Phenotypic Rescue of a Peripheral Clock Genetic Defect via SCN Hierarchical Dominance

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1 Cell, Vol. 110, , July 12, 2002, Copyright 2002 by Cell Press Phenotypic Rescue of a Peripheral Clock Genetic Defect via SCN Hierarchical Dominance Matthew P. Pando, 1,3 David Morse, 1,4 Nicolas Cermakian, 1,5 and Paolo Sassone-Corsi 1,2 1 Institute de Génétique et de Biologie Moléculaire et Cellulaire CNRS-INSERM-ULP 1 rue Laurent Fries Illkirch, Strasbourg France Summary The mammalian circadian system contains both cen- tral and peripheral oscillators. To understand the com- munication pathways between them, we have studied the rhythmic behavior of mouse embryo fibroblasts (MEFs) surgically implanted in mice of different genotypes. MEFs from Per1 / mice have a much shorter period in culture than do tissues in the intact animal. When implanted back into mice, however, the Per1 / MEF take on the rhythmic characteristics of the host. A functioning clock is required for oscillations in the target tissues, as arrhythmic clock c/c MEFs remain arrhythmic in implants. These results demonstrate that SCN hierarchical dominance can compensate for severe intrinsic genetic defects in peripheral clocks, but cannot induce rhythmicity in clock-defective tissues. Introduction factors, CLOCK and BMAL1 (Gekakis et al., 1998; Kume et al., 1999), both members of the bhlh-pas family (Hogenesch et al., 1998; King et al., 1997). The CLOCK:BMAL1 heterodimer binds to E box elements in the promoters of clock genes and clock-controlled genes (Gekakis et al., 1998; Jin et al., 1999; Ripperger et al., 2000; Travnickova- Bendova et al., 2002). CLOCK:BMAL1 drives the rhythmic transcription of both Period (Per) and Cryptochrome (Cry) genes, an event that initiates the expression of proteins involved in both positive and negative feedback loops (Gekakis et al., 1998; Shearman et al., 2000). The Cry gene products are inhibitors of CLOCK:BMAL1- mediated transcription through direct protein-protein interaction (Griffin et al., 1999; Kume et al., 1999) and are essential to the negative feedback loop (Griffin et al., 1999; Kume et al., 1999; Okamura et al., 1999; van der Horst et al., 1999; Vitaterna et al., 1999). The per2 gene product functions as a positive regulator of Bmal1 ex- pression (Oishi et al., 1998; Shearman et al., 2000). As BMAL1 is the likely limiting factor in CLOCK:BMAL1 heterodimer formation, its accumulation may drive the reinitiation of Per and Cry transcription. The decrease in Bmal1 expression in the SCN of Clock mutant and Per2 mutant mice (Shearman et al., 2000) may thus be due to the decreased PER2 levels in these mice. Peripheral tissues display oscillations in Per expres- sion similar to those of the SCN, but with a 3 9 hr phase delay (Cermakian et al., 2001; Oishi et al., 1998; Zylka et al., 1998), and these oscillators in culture dampen much faster than do SCN neurons (Abe et al., 2002; Allen et al., 2001; Balsalobre et al., 1998; Welsh et al., 1995; Yamazaki et al., 2000). Additionally, peripheral oscillations in Per2 are lost in SCN-lesioned animals (Sakamoto et al., 1998). These observations suggested that periph- eral clocks may require SCN-derived signals to drive or synchronize their oscillations. The control of peripheral clocks by the SCN is thought to occur through a combi- nation of neuronal and humoral signals (Allen et al., 2001; Balsalobre et al., 2000; Buijs and Kalsbeek, 2001; Le- Sauter and Silver, 1998; McNamara et al., 2001; Ueyama et al., 1999). While studies with serum-shocked MEFs from normal and Cry mutant mice indicate that several critical com- ponents constituting a peripheral clock are identical to those operating in the SCN (Balsalobre et al., 1998; Ya- gita et al., 2001, 2000), several observations suggest that significant differences exist. First, Bmal1 does not oscillate in the SCN of Clock mutant mice, but oscillates with reduced amplitude in the periphery (Oishi et al., 2000). This indicates that CLOCK is essential for Bmal1 The circadian system is responsible for regulating a wide variety of physiological and behavioral rhythms (Cer- makian and Sassone-Corsi, 2000; Dunlap, 1999; Pittendrigh, 1993; Reppert and Weaver, 2001; Young and Kay, 2001). Classically, the circadian system has been thought to be based on centralized clock structures. The discovery that both vertebrates and invertebrates have a widely dispersed circadian timing system challenged this view (Balsalobre et al., 1998; Giebultowicz et al., 2000; Plautz et al., 1997; Tosini and Menaker, 1996; Whitmore et al., 2000, 1998; Yamazaki et al., 2000). Indeed, many tissues contain intrinsic oscillators, and possibly every cell in a given tissue contains an autonomous clock. The mammalian central clock is located in the suprachiasmatic nucleus (SCN) within the anterior hypothalamus (Klein et al., 1991). The neurons of the SCN contain cell- autonomous, self-sustained circadian oscillators (Welsh et al., 1995), whose molecular basis involves several interconnected positive and negative transcriptionaltranslational feedback loops (Shearman et al., 2000). transcription only in the context of the SCN. Second, The central oscillator is driven by two transactivating two studies have implicated MOP4/NPA2, a homolog of CLOCK not expressed in the SCN in the regulation of 2 Correspondence: paolosc@igbmc.u-strasbg.fr vascular (McNamara et al., 2001) and forebrain clocks 3 Present address: ExonHit Therapeutics, 217 Perry Parkway, Build- (Reick et al., 2001). Thus, there may be tissue-specific ing 5, Gaithersburg, Maryland differences in the molecular composition of the circa- On leave from the Département des Sciences Biologiques, Unidian clock, and clock components that have subtle efversité de Montréal, 4101 Sherbrooke est, Montréal (QC) H1X 2B2, Canada. fects on central clock function may play a more promi- 5 Present address: Douglas Hospital Research Center, McGill University, 6875 LaSalle Blvd, Montreal (QC) H4H 1R3, Canada. nent role in the regulation of peripheral clocks and vice versa.

