Author response: Differential effects of light and feeding on circadian organization of peripheral clocks in a forebrain Bmal1 mutant
2014-12-16

Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract In order to assess the contribution of a central clock in the hypothalamic suprachiasmatic nucleus (SCN) to circadian behavior and the organization of peripheral clocks, we generated forebrain/SCN-specific Bmal1 knockout mice by using floxed Bmal1 and pan-neuronal Cre lines. The forebrain knockout mice showed >90% deletion of BMAL1 in the SCN and exhibited an immediate and complete loss of circadian behavior in constant conditions. Circadian rhythms in peripheral tissues persisted but became desynchronized and damped in constant darkness. The loss of synchrony was rescued by light/dark cycles and partially by restricted feeding (only in the liver and kidney but not in the other tissues) in a distinct manner. These results suggest that the forebrain/SCN is essential for internal temporal order of robust circadian programs in peripheral clocks, and that individual peripheral clocks are affected differently by light and feeding in the absence of a functional oscillator in the forebrain. https://doi.org/10.7554/eLife.04617.001 eLife digest Jet lag is a common experience when flying long distance. This disorientating phenomenon occurs when our internal ‘body clock’ remains set to the time zone where the plane departed and fails to reset to the new local time. Our internal clock actually consists of a series of clocks—each of which is based upon groups of genes that are switched on and off at different times of the day and night. There is a master clock in our brain and a series of peripheral clocks in our other organs and tissues. The master clock is thought to coordinate the peripheral clocks, which in turn control the fluctuating activity of a specific organ in response to the time of day. To further investigate the master clock, a typical approach has been made to disable it by deleting the genes for its components. But some of these deletions can cause abnormalities in mice and some are lethal. To get around these problems, Izumo, Pejchal et al. have devised a way to delete a molecular component of the master clock only in the mouse's brain. Izumo, Pejchal et al. used this approach to specifically disable the mouse's master clock and, unlike mice that completely lack the Bmal1 gene, mice with the brain-specific deletion were as healthy and lived as long as normal mice. A molecular probe was used to monitor the peripheral clocks in different organs and tissues of these mutant mice, and revealed that, without a working master clock, the peripheral clocks were no longer synchronized. Izumo, Pejchal et al. found that the lost synchrony could be partially restored by training the mice to adapt to cycles of light and dark and feeding schedules. Following on from the work of Izumo, Pejchal et al., one of the next challenges is to understand how the master clock communicates with the peripheral clocks in different organs and tissues around the body. https://doi.org/10.7554/eLife.04617.002 Introduction The suprachiasmatic nucleus (SCN) in the hypothalamus is the primary regulator of daily rhythms of behavior and physiology in mammals (Meijer and Rietveld, 1989; Ralph et al., 1990; Sujino et al., 2003; Welsh et al., 2010), yet the majority of tissues in the body possess autonomous cellular oscillators (Yamazaki et al., 2000; Abe et al., 2002; Nagoshi et al., 2004; Welsh et al., 2004; Yoo et al., 2004; Abraham et al., 2005). The circadian clock in the SCN as well as in other mammalian cells is composed of interacting positive- and negative-transcriptional and post-translational feedback loops involving Clock and Bmal1 transcription factors and their target genes, Period (Per1, 2, and 3) and Cryptochrome (Cry1 and 2) (King et al., 1997; Gekakis et al., 1998; Hogenesch et al., 1998; Kume et al., 1999; Lee et al., 2001; Lowrey and Takahashi, 2004, 2011; Huang et al., 2012; Mohawk et al., 2012). Extensive evidence suggests that the SCN controls not only behavioral rhythms but also the