Rhythmic expression of cryptochrome induces thecircadian clock of arrhythmic suprachiasmaticnuclei through arginine vasopressin signalingMathew D. Edwardsa, Marco Brancaccioa, Johanna E. Cheshama, Elizabeth S. Maywooda, and Michael H. Hastingsa,1

aDivision of Neurobiology, Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 0QH, United Kingdom

Edited by Joseph S. Takahashi, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 26, 2016(received for review September 25, 2015)

Circadian rhythms in mammals are coordinated by the suprachiasmaticnucleus (SCN). SCN neurons define circadian time using transcriptional/posttranslational feedback loops (TTFL) in which expression ofCryptochrome (Cry) and Period (Per) genes is inhibited by their proteinproducts. Loss of Cry1 and Cry2 stops the SCN clock, whereas individualdeletions accelerate and decelerate it, respectively. At the circuit level,neuronal interactions synchronize cellular TTFLs, creating a spatiotem-poral wave of gene expression across the SCN that is lost in Cry1/2-deficient SCN. To interrogate the properties of CRY proteins requiredfor circadian function, we expressed CRY in SCN of Cry-deficient miceusing adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2::EGFP under aminimal Cry1 promoterwas circadian and rapidly inducedPER2-dependent bioluminescence rhythms in previously arrhythmicCry1/2-deficient SCN, with periods appropriate to each isoform.CRY1::EGFP appropriately lengthened the behavioral period inCry1-deficient mice. Thus, determination of specific circadianperiods reflects properties of the respective proteins, independentlyof their phase of expression. Phase of CRY1::EGFP expression wascritical, however, because constitutive or phase-delayed promotersfailed to sustain coherent rhythms. At the circuit level, CRY1::EGFPinduced the spatiotemporal wave of PER2 expression in Cry1/2-deficient SCN. This was dependent on the neuropeptide arginine va-sopressin (AVP) because it was prevented by pharmacological block-ade of AVP receptors. Thus, our genetic complementation assayreveals acute