2 Cell 108 primary mouse embryo fibroblasts (MEF) from Per1 / Disruption of Per1 results in subtle phenotypic more severe in MEFs than in the SCN. While differences changes in circadian function (Bae et al., 2001; Cermakian in gene function and expression between SCN and pe- et al., 2001; Zheng et al., 2001). Per1 mutant mice ripheral clocks have not been extensively described, are able to entrain to standard light:dark (LD) cycles, mild divergences have been reported (Oishi et al., 2000). display a modest shortening of the free running circa- Here we have uncovered a profound difference, manifested dian period, a slight decrease in oscillator precision, by the dramatically shortened intrinsic period in and no significant alterations of clock gene expression the peripheral clocks of Per1 / mice. in the SCN. Interestingly, rhythmic expression of clock When MEFs from Clock c/c mutant mice were subjected genes in peripheral tissues appeared slightly delayed in to the same serum-shock treatment (Figure 1C), we consistently the Per1 mutant mice, suggesting a specific role for observed peaks of Per2 expression 28 and 56 Per1 in peripheral clocks (Cermakian et al., 2001). hr following the serum shock (Figure 1D). These data fit We show here that in drastic contrast to the phenotype poorly to a cosine curve (r 0.5) but are suggestive of of the Per1 null mice, Per1-deficient peripheral oscilla- a 28 hr period. This expression profile is reminiscent of tors placed in culture display an intrinsic period of only the extended activity period observed in heterozygous 20 hr. This implies that the function of PER1 is different Clock mutants and initial free-running rhythms for homozygous in SCN neurons and in peripheral tissues. We have exploited Clock mutants in constant darkness (DD; Vitain this difference to investigate the functional de- terna et al., 1994). However, if these data do reflect clock pendence of the peripheral clocks on the central pacemaker. function in these MEFs, this clock functions poorly, as We show that, under normal physiological judged from a comparison of the amplitude of the Per2 conditions, the SCN is able to rescue or compensate oscillations in the Per1 / and Clock c/c MEFs (Figure 1D). for genetic defects affecting the period of peripheral clocks. Using MEFs encapsulated in a collagen disk and Differential Induction of Per2 by Serum Shock implanted surgically into a host animal, we show that An interesting feature of these in vitro oscillations is the peripheral clocks are subordinated to the dominance clear difference in the kinetics of Per2 induction between exerted by the central clock and exhibit the characteris- the three MEF genotypes. To directly compare the varitics of the host SCN. ous MEFs, RNA levels for the first five time points of the wt, Per1 /, and Clock c/c samples were normalized to Results each other. The normalized RNAs were simultaneously processed and run next to each other on a single gel Unmasking a Short Period Peripheral Clock (Figure 1E). In wt MEFs, the abundance of Per2 tran- Phenotype in Per1 / Mice scripts increases gradually over the first 2 hr and is then We have recently reported that mice lacking the Per1 downregulated by 8 hr postinduction. In Per1 / MEFs, gene display an almost normal circadian phenotype, basal expression of Per2 (at time zero) is higher than in characterized by rhythms in locomotor activity approxiamplitude wt MEFs, induction is more rapid with a slightly higher mately an hour shorter than their wild-type (wt) littermates and downregulation occurs earlier (Figure 1F). (Cermakian et al., 2001). The only other disbasal This suggests that Per1 is involved in modulating both cernible phenotype in these Per1 mutant mice is a slight and signal-induced Per2 expression levels. Finally, phase delay in Per2 gene oscillation in peripheral tiscompared in the Clock c/c MEFs, Per2 activation is slightly delayed sues. This suggested that Per1 might play a specialized to what is seen in the wt MEFs, and the ampli- role in peripheral clocks. To test this, we established tude of the induction is significantly reduced (Figures 1E and 1F). This is similar to observations made in the embryos as a model for peripheral cell oscillators (Yagita SCN of Clock c/c mutants, where both Per1 and Per2 et al., 2001). For comparison, we also established MEFs transcripts are expressed at a reduced level (Jin et al., from wt embryos and Clock c/c mutant embryos. 