputs, which affects RNA and proteins contribute to the oscilla- its own oscillation via feedback, and which tion? What are the details of inhibition and responds to zeitgeber signals. A machinery activation? How is the loop affected by zeit- generating rhythmicity has to involve neg- geber signals (see section 2.5)? The hard ative feedback; this has long been known work of molecular biochemistry began to from modelling oscillations mathematically fill in the details about the progression of (for references, see [90]).). this autoregulating negative feedback loop. Are these criteria fulfilled for per and frq? Null mutants are arhythmic [22, 116] 2.3.1. The circadian time range and in free-running conditions mRNA and protein levels oscillate rhythmically [23, Both PER [23] and FRQ [30] are phos- 30]. Innovative experiments with inducible phorylated in a time-dependent manner. This or constitutive promoters (rhodopsin and progressive phosphorylation, together with heat shock promoters in Drosophila and the the lag of 4-6 h between mRNA and pro- quinic acid inducible qa-2 promoter in Neu- tein peaks [30, 65], are thought to be respon- rospora) allowed the remaining questions sible for the circadian time range generated by the feedback loop. Strong support for the dent, and that PER and TIM form a com- phosphorylation hypothesis came with the plex (thereby stabilizing monomers) which discovery of a new clock gene. Various alle- is necessary for both proteins to enter the les of doubletime (dbt) produce phenotypes nucleus [116]. It became clear that both con- similar to per and tim mutants; dbt is, how- tributed to the negative feedback. ever, different in two important qualities. Unlike in the former two genes, the mRNA 2.3.4. The activating process is not rhythmic (though DBT is essential for rhythmicity) and null mutations are lethal After clock was cloned in the mouse [6, [47, 80]. DBT is a casein kinase I homo- 46], a search for partners of the CLOCK logue [80] and is responsible for the phos- protein began. The rationale for this search phorylation of PER. Gene dosage studies [32] was based on the following results: indicate that dbt function negatively corre- i) analysis of the Drosophila per-promoter lates with period [116]; thus, phosphoryla- revealed a short enhancing sequence (E-box, tion contributes to the circadian period CACGTG) that was responsible for the length. robust rhythmic transcription of per [33]; ii) basic-helix-loop-helix (bHLH) tran- 2.3.2. Transcription and turnover scription factors are known to bind to E-boxes, but only when they form het- Theoretically, the degradation kinetics erodimers with a partner protein; and iii) of mRNA and protein are crucial i) for the CLOCK itself contains a bHLH motif [32]. system to oscillate, ii) for it not to damp, Using the two-hybrid system, a protein bind- and iii) for the length of the period. So far, ing to CLOCK was found and its gene was little is known about the degradation kinet- cloned. By sequence homology, it was iden- ics in the different model systems. A time- tified as bmall (brain and muscle arnt-like of-day-specific, i.e. cyclic, degradation has protein 1) [32, 43], an isoform of the inde- been suggested for per mRNA [21 Results pendently cloned mop3 (members of the PAS also strongly suggested that the rhythm in superfamily) [38]. These discoveries finally mRNA levels (of per, tim and frq) are due to ’closed the circadian loop’ [19, 32] predicted changing transcription rates, rather than due 8 years before. Two recently identified clock to controlled degradation. Recently, nuclear mutants in Drosophila, cycle and jerk, turned run-on experiments were able to correlate out to be homologues of bmall and clock, per and tim rhythmicity with de novo tran- respectively [5, 93]. They also bind as het- scription [105]; yet, per cycling is not only erodimers to the E-box of the Drosophila controlled at the transcriptional level [16, per promoter [19], thereby activating per 29, 105]. transcription. Experiments using promoters of Drosophila per and tim as well as of mouse showed that of the 2.3.3. The inhibiting process perl , binding CLOCK/BMAL1 heterodimer is necessary From the ’simple’ molecular clock model, as the activating element for per and tim RNA [19, 32], while interac- the role of PER as an inhibitory element rhythmicity tions of PER/TIM heterodimers with was known but what was the role of TIM CLOCK/BMALl are in this negative feedback? tim was discov- inhibitory. ered independently by two approaches - Within a few years, the missing elements once by mutagenesis, as described above, of the ’simple’ molecular clock model had and once in a search for protein partners been discovered and numerous homologues binding to PER (using the two-hybrid sys- were found indicating that the molecular tem) [31]. Subsequent experiments showed mechanisms of the circadian system have that per and tim oscillations are interdepen- been conserved between insects and mam-