circadian programs of peripheral tissues (Yoo et al., 2004; Guo et al., 2005, 2006; Saini et al., 2013; Yamaguchi et al., 2013). However, much remains unclear regarding the relationship between the central clock and peripheral oscillators, which can be entrained by many different stimuli (Stokkan et al., 2001; Buhr et al., 2010; Saini et al., 2012). To clarify the contribution of a central clock to the circadian rhythms of peripheral tissues, we sought to find a way to disable the molecular oscillators in the brain without affecting the genetic components in the rest of the body. Traditionally, a functional role of a gene has been investigated by a gene targeting strategy, which eliminates the gene from embryonic stem cells so that it is inactivated ubiquitously in mice. While this approach provides a powerful method to study the function of genes in vivo, it cannot be applied to genes that affect developmental or metabolic processes crucial for survival. Moreover, even if the knockout mice survive to adulthood, some lines suffer from systemic conditions and hence require special handling in experiments. For instance, although a global knock out of Bmal1 (Bmal1−/−) completely eliminates circadian rhythms (Bunger et al., 2000), the animals suffer from morbid conditions including arthropathy (Bunger et al., 2005), sterility (Alvarez et al., 2008; Boden et al., 2010), defects in glucose homeostasis (Rudic et al., 2004; Lamia et al., 2008; Marcheva et al., 2010), premature aging, and decreased lifespan [their average lifespan is 37 weeks (Kondratov et al., 2006; Sun et al., 2006)]. Possibly due to the pleiotropic effects of Bmal1, results of studies such as food entrainment behavior with Bmal1−/− mice have been controversial (Fuller et al., 2008; Mistlberger et al., 2008; Fuller et al., 2009; Pendergast et al., 2009; Storch and Weitz, 2009; Mistlberger et al., 2009a, 2009b; Mieda and Sakurai, 2011). To circumvent these problems, conditional knockout techniques utilizing Cre recombinase and loxP sequences have been applied widely in the nervous system (Gavériaux-Ruff and Kieffer, 2007; Taniguchi et al., 2011). However, until recently, Cre drivers that can efficiently recombine a floxed allele in the majority of SCN neurons have not been identified (Mieda and Sakurai, 2011; Musiek et al., 2013 but see Husse et al., 2011). In this study, we examined the ability of a forebrain-specific Cre driver to excise floxed alleles of Bmal1, a critical component of the molecular oscillator, specifically from brain regions including the SCN. The resulting knockout mice were devoid of BMAL1 expression in the forebrain/SCN but not in other peripheral tissues. Unlike Bmal1 global knockout mice, Bmal1 forebrain knockouts did not display detectable defects in reproduction, activity, body weight, or life span, thereby providing an ideal model to study circadian physiology in the absence of a central clock. These genetically engineered mice are better experimentally controlled than mice that receive SCN lesions, which is a classic method to induce arrhythmic behavior in rodents (Meijer and Rietveld, 1989). With SCN lesions, precise removal of the SCN tissue varies from mouse to mouse and requires laborious confirmation of the excision. In addition, ablation of the SCN destroys neuronal networks and communication pathways, rendering the network responses and mechanisms incapacitated. With forebrain-specific Bmal1 knockout mice, we examined the impact of defunct brain clocks on the organization of peripheral oscillators as well as their response to environmental signals such as light and food. Cre-mediated excision of Bmal1 in the forebrain/SCN confers complete arrhythmicity to circadian behavior. In contrast to the abolition of rhythms in the SCN, all peripheral tissues in these mice sustained circadian rhythmicity; however, they lacked phase coordination and normal amplitude in constant darkness. The synchrony and the oscillatory amplitude of peripheral rhythms in these mice were completely rescued by exposure to light dark cycles but not fully in time-restricted feeding experiments. The feeding