1999) and their induction is blunted (Shearman and To determine if Per1 is an essential clock component Weaver, 1999). It also confirms that functional CLOCK of peripheral oscillators, we employed a slight variation protein is essential for proper Per2 transcriptional acti- of the serum-shock method described by Balsalobre et vation in peripheral oscillators. al. (1998) to initiate circadian gene transcript cycling in our MEFs. We observe a clear circadian oscillation of Hierarchical Dominance and Phenotypic Rescue Per2 gene expression upon serum shock of normal by the Central Clock MEFs (Figure 1A), as previously described for a variety To test if the central clock could compensate for the of cell lines (Balsalobre et al., 1998; McNamara et al., Per1 / defect in peripheral clocks, we had to reestab- 2001; Yagita et al., 2001). The average ( SD) of three lish a link between the SCN and the peripheral clocks experiments can be fit to a cosine with a period of 23.4 in the MEFs. We predicted that Per1 / MEFs in an 1.2 hr (r 0.77; Figure 1D). environment where they could receive and interpret Serum shock of Per1 / MEFs yielded a surprising SCN-controlled signals would show near normal circaresult (Figure 1B). Instead of the slightly shorter 23 hr dian oscillations of Per2 gene expression. To accomplish period predicted by the activity rhythms of Per1 / mice this, we established the implant procedure deperiod (Cermakian et al., 2001), the peripheral clocks within the picted schematically in Figure 2. Per1 / MEFs produced Per2 oscillations with a much The implantation procedure was first tested on shorter period. The period calculated by fitting of an Per1 / mice. Prior to implantation, the mice were average of three experiments to a cosine was 20 1 housed individually and entrained to a 12 hr light: 12 hr hr (r 0.73). Clearly, the effect of Per1 ablation is much dark (LD) cycle for at least two weeks. Mice were then

3 Communication between SCN and Peripheral Clocks 109 Figure 1. The Period of Per2 Oscillations in Serum-Shocked MEFs Is Genotype-Dependent Total RNA was isolated at the indicated time points from mouse embryo fibroblasts (MEFs) placed in serum free medium following a 2 hr exposure to 50% horse serum. (A C) RNase protection assay (RPA) of Per2 gene expression in serum-shocked (A) wild-type (WT) MEFs, (B) Per1 / MEFs, and (C) Clock c/c MEFs. (D) The Per2 oscillations were quantitated by densitometry and normalized to the highest value (100%). The mean and SD of three independent experiments is shown for all cell types with the data from WT or Per1 / MEFs fit to a cosine curve. (E) RPA of Per2 and -actin expression in WT, Per1 /, and Clock c/c MEFs following serum shock. RNA levels were equalized between all experiments to allow amplitude comparisons. (F) Per2 expression levels were quantified by densitometry and normalized to -actin. Shown are the average and standard deviation of three independent samplings for each time point. For all RPAs, 2 g E. coli trna (t) serves as a negative control reaction and the relative amounts of total RNA used for all samples are shown. placed in DD for three days and the Per1 / MEF-colla- what will be the period of that oscillation?; and (3) will gen disks were implanted on the fourth day in DD (between the resulting oscillation be in phase with host organs what corresponded to ZT1 and ZT3 of the previ- and tissues? Figure 3A shows RNase protection analysis ous LD cycle). At 4 hr intervals during days four and five of total RNA from wt host kidney and skeletal muscle, postimplantation, implants were recovered and skeletal and total RNA from Per1 / MEF-collagen implants recovered muscle and kidney samples were taken from each animal. from the same wt hosts. Kidney and skeletal Total RNA isolated from each tissue was then muscle display robust oscillations in Per2 expression probed to determine Per2 expression levels. with a period of approximately 24 hr in keeping with Three important questions could be addressed by this previous data (Cermakian et al., 2001). initial experiment: (1) will Per1 / MEFs, that had not Strikingly, the oscillation in Per2 expression in the received a serum shock, begin to oscillate when placed Per1 / MEF-collagen implant samples has a period and in a wt host animal?; (2) if they do begin to oscillate, phase equivalent to what is observed for the host tissues Figure 2. Schematic of the Implantation Procedure Following the production of MEFs, the cells are resuspended in a medium supplemented with collagen. After approximately 12 hr, a durable collagen matrix forms around the MEFs that confines them in a disk, or implant, with a diameter of approximately 1.5 cm. This collagen disk is then implanted subcutaneously on the back of a host mouse. After several days, implants and mouse tissues are collected for RNA purification and analysis.