mals. Table I summarizes those genes that BMAL1/CLOCK constitutes the transcrip- led to the full description of the molecular tional activator of the per and tim promoter. circadian loop in animals as well as those Their products, PER and TIM, also form a which are candidates for the completion of heterodimer, necessary for their translocation the circadian loop in micro-organisms and to the nucleus and for inhibition of their higher plants. First insights into the func- transcription via interaction with BMAL1/ tion of clock genes came from structural CLOCK. Thus, the function of clock genes, The PAS domain similarities (table In. described so far, is to control transcription. (named after the three Drosophila genes Most recently, it has been shown how an PER, ARNT, SIM), which mediates pro- output rhythm in the mammalian SCN is tein-protein interaction, was one of the first controlled by this autoregulating negative common elements recognized [18]. Fur- feedback loop [45]. The promoter of the thermore, domains and DNA-binding vasopressin gene, is also activated by the or sequences controlling cytoplasmatic BMAL1/CLOCK complex (again via an E- nuclear localization and indi- (CLS NLS) box) and is, thus, controlled in the same way cate the involvement of heterodimers that as the clock components themselves. An are translocated to the nucleus at some part aspect of the circadian regulation of the circadian to control important cycle transcrip- of is the fact that the tion. vasopressin vasopressin promoter and its expressed peptide do not constitute a negative feedback and are, thus, clearly downstream of the mechanism that 2.4. The current models generates the circadian rhythmicity. Based on the vasopressin model, it will be inter- The detailed model for the molecular cir- esting to see how the circadian clock can cadian oscillator in animals (figure 2A) is control different output rhythms that do not based on two pairs of heterodimers. oscillate in phase. Phase specificity may be achieved by further transcriptional elements Drosophila [76]. A clock-driven feedback that are activated at other circadian phases. loop involving an RNA binding protein was In mammals, this could involve different found in Arabidopsis [35], which can be per genes (mPerl and mPer2) which oscil- regarded as a ’slave’ to the circadian feed- late out of phase by 4 h [4]. There are, of back loop. It could act as a gear in the cir- course, many other scenarios by which out- cadian pathway determining a specific phase put rhythms could be regulated at different of the output. circadian times. For example, all output rhythms so far described in the marine alga Although the basic molecular mecha- Gonyaulax, are controlled at the transla- nisms of the circadian loop in animals tional level [86] with a constitutive expres- involve the same elements, there are sev- sion of the respective RNAs. Another pos- eral interspecific differences. The mam- sibility is the existence of ’slave oscillators’, malian per RNAs peak during the day phase a concept that was first advocated by Pit- [4, 45, 104, 109, 112], but they reach their tendrigh for the eclosion rhythm in maxima during the night in Drosophila [34, 65]. In the fly, tim mRNA levels cycle [42, the 24-h cycle of the environment is another 101], while they are constitutive in the [88]. Several experiments over the last few mouse [96, 120]. CLOCK is weakly rhyth- years have addressed the molecular mecha- mic in flies but it is constitutive in the mouse nism of light entrainment. In some cases, [53]. BMALI is robustly rhythmic in the the distinction between oscillator and light rat SCN [39] but is constitutive in input pathway is difficult to make. The Drosophila S2 cells in culture [19]. Thus, mouse perl promoter, which has kinetics in mammals and in flies both the inhibitory reminiscent of an oscillator component, con- and the activating heterodimers are rhyth- tains light regulative elements [4, 104] and mic (rhythmicity of the functional element, is induced by light similar to immediate the heterodimer, requires only one rhyth- early genes [113]. Mouse per2 is also light- mic partner). In mammals, BMALl/CLOCK inducible, but the kinetics are much slower is rhythmic owing to the rhythmicity of [4, 104]. Light regulation of the per pro- BMAL1, in flies owing to the cycling of moter has not been shown in Drosophila, CLOCK. In mammals, PER/TIM is rhyth- where photic entrainment is mediated via a mic owing to PER and in flies owing to rapid light-dependent degradation of TIM, both. These statements, of course, rely on thereby, also destabilizing PER [42, 73]. the demonstration that rhythmicity of RNA The differences in temporal expression of also corresponds to rhythmicity of protein, clock genes (see section 2.4) reflect the dif- which has yet to be shown for some of the ferences in regulation by light. components. But what makes the activating elements rhythmic? In