cues appear to control the phase expression of the peripheral clocks in a tissue-specific way and in a distinct manner from light. These results demonstrate that an intact central clock plays an essential role in driving normal circadian behavior as well as synchronized and robust circadian programs in peripheral clocks and that light and feeding act on individual peripheral clocks differently in the absence of a functional oscillator in the SCN. Results Forebrain/SCN-specific deletion of Bmal1 Bmal1 (also known as Arntl, Mop3) is one of the essential components of the molecular oscillator, unique in that the single knockout of Bmal1 can confer arrhythmicity both at the behavioral and molecular levels (Bunger et al., 2000). To generate a forebrain-specific knockout of circadian rhythms, we crossed floxed Bmal1 (Bmal1fx/fx) mice (Johnson et al., 2014) to Camk2a::iCreBAC mice (CamiCre or Cre) (Casanova et al., 2001) and produced CamiCre+; Bmal1fx/fx (that we denote as ‘Brain’ Knockout, BKO, defined as a forebrain-specific knockout) as well as Bmal1fx/fx (Fx/Fx), CamiCre+ (Cre), and CamiCre+; Bmal1fx/+ (Het) control mice. Camk2a::iCreBAC mice were used because their Cre expression is faithful to the endogenous Camk2a gene expression pattern: it is neuron-specific, forebrain-enriched (Casanova et al., 2001), and expressed highly in the SCN as observed in Cre-dependent fluorescence reporter mice, CamiCre+; tdTom+ (Figure 1A–E). Although there are more than 37 different Camk2a::Cre transgenic lines reported in Mouse Genome Informatics database (http://www.informatics.jax.org/searchtool/Search.do?query=Camk2a-cre&page=featureList), the majority do not appear to express well in the SCN (e.g., http://connectivity.brain-map.org/transgenic/experiment/81162458), likely because of shorter promoter regulatory sequences and position effects. The other advantage of this Cre driver is that its Cre expression is post-natal and starts at P3 (Casanova et al., 2001), thus reducing potential developmental effects. Bmal1fx/fx mice were generated by inserting loxP sites into the introns surrounding exon 4, which codes for the DNA-binding bHLH domain (Johnson et al., 2014). Upon Cre recombination, exon 4 is deleted in BKO brain tissues that express Camk2a::iCre. The predicted BMAL1 sequence encounters a STOP codon after a stretch of 18 non-native residues following native coding exon 3. The resulting protein is 91 amino acids long (vs 626 amino acids of native BMAL1) and is almost identical to the truncated BMAL1 protein of Bmal1−/− mice (Bunger et al., 2000); both proteins contain no known functional domains and do not accumulate to a detectable level. Figure 1 with 2 supplements see all Download asset Open asset CamiCre+; Bmal1fx/fx mice are forebrain/SCN-specific Bmal1 knockouts. (A) Dorsal view of a CamiCre-; tdTom+ control brain. Bright field. Scale bar: 1 mm. (B) Dorsal view of a CamiCre+; tdTom+ brain. Bright field. (C) Ventral view of (B). (D) Fluorescence image of a coronal brain section containing the SCN from a CamiCre+; tdTom+ mouse. (E) Confocal image of the SCN showing tdTomato expression in the CamiCre+; tdTom+ mice. Scale bar: 50 μm. (F, G) Coronal brain sections at the level of the SCN, prepared from mice sacrificed every 4 hr starting at CT2 following 2 days of DD, were hybridized in situ to examine Bmal1 (F) and Per2 (G) mRNA levels in CamiCre+; Bmal1fx/fx (BKO) mice (n = 3, except n = 4 for CT10, 14, 18) and Bmal1fx/fx (Fx/Fx) control littermates (same as BKO, except n = 1 for CT18 Bmal1 and n = 3 for CT18 Per2). Below each coronal brain section is a close-up of the SCN. (H, I) Optical density graphs of the in situ time course data displayed in (F) and (G). Shown are mean ± SEM, with significant effect of genotype in (H) [F1,46 = 40.7, p < 0.0001] and (I) [F1,44 = 1148, p < 0.0001] by GLM ANOVA. Tukey–Kramer multiple comparison post-tests (p ≤ 0.05) showed that in contrast to Fx/Fx littermates, neither Bmal1 nor Per2 mRNA is rhythmic in BKOs. (J) Immunohistochemistry for BMAL1 on SCN-containing coronal sections of Fx/Fx (n = 3), BKO (n = 3), and Bmal1−/− (KO, n = 1) mice sacrificed