4 Cell 110 Figure 3. Rescue of the Short Period Per1 / Peripheral Clock Phenotype In Vivo Collagen-embedded MEFs were prepared and implanted subcutaneously into mice. At each indicated time postimplantation skeletal muscle, kidney, and implant samples were harvested from a single mouse and total RNA was isolated. (A C) RPA of Per2 expression in (A) Per / host tissues and a Per1 / MEF-collagen implant, (B) Per / host tissues and a Per1 / MEF-collagen implant, and (C) Per / host tissues and a Clock c/c MEF-collagen implant. (D) Densitometric scans of the kidney and implant Per2 oscillations in (A) (blue curves) and (B) (red curves) were normalized to the highest value in the kidney (100%) and plotted as a function of time after implantation. trna (t) is used as a control, and the relative amounts of total RNA shown. (Figures 3A and 3D). We conclude that humoral signals the same wt tissues (compare Figures 1A and 1B). Finally, present in the host can indeed initiate and maintain an SCN lacking Per1 is still able to generate hupresent circadian oscillations in unstimulated MEFs and can moral signals capable of imposing a near normal periodicity thus compensate for the Per1 genetic defect in the implanted on a genetically defective peripheral clock. The peripheral clock. ability of the SCN to impart its periodicity on a peripheral A similar experiment was performed using Per1 / clock, which intrinsically would have a 20 hr period, is MEF-collagen implants introduced into Per1 / mice a striking example of hierarchical dominance within the (Figure 3B). Again, robust Per2 oscillations were observed mammalian circadian system. in the kidney and skeletal muscle of the Per1 / Lastly, the implantation experiment was varied by in- mice. The period of these oscillations is approximately troducing Clock c/c MEFs into a Per1 / host (Figure 3C). 24 hr, as expected from the mild circadian phenotype of Although the host tissues display the expected rhythmicity this mutation (Figure 3D). Importantly, Per2 expression of Per2 expression, there is no overt 24 hr periodthis again oscillates in the Per1 / MEF-collagen implants icity in the implant. Clearly, a robustly functioning clock with a periodicity and phase identical to that of the host. is essential if the implant is to exhibit rhythmic Per2 These results establish that Per1 has profoundly dif- expression. This in turn suggests that the Per2 rhythmicity ferent circadian functions depending on where it is expressed. observed in the Per1 / MEF implants is not simferent In the SCN of our Per1 / mouse model, it is clear ply driven but entrained by the SCN. Thus, the interaction that Per1 does not play a critical role in light-dependent between implant and host is more subtle than a signaling and entrainment. These Per1 / mice readily direct causal link between timing cues originating from entrain to an LD cycle and respond normally to phaseshifting a central pacemaker and their target. The timing cues light pulses given during the night phase (Cer- do exist but must be interpreted by the implants own makian et al., 2001). In addition, Per1 plays only a minor clock in order to act as a Zeitgeber. role in central clock function and timing, as evidenced We conclude from these observations that Per1 is by the almost normal locomotor rhythm of the Per1 / not an integral part of the core oscillatory machinery in mice. The peripheral tissues examined in the Per1 / peripheral clocks, because rhythmicity in gene expression mice thus display the same phase as that observed in is conserved in Per1 mutants (Figures 1B and 3)

5 Communication between SCN and Peripheral Clocks 111 (Cermakian et al., 2001). Furthermore, as the short pe- In contrast to their arrhythmicity in DD, Clock c/c mice riod phenotype caused by the deletion of Per1 in the maintained under LD conditions show clear activity periphery is revealed only when the tissue is deprived rhythms (Vitaterna et al., 1994). In agreement with these of signals from the SCN (compare Figures 1A and 3), observations, we find that the Per2 levels in Clock c/c Per1 may perhaps be responsible for intrinsic peripheral peripheral organs display a clear 24 hr periodicity (Figure clock accuracy or timing. 4D). However, there were two unusual features to these oscillations. First, while the Per2 transcript accumula- Phenotypic Inheritance by Peripheral Oscillators tion in these mice appeared similar in phase to wt mice, To explore the extent to which the central clock can the decrease in Per2 levels in Clock c/c mice does not impose its rhythmic period on peripheral oscillators, occur until the onset of the light period (Figure 4F). We Per1 / MEF-collagen implants were introduced into had originally predicted that the decrease in transcripmice heterozygous and homozygous for the Clock c mu- tional activity of the mutant CLOCK protein in vitro (Gektation. Per2 oscillations in the kidney, skeletal muscle, akis et al., 1998) might result in a delay in the phase of and Per1 / MEF-collagen implant samples collected Per2 accumulation, but our results suggest instead that from the Clock / mice are nearly identical to what was the Clock c mutation may affect the ability of PER or CRY seen in the Per1 / and Per1 / mice, as expected (com- to properly inhibit the transcriptional activity remaining pare Figures 3A and 4A). The period of Per2 oscillations in the mutant CLOCK in vivo. Second, given this Per2 in the Clock / kidney and implant obtained by curve rhythm in Clock c/c tissues in LD, the arrythmicity of fitting are hr (r 0.91) and hr Clock c/c MEFs in a wt host kept under DD was surprising (r 0.98), respectively (Figure 4E). (Figure 3C). This drastic difference underscores the im- Clock /c mice have free-running periods significantly portance of masking by light, which appears to affect longer than 24 hr (Vitaterna et al., 1994). We applied the the activity patterns of the animal through a pathway same implantation procedure described above to the independent of the SCN (Redlin and Mrosovsky, 1999). Clock /c mice, and predicted a longer period in Per2 Lastly, and even more interesting, the Per2 expression oscillation if the same central timer controlled