Drosophila, The rapid light induction of the mam- CLOCK is absent in tim° and pero flies [53], malian perl gene is analogous to the rapid thus constituting another possible feedback induction of frq mRNA after exposure to loop, while this interaction has not yet been light [17]. In Neurospora, almost all light shown in mammals. responses are induced by blue light. Two sets of mutants, white collar-1 and white Unlike in the details of the tran- animals, collar-2 (wc-1 and wc-2) [20] lack all char- scriptional feedback loop have not been for- acterized responses to blue light, including identified in Neurospora, sev- mally though circadian entrainment [92]. Thus, it is con- eral candidates exist The good (figure 2B). ceivable that a discrete conservation of elements between single, photorecep- striking tor system is responsible for light reception insects and mammals does not apparently in Neurospora. The white collar mutants extend to fungi, which raises the important are arhythmic in constant conditions, even question of analogy versus homology among following synchronization with a tempera- the circadian systems across different phyla. ture [ 18]. Both are DNA- Will the that pulse WC-proteins strategies divergent organisms binding transcription factors responsible for be similar, or will there be developed impor- different aspects of positive induction in tant differences? What specific biochemi- light responses [11, 57], cal/metabolic mechanisms will be to including frq adopted [18]. The absence of WC-1 accommodate needs accord- expression species-specific induction of both frq and itself to life and niche? impairs light ing cycle, spatial temporal (thus, wc-1 transcription is positively autoregulated). In the absence of WC-2, wc-1 is not light-inducible but frq is, how- 2.5. Shedding light on the clock ever, with altered baseline and saturation levels [18]. Recently, a wc-2 allele was The endogenous generation of circadian described with an altered period and defec- rhythmicity is one of the important quali- tive temperature compensation (Colette M., ties of circadian systems. Synchronization to Dunlap J., pers. comm.). One interpretation of the white collar findings is that they are mutants [108]. In contrast, when total head oscillator components [22]. They could, extracts are analysed for per and tim or when however, also be part of the circadian light their expression is recorded from whole flies input pathway and still lead to arhythmicity via a bioluminescence reporter gene [78], of the oscillator (see section 3.1). they are arhythmic [108]. Besides the lat- In higher plants, phytochromes had long eral neurons, circadian oscillators are present been candidates for circadian photorecep- throughout the entire fly [79] and these to become in the tors [62] but they could account only for appear arhythmic cry mutant. biochemical part of the circadian light responses [70]. In Although photorecep- algae, for example, two independent light tion remains to be demonstrated for the ani- inputs with different spectral sensitivities mal cryptochromes, cry plays some important role in circadian Its have opposite effects on the circadian clock light reception. results in [84]. The identification of a class of blue overexpression stronger responses brief light-sensitive receptors in plants, the cryp- to light pulses compared to wild-type, tochromes, led to discoveries well beyond while these responses are absent in cry the plant kingdom. Cryptochrome (cry) is mutants [26]. The activity rhythm of the homologous (by sequence) to the DNA mutant, however, remains entrainable to repair enzyme DNA-photolyase, but lacks light/dark cycles, maybe via feed back from DNA-repair function. Its role in light recep- light/dark-driven activity (Rosbash, pers. tion was first discovered in connection with comm.). These results also substantiate that hypocotyl elongation [2]. Cryptochromes the Drosophila activity rhythm is controlled are flavin-binding, redox-sensitive, soluble primarily by the lateral neurons [36] and proteins [1, 56]. Loss of either of the cryp- not by any of the numerous other oscilla- tochrome genes cryl or cry2 in Arabidopsis tors [79]. results in period changes of the free-running rhythms in constant blue light. Simi- As in Arabidopsis, two photolyase/cryp- lar to the phytochrome mutant phyA, cryl1 tochrome homologues were found in mice exhibits a longer period at low blue light and humans, cry] and cry2 [40, 113]. mCryl fluences compared to wild-type, consistent expression in the mouse SCN is circadian. with the physical association of phyA and Mouse strains lacking mCry2 are still highly cryl [3]. Period is less affected in both phyB sensitive for phase shifting by light pulses and cry2 [107] suggesting different mecha- and show altered periods in the circadian nism than for the responses via phyA and activity rhythm [113]. It had already been cry 7. shown in mammals that circadian entrain- Recently, homologues