at ZT16. Captured with an 20× objective. (K, L) Western blot analysis of BMAL1 in BKOs sacrificed at ZT16. (K) Western blot of various tissues in Fx/Fx (n = 2), BKO littermate (n = 2), and KO (n = 1) mice. (L) Western blot of frontal cortex, cerebellum, and liver in Cre (n = 2), Fx/Fx (n = 2), BKO (n = 3), and KO (n = 2) mice. F. CTX: frontal cortex, Cer: cerebellum. https://doi.org/10.7554/eLife.04617.003 In contrast to global Bmal1 knockouts, the reproduction, longevity, and health of BKO mice were as robust as that of wild-type mice. The fertility of both males and females was indistinguishable from wild-type. At 2 months and 4–6 months of age, the body weights of BKO were found to be nearly identical to Fx/Fx, Cre, and Het mice (Figure 1—figure supplement 1). In order to assess the deletion of Bmal1 in the SCN, we examined the circadian mRNA expression pattern of Bmal1 and Per2, a key Bmal1 target gene, by in situ hybridization after 2 days of constant darkness (DD). At all seven time points taken over a period of 24 hr, BKOs showed a low level of Bmal1 and Per2 mRNA in the SCN as well as in the entire coronal brain section (Figure 1F,G). In more detailed analysis, control mice (Cre, Fx/Fx) showed intense mRNA expression of Bmal1 in the SCN at its peak time (CT18) as well as circadian fluctuation (CT18 vs CT6) (Figure 1F,H; Figure 1—figure supplement 2). Accordingly, Per2 mRNA was also robustly expressed in the SCN in a circadian manner (Figure 1G,I; Figure 1—figure supplement 2). Het mice showed reduction in the Bmal1 mRNA abundance, yet cycling of Per2 mRNA was retained in the SCN (Figure 1—figure supplement 2), indicating that 50% wild-type Bmal1 mRNA is sufficient to sustain Per2 expression in the SCN. In contrast, in BKO mice, Bmal1 mRNA was reduced ∼90% in the SCN (Figure 1F,H; Figure 1—figure supplement 2), and Per2 mRNA cycling was significantly attenuated (∼20% WT levels at CT10, see Figure 1G,I). To confirm that no BMAL1 protein was expressed in the SCN of BKOs, we performed immunohistochemistry in the SCN at Zeitgeber Time (ZT) 16. In contrast to the intense BMAL1 staining in the SCN of Fx/Fx controls, BMAL1 staining in the BKO and global Bmal1 knockout SCN was reduced to background levels (Figure 1J). To further characterize the tissue specificity of the BKOs, we used Western blot analyses to measure BMAL1 protein levels in various brain and body tissues at ZT16 (Figure 1K,L). In contrast to Fx/Fx and Cre controls, BMAL1 (∼69 kD) was detected only faintly in BKO frontal cortex and olfactory bulb (Figure 1K), likely due to glial and non-neuronal expression of BMAL1 since Camk2a is not expressed in glial cells (Lin et al., 1987). Levels of BMAL1 in the cerebellum of BKOs were similar to Fx/Fx and Cre control mice (Figure 1L). Outside of the brain, like Fx/Fx mice but unlike global Bmal1−/− mice, BKOs showed equivalent BMAL1 expression levels in the liver, spleen, kidney, heart, and lung (Figure 1K). Taken together, these results confirm the forebrain specificity of our Bmal1 conditional knockouts. Lack of circadian rhythms in BKOs To assess whether forebrain/SCN Bmal1 is necessary for normal circadian behavior, we examined circadian rhythms of locomotor activity in Cre, Fx/Fx, Het, and BKO mice in a 12 hr:12 hr light:dark (LD) cycle followed by DD and constant light (LL) conditions (Figure 2). In both DD and LL, control mice (Cre, Fx/Fx, and Het) expressed robust circadian rhythms of activity (Figure 2A,C,D). The locomotor activity of Het mice was indistinguishable from that of the other controls. In contrast to controls, Bmal1 brain knockouts exhibited no circadian rhythm of activity in DD or in LL (Figure 2B–D). Out of 31 BKO mice, none had a period in the circadian (18–30 hr) range in DD. In LL, only 2 out of 21 mice had a period in the circadian range (24.1 and 20.9 hr). However, they showed extremely low amplitudes of circadian activity (0.025 and 0.019, respectively, fraction of power in the circadian range, where 1.0 represents the total power in the normalized power spectrum), and spectral analysis using Fast Fourier Transform (FFT) did not