locomotor pattern of wt MEF implants in these Clock c/c animals rhythms and rhythmic gene expression in peripheral tis- appears normal. In agreement with our previous results, sues. Indeed, the peak of Per2 expression is altered in the onset of Per2 accumulation in the implant mimics the kidney and skeletal muscle of Clock /c animals (Fig- that observed for other host tissues. However, in these ure 4B). Strikingly, the Per1 / MEF-collagen implants wt MEFs, the CLOCK protein is fully functional and the also display the long-period phenotype of the Clock /c decrease in Per2 is typical of wt animals. The masking mice (Figure 4B). Curve fitting shows that the Per2 effect of the LD cycle thus results in Per2 rhythms with rhythm has a similar period in the kidney ( hr, periods dependent on the Zeitgeber and waveforms der 0.92) and in the implant ( hr, r 0.63), both pendent on the genotypes of tissues. of which are longer than in the Clock / hosts (Figure 4E). Thus, not only the intrinsic 20 hr period of the Per2 Daytime Feeding Entrains MEF Clocks oscillation in Per1 / MEFs can be entrained by an SCN to a 24-Hour Rhythm with an approximately 24 hr period, it can also be en- LD cycles are able to entrain Per2 rhythms in peripheral trained to the even longer period of the Clock c heterozytissues even in mice whose clock is functioning poorly. gotes. These observations further support our con- Given that the masking effect of light can result in an tention that the SCN can mask genetic defects in effective restriction of feeding to the nocturnal phase, peripheral clocks and reinforce the idea of SCN domiand that restricted feeding can entrain oscillations in nance over the intrinsic rhythmic properties of peripheral oscillators. peripheral tissues, it is possible that the rhythmicity ob- After several days in constant darkness, Clock c/c mice served in the tissues of Clock c/c mice might have resulted display arrhythmicity in activity (Vitaterna et al., 1994) from feeding only during the dark phase of the cycle. and SCN neuronal firing (Herzog et al., 1998). Can cells To test directly the impact of restricted feeding protocapable of producing rhythmic oscillations do so in the cols on Per2 expression in the MEF implants, we pro- presence of an SCN that cannot? Clock c/c mice, which vided access to food only during the light phase of a have been maintained under DD for 12 days, have ranaffected by restricted feeding and maintained its normal LD cycle (Figure 5A). As expected, the SCN was not dom levels of Per2 expression in the kidney and skeletal muscle (Figure 4C). Intriguingly, there is some correlaas predicted (Damiola et al., 2000; Stokkan et al., 2001), phase with respect to the LD cycle (Figure 5B). Again tion between the expression levels of Per2 observed at certain time points, for example at 88 hr postimplantarespect the phase of Per2 expression in the liver is inverted with tion. It is possible that improperly regulated signals may to the normal phase under LD cycles (Figure still be originating from a randomly firing SCN, causing 5A). Liver is the tissue most susceptible to entrainment global fluctuations in Per2 expression in many peripheral by restricted feeding, but other tissues such as kidney tissues. We also observe that the Per1 / MEFs imgree. and muscle also show the phase reversal to some de- planted in a Clock c/c host exhibit the same arrhythmic The important feature of this experiment lies in phenotype displayed by the kidney and skeletal muscle. Per2 expression in the implant. Four days after implanta- Therefore, in the absence of directive signals from the tion in animals entrained by food restriction, Per1 / SCN, even cells that do contain a robust circadian oscilmately implants display a peak in Per2 expression at approxilator do not display a discernable rhythm. This experihost the same time as other peripheral tissues of the ment also indicates that the implantation procedure by (Figure 5A). The period of the implant is difficult to itself is not sufficient to initiate rhythmicity in an implant. determine, presumably due to the fact that feeding is a

6 Cell 112 Figure 4. The Rhythmic Phenotype of the Implant Is Dictated by Circadian Phenotype of the Host Collagen-embedded MEFs were prepared and implanted subcutaneously into mice. At each indicated time postimplantation, skeletal muscle, kidney, and implant samples were harvested from a single mouse and total RNA was isolated. (A D) RPA of Per2 expression in (A) Clock / host tissues and a Per1 / MEF-collagen implant, (B) Clock /c host tissues and a Per1 / MEFcollagen implant, (C) Clock c/c host tissues and a Per1 / MEF-collagen implant under DD, and (D) Clock c/c host tissues and a Per1 / MEFcollagen implant under LD. The light regime for each experiment is shown either by a black rectangle (constant darkness) or by alternating white and black rectangles (LD 12:12) immediately above the panels. (E) Densitometric scans of the kidney and implant Per2 oscillations in (A) (blue curves) and (B) (red curves) were normalized to the highest value in kidney (100%) and fit to a cosine curve. (F) Densitometric scans of the Per2 oscillations in the Per1 / MEF implant (green curve) fit to a cosine curve and the kidney (purple curve) of Clock c/c hosts maintained under LD conditions. trna (t) is used as a control and the relative amounts of total RNA shown. Implant Integrity A potential caveat to the results described above is the possibility that host cells could infiltrate the MEFcollagen implant, resulting in misleading results. How- ever, at the time points used for the experiments presented in this study, no gross infiltration of the implant by surrounding host tissues was observed by macroscopic examination. To completely rule out this potential prob- poor Zeitgeber for MEFs, as it is for the muscle. However, if the implant were not entrained by the feeding regime, it would be unlikely to display a prominent maximum at the same time as other tissues. Thus, uncoupling of the implant clock from the SCN did not unmask the phenotype observed in cell culture. Instead, food restriction to daytime actively entrains the clock intrinsic to the MEFs.