of the DNA pho- ment was transduced through the eyes, but - tolyase and plant cryptochrome family have as in actively swimming algae - not by the used for orientation been found in insects [26, 108] and mam- photoreceptors spatial mals, including humans [40, 113], and it [88]. The finding in mammals is based on the known ocular has been postulated that they constitute a eliminating receptor types conserved type of circadian photoreceptor by molecular, cell-specific methods without the of the circadian across several phyla. However, the story is losing ability sys- complicated [60]. Unlike PER and TIM, tem to respond to light (measured both for Drosophila cry is considered to be an ele- phase shifting and melatonin suppression) ment of the circadian light transduction path- [28, 61 ]. Thus, due to the redundancy in cir- way that is under the control of the circa- cadian photic input, the direct involvement dian oscillator [26]. Locomotor activity as of a gene product as a receptor has to be well as tim and per expression in the lateral tested using strains as genetic backgrounds neurons, the circadian pacemakers in the that are already impaired in other receptor Drosophila [36] brain, are rhythmic in cryb candidates. 3. RECONSTRUCTION they, therefore, have been predominantly used to find out whether genes are involved 3.1. A complicated assignment in circadian mechanisms. They are, however, artefacts of laboratory experiments Like many other functions in biology, and do not reflect the which was circadian can be as com- reality systems regarded for the evolution of circadian and responsible plex pathways, integrating exogenous clocks. A of the circadian rules that information and cel- majority endogenous regulating have been lular and developed by physiological systemic processes accordingly. research deal with the behaviour of circa- At their sensory end, information about the dian systems under zeitgeber conditions [9, cyclic environment is received and trans- 68, 77] and help us understand how endoge- duced to the mechanisms that produce the nous are entrained (rather than syn- circadian which will then con- rhythms rhythmicity chronized in a trol the different of the by being driven) very sys- output rhythms tematic into the structure of This involve feed- way rhythmic organism. pathway may the environment. The interactions of the back not within the oscillator but also only molecular oscillator will even- in the and Furthermore, components inputs outputs. have to all the characteristic themselves can be under circadian tually explain inputs features which have been described in detail control [26, 89] and feed back outputs may by physiological research (including the to the oscillator [87]. Due to the complexity more ’esoteric’ features such as after-effects, of this pathway, all elements involved could or internal [9, be as well as splitting desynchronization theoretically rhythmic produce 77, the arhythmicity when their function is 86]). Conversely, physiological methods and can be used to elu- mutation of a mak- protocols destroyed (e.g. by gene), cidate the role and function of the molecu- the of clock and their ing assignment genes lar all circadian within the circadian dif- components. Although sys- products pathway tems ficult. In addition, physiological experiments strongly respond to light signals, they can also be entrained by non-photic stim- have shown that single cells can contain uli. These can also be an important tool in more than one circadian oscillator [86], so probing the function of the different molec- that mutations of genes that produce key ular components. elements within one of the oscillators may not necessarily lead to an arhythmic phe- We have addressed the difficulties shown notype. in our theoretical model, described above, We have shown theoretically that ele- experimentally using the Neurospora model ments of input pathways can comply with all system. In a series of experiments [68], we of the criteria also used to characterize ele- were able to show that Neurospora strains ments of the oscillator When differ- [90]. that are impaired or non-functional for FRQ ent properties are assigned to an input ele- retain qualities characteristic for circadian ment in a mathematical model (thereby systems. Namely, all Neurospora period alleles of a involved in simulating gene sig- mutant frq strains, as well as those deficient nal transduction to the circadian oscillator as for FRQ protein (e.g. frq9, which cannot well as their and overproduction induction), produce functional FRQ), are entrainable the different resulting rhythm adopts periods, by temperature cycles. During this temper- becomes or with arhythmic, responds phase ature entrainment of spore formation, frg9 shifts section (see 2.2). mRNA remains arhythmic at high levels. When temperature cycles of different periods are the different strains 3.2. 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