detect periodicity in the circadian range in any of the BKOs under either DD or LL conditions (Figure 2D). Figure 2 Download asset Open asset Complete loss of circadian rhythmicity of Bmal1 brain knockout mice in constant darkness and constant light. (A, B) Representative actograms of daily wheel-running activity of Cre, Fx/Fx, Het, and BKO mice. Activity records were double plotted, with each day being represented beneath and also to the right of the preceding day. Horizontal black and white bars at the top of each actogram represent lights off and on, respectively. Mice were housed in LD, released into DD for 4 weeks, returned to LD for 2 weeks, released into LL for 4 weeks, and then returned to LD. GLM ANOVA and Tukey–Kramer multiple comparison post-tests were used to compare genotypes tested. BKOs showed no significant periodicity in DD or LL. (C–E) Period (C), amplitude of circadian rhythm and activity levels (E) in Cre mice (n = for DD, 12 for LD, 12 for Fx/Fx mice (n = 31 for DD, 18 for LD, 18 for Het mice (n = 31 for DD, for LD, for and BKO mice (n = 31 for DD, for LD, 21 for graphs mean ± (C) period was using for days of DD or LL. A significant effect of genotype on period was found for both DD = p < 0.0001] and LL = p < In DD, Cre mice were found to have a longer period than the other control groups ≤ In LL, Het mice were found to have a shorter period than the other control groups ≤ all control groups showed a period in both DD and LL similar to that reported for WT mice. (D) DD and LL amplitude of circadian rhythm represented by in the circadian A significant effect of genotype on amplitude was found for both DD = p < 0.0001] and LL = p < with BKO mice significantly amplitude ≤ 0.05) in both DD and LL. (E) daily DD, LD, and LL activity in 24 BKO activity levels were found to be significantly than of controls in DD = p < 0.0001] ≤ 0.05) and LL = p = ≤ 0.05) but not in LD. total daily activity of BKOs was significantly than that of control mice both in DD and LL (Figure studies have found that the locomotor activity of Bmal1−/− mice is decreased wild-type levels (Bunger et al., and that transgenic BMAL1 expression in this activity et al., Taken together, these results suggest that a peripheral in BMAL1 is for the low activity levels that had been reported in global Bmal1−/− mice. In BKOs do not have this peripheral so that is the absence of a central circadian which activity the and likely to an in levels of to light cycles and of the between BKO and mice is that BKOs display entrainment to light/dark cycles because BKOs are from of to the SCN which occurs in (Figure 2). However, the activity of BKOs in LD was and on average than controls. a activity level was in BKOs ± of total daily to the control groups ± average of the control groups ± (Figure supplement 1). it was unclear whether the activity of Bmal1 brain knockouts around the light/dark was due to entrainment of a circadian or to a in In order to whether Bmal1 brain knockouts to we used a Figure The activity of Cre, Fx/Fx, and Het mice the were indistinguishable from the activity in LD (Figure In contrast, none of the BKO mice used in this study entrained to the (Figure 3 BKO mice showed activity that their activity in DD (Figure 2 BKO mice displayed a activity pattern (Figure and BKO mice exhibited activity that was between these (Figure Unlike control mice, BKO mice their activity between the day and the of the (Figure In controls of their activity the to only for BKO mice (Figure The of activity that BKOs day vs was not significantly This of entrainment to is of the entrainment behavior of which as a of circadian oscillators that with phase to the and we that entrainment in Bmal1 brain knockout mice is under with the that the of phase entrainment be this suggests that the periodicity in LD in BKOs from entrainment of a of oscillators in which phase control is Figure 3 with 1 supplement see all Download asset Open asset entrainment and are in Bmal1 brain knockout mice. (A, B) Representative actograms of daily wheel-running activity of Cre, Fx/Fx, Het, and BKO mice. Mice were housed in LD, released into DD