7 Communication between SCN and Peripheral Clocks 113 Figure 5. Entrainment of the Per2 Rhythm in the Implant by Daytime Restricted Feeding Collagen-embedded MEFs were prepared and implanted subcutaneously into mice. At each indicated time postimplantation, liver, skeletal muscle, kidney, and implant samples were harvested from a single mouse and total RNA was isolated. Brains were also harvested from the same mice and processed for in situ hybridization. Mice under a 12:12 LD cycle were entrained to daytime feeding for ten days prior to implantation and maintained under this schedule (shown schematically above A) for the duration of the experiment. (A) RPA of Per2 expression in WT host tissues and a Per1 / MEF-collagen implant. trna (t) is used as a control, and the relative amounts of total RNA shown. (B) In situ hybridization of serial sections from each brain with either a VIP or a Per1 probe at the indicated times postimplantation. lem, the fluorescent marker PKH26 was incorporated is likely to shed light on how these two types of oscillators into the cytoplasmic membrane of the MEFs prior to interact. There could even be subtle or significant implantation. This marker can be visualized using a fluo- differences between peripheral oscillators, which would rescence microscope following the retrieval of the MEFcollagen allow differential interpretation of signals originating implant from a host animal. from the SCN and the environment. Figure 6 shows sections of Per1 / MEF collagen implants We have demonstrated that striking differences exist that were placed subcutaneously into Per1 / between central and peripheral oscillators. Per2 oscilla- and Clock /c host animals. The implants were retrieved tions in Per1 / MEFs have a much shorter period than at a time corresponding to the final time point taken in in wt cells, indicating that Per1 has a drastically different each of the implantation experiments performed. Following function in central versus peripheral clocks. One possi- sectioning, samples were counterstained with ble explanation for this is that some functional redun- DAPI and analyzed. Skeletal muscle was harvested from dancy is present within the SCN and not within periphthe same animal as the implant for a negative control. eral clocks. In the SCN, Per1 appears to suppress Per2 MEFs are distinguished from host cells by the colocali- basal gene activity. In the absence of Per1, Per2 expreszation of red fluorescence with blue DAPI stained nuclei, sion peaks at a faster rate and with greater amplitude. as host cell nuclei do not exhibit any red fluorescence. Indeed, PER2 expression is increased in peripheral tis- The merged images show clear colocalization of red sues of Per1 null mice (Zheng et al., 2001). Because PKH26 staining with virtually all the DAPI stained nuclei PER2 positively regulates Bmal1, improperly regulated in the implant (Figure 6). Quantification of several fields Per2 activation may prematurely initiate the beginning from serial sections through implants showed that 98% of the subsequent cycle, thus giving rise to the short of the nuclei colocalized with PKH26 red fluorescence. period phenotype. It is likely that most if not all of the nuclei that did not The model of SCN control over physiology and behavcolocalize with red fluorescence were due to sectioning ior through neuronal and humoral outputs (Allen et al., and labeling variability. Therefore, we conclude that the 2001; Balsalobre et al., 2000; Buijs and Kalsbeek, 2001; observed Per2 expression results from the implanted LeSauter and Silver, 1998; McNamara et al., 2001; Ue- MEFs and not from contaminating host cells. yama et al., 1999) provided the framework for elucidating the mechanisms that regulate the interplay between the Discussion SCN and peripheral clocks. SCN-lesioned animals lose a variety of behavioral and physiological rhythms which The understanding of the physiological and functional can be partially restored with SCN transplants (Ralph et relationship between central and peripheral clocks is al., 1990; Silver et al., 1996). essential in circadian biology. Characterization of any By transplantation of MEFs we have revealed the differences between the SCN and peripheral oscillators dominance of the SCN over the activity of peripheral

8 Cell 114 Figure 6. MEF-Collagen Implants Maintain Their Integrity after 5 Days in the Host Per1 / MEFs were labeled with PKH26 prior to being incorporated into a collagen implant. Recovered implants and control skeletal muscle were sectioned, stained with DAPI, and examined with a fluorescence microscope. DAPI and PKH26 staining of Per1 / MEF-collagen implants harvested 5 days after implantation in a Per / host mouse (A) or in a Clock /c host mouse (B). No PKH26 staining is observed in skeletal muscle from the host. clocks. In the absence of external environmental cues, expect, is most susceptible. Our Per1 / MEF implants Per1 / MEFs take on a 24 hr period in wt hosts, a behave more similarly to muscle in this regard. Moreslightly longer period in Clock /c hosts, and an arrhyth- over, MEF implants acquire the same period of Per2 mic phenotype in Clock c/c hosts (Figure 4). The adoption oscillation as other peripheral tissues, i.e., 24 hr and not by the MEFs of the phenotypic characteristics of host 20 hr. Altogether, these data suggest that restricted SCNs illustrates the striking hierarchical dominance existing feeding does not simply uncouple peripheral oscillamals. between central and peripheral clocks in mam- tors from the SCN. This treatment instead acts through This strict hierarchy does not appear to operate an active mechanism, producing a bona fide signal that in Drosophila, as shown by maintenance of the phase can produce phase changes in some tissues. in transplanted excretory tubules into a host fly maintained It is important to emphasize that clock gene oscillation on an opposite cycle (Giebultowicz et al., 2000). in the periphery requires a functional oscillator. This can Additional evidence comes from studies on the olfactory be deduced from the lack of rhythmicity in Clock c/c MEFs response rhythms in the antennae, where local per defi- implanted in a wild-type host (Figure 3C). While SCNderived ciency results in arrhythmicity (Krishnan et al., 1999). signals can sustain oscillations in a heavily Environmental cycles seem to directly entrain peripheral damped peripheral oscillator, they are insufficient to clocks without the need of a dominant pacemaker induce oscillations in cells without intrinsic rhythmic ca- (Plautz et al., 1997). Similarly, peripheral tissues in zebra- pacity. How then can the rhythmicity in tissues of Clock c/c fish show independence from central clocks (Cermakian mice maintained under LD conditions be explained? An et al., 2000; Whitmore et al., 1998) and are light-respon- intriguing possibility is that light may be directly responsible sive (Whitmore et al., 2000). Peripheral circadian photoreception for neuronal of hormonal signals which act synersive has not been demonstrated in mammals, but gistically with SCN derived signals to force oscillations feeding schedule is a potent peripheral Zeitgeber (Damiola even in cells without oscillatory capability. et al., 2000; Stokkan et al., 2001). Three prominent pathways are directly or indirectly Under restricted feeding, the SCN can be uncoupled involved in the entrainment of central and peripheral from peripheral clocks, which become directly entrained clocks (Figure 7). Light entrainment, where photic stimuli to an environmental stimulus (Damiola et al., 2000; Stokkan are transmitted through the retina to the SCN, is essen- et al., 2001). We have placed implants into mice tial (Figure 7A). Once entrained to environmental light subjected to a daytime restricted feeding regime (Figure cycles, the SCN acts as a robust self-sustained oscillator 5). As noted previously, different tissues have different that resets or synchronizes peripheral clocks so that degrees of susceptibility to food. Liver, as one might their oscillations will be in phase with the environment

9 Communication between SCN and Peripheral Clocks 115 Figure 7. Schematic of Entrainment Mechanisms for Peripheral Oscillators in the Mouse Light, activity, and feeding schedule can all affect circadian rhythmicity of the SCN and/ or the peripheral tissues (A C). We have combined these pathways into a unified model (D), which also includes postulated influence of activity rhythms on feeding schedule and SCN-independent effects of light on peripheral tissues. (Brown and Schibler, 1999; Yamazaki et al., 2000). When activity and feeding rhythms results in a consequent isolated, peripheral oscillators lose overt rhythmicity entrainment of peripheral clocks. Alternatively, these after several cycles (Abe et al., 2002; Yamazaki et al., pathways may be acting on peripheral clocks in parallel 2000). However, entrainment of these peripheral clocks and could thus be either synergistic or competitive. depends on the presence of a functioning oscillator in However, our results with the Clock mutant argues these tissues (Figure 3C). against the latter possibility, as Clock c/c mice in LD dis- Other cues can affect the SCN. A feedback regulation play Per2 rhythms in peripheral organs. Moreover, a exists between the SCN and activity (Figure 7B). The comparison of the results obtained in these mutant mice central clock can be reset by arousal (Maywood et al., and with Clock c/c MEF implants in wt host suggests that 1999; Mrosovsky, 1996). In addition, the SCN regulates light may have an effect on the periphery through a behavioral rhythms through projections to other hypo- pathway circumventing the SCN. Recent observations thalamic nuclei (Abrahamson et al., 2001; Aston-Jones that suggest direct connections from the retina to hypo- et al., 2001). Recent data indicate that this may be thalamic nuclei