for 4 weeks, returned to LD for 2 weeks, to a for 4 weeks, returned to LD for 2 weeks, and then switched to an cycle for 3 Shown are days of the and days of LD activity of Fx/Fx (n = and BKO littermates (n = (C) for individual (D) the average for the entire data represents a for (E) activity ± hr of day and hr of of In contrast to Fx/Fx littermates, BKOs are day vs night. < by (F) of BKO (n = and Fx/Fx littermates (n = specific hr light of LD activity ± each light is by the total of that the dark the data for each genotype represents the of activity between the of light and the of There was a significant between the genotype and time using GLM ANOVA = p < days and were found to be significantly different by Tukey–Kramer multiple comparison post-tests ≤ (G) Representative actograms of daily wheel-running activity of Fx/Fx and BKO These are the mice as in (A, and respectively. are in to the of the LD cycle as well as to the of wheel-running activity under this that, after 1 of this phase are (H) Representative amplitude power of Fx/Fx and BKO mice, from analyses performed on the activity records in (G). The for both genotypes were in the range, to the LD The peak for the controls was in the circadian (18–30 hr) range of the BKOs was found to have a period in the circadian range (I) of amplitudes in the range and in the circadian range for the Fx/Fx and BKO mice as in to controls, BKOs had significantly amplitude in the range < by and significantly amplitude in the circadian range < by the and LD results both some of in BKOs, we next whether ability of light to locomotor in Bmal1 brain knockouts was To assess in BKO mice, we used an LD cycle et al., (Figure Mice are to to LD thus the and the effects of light to be this for a the of all of a circadian cycle (Figure BKOs exhibited significantly activity the light of LD ± of total activity vs ± for Fx/Fx littermates, p < by Fx/Fx mice display more activity the light of LD because the circadian clock is activity the of light (Figure While Fx/Fx mice have an circadian component of activity under LD BKOs have none (Figure Figure supplement 1). from this when we examined the time course of the it became that the of BKOs is to controls (Figure a of 50% to BKOs to the of the light actograms on a time in of this in BKOs (Figure supplement 1). These entrainment and studies to that BKOs lack an endogenous clock but also some synchrony and in peripheral tissues of BKOs To the temporal organization of peripheral oscillators at an we crossed the BKOs to mice (Yoo et al., and the signals from the SCN and other tissues in the body. with the robust of the control SCN, the SCN of BKOs exhibited expression with but detectable rhythms, which were to 24 hr ± hr in Fx/Fx control SCN vs ± hr in BKO SCN for mean period ± Figure These rhythms are likely to be from glial cells neurons in which Cre-mediated excision of floxed Bmal1 was Figure or from that we have observed in the SCN of global Bmal1 knockouts et al., expression in the hypothalamus in BKOs was also significantly In contrast to the SCN and peripheral tissues retained circadian rhythms (Figure with the normal expression of BMAL1 in peripheral tissues (Figure 1K). Figure 4 Download asset Open asset of circadian expression of in the forebrain/SCN knockout mice. (A) Representative records of of circadian expression from various tissues in Fx/Fx and BKO mice. were prepared from mice in LD. are the LD cycle of day Shown are days of after except for the SCN for which was on day Period of various tissues from Fx/Fx control and BKO mice in LD (B) and DD The is on Shown are mean period ± < < by These to the forebrain-specific knockout mice to the relationship between the central and circadian of peripheral tissues. studies have a similar by the SCN (Yoo et al., 2004; et al., 2012; Saini et al., 2013). Unlike mice, however, BKOs intact networks and normal or levels of behavioral activity in LD and in constant conditions. This new analysis to whether deleting BMAL1 the molecular in the without the of the SCN, is sufficient to affect the organization of peripheral of mice littermate control and were