regulating activity (Kramer et al., 2001) achieved, at least in part, through SCN-borne TGF and strengthen this view. Dissection of the entrainment path- signaling through EGF receptors in neurons of the sub- way into its various components is important to acquire paraventricular zone (Kramer et al., 2001). Activity a complete understanding of the mechanisms involved rhythms may also have direct or indirect effects on the in central and peripheral clock regulation. entrainment of peripheral clocks through, for example, What signals are generated by normal or mutant SCNs the timing of food absorption. that may regulate peripheral clocks? These may be involved Restricted food availability has been shown to act as in phase setting, driving peripheral oscillations, a Zeitgeber for peripheral clocks (Figure 7C; Damiola et inhibiting these pathways, or a combination of these. al., 2000; Krieger et al., 1977; Stokkan et al., 2001). Here, Glucocorticoids (Balsalobre et al., 2000; Le Minh et al., we demonstrate that this occurs through an active 2001) and nuclear hormones (McNamara et al., 2001) mechanism and not a simple uncoupling of peripheral have been implicated in regulating the phase of periph- clocks from the central pacemaker. Two possibilities eral clocks. In the present study, we have described a may explain how entrainment by food restriction can powerful and highly flexible implantation procedure that circumvent the otherwise strong link between the SCN may prove useful. It will allow a variety of combinations and the periphery. First, the SCN could affect peripheral of host and peripheral tissue genotypes to be tested in clocks primarily through the activity rhythms and food a straightforward and efficient manner. Instead of using intake; due to SCN function, nocturnal animals are more tissue-specific knockouts, one could use implant with active at night and thus eat mostly during this period. the appropriate signaling molecule mutated. In addition This nocturnal feeding entrains peripheral clocks through to flexibility, this approach allows the comparison, within a mechanism similar to the one involved in the daytime a single host animal, of clock gene expression in the restricted feeding experiments. In this case, daytime implant (a model for peripheral clocks) to that present restricted feeding would simply short circuit the SCN- in a variety of peripheral tissues. periphery connection, by imposing the time of food intake and therefore the signal for peripheral tissue clock Experimental Procedures resetting. A second way by which the SCN could influence peripheral clocks is through neuronal or humoral Cell Culture Mouse embryo fibroblast cultures were established by harvesting pathways linking the hypothalamus to the periphery in embryos at embryonic day 13. Head and liver removed, the reparallel to the activity-feeding pathway. This would maining embryonic tissue was passed five times through an 18- imply that the entrainment by food restriction is domi- gauge needle. The disrupted tissue was plated in DMEM 10% nant over other entrainment pathways, for at least some FCS (GibcoBRL) and cultured at 37 C in5%co 2. Cultures were tissues, as under these conditions the SCN and other passed 1:3 after reaching confluency and discarded after reaching oscillators appear uncoupled. passage 10. Serum shock was as described (Balsalobre et al., 1998) except These three pathways (Figure 7A 7C) can be intethat confluent MEFs were grown for three days in DMEM 1% FCS grated in a unified view that takes our results into ac- prior administration of DMEM 50% horse serum (GibcoBRL). count (Figure 7D). Light entrains SCN rhythms, and the SCN in turn synchronizes peripheral tissues, as time of In Situ Hybridization and RNase Protection Assays food administration and perhaps activity do. An interest- All riboprobes were generated using an in vitro transcription kit ing possibility is that the control exerted by the SCN over (Promega). In situ hybridization on frozen brain sections was as

10 Cell 116 described (Cermakian et al., 2001). Probes cover nt of Per1 ORF and nt of Genebank VIP sequence X RNase protection assays (RPA) were as described (Cermakian et al., 2001). References Abe, M., Herzog, E.D., Yamazaki, S., Straume, M., Tei, H., Sakaki, Y., Menaker, M., and Block, G.D. (2002). Circadian rhythms in isolated brain regions. J. Neurosci. 22, Formation of Collagen Implants Abrahamson, E.E., Leak, R.K., and Moore, R.Y. (2001). The suprachi- Collagen implants were formed as described (Palmer et al., 1989; asmatic nucleus projects to posterior hypothalamic arousal sys- St. Louis and Verma, 1988). Briefly, the listed components were tems. Neuroreport 12, combined in the following order: 1.5 ml of 2 DMEM (GibcoBRL), 1.5 ml of FCS (GibcoBRL), 1.5 ml of 3 mg/ml rat tail collagen (Sigma), Allen, G., Rappe, J., Earnest, D.J., and Cassone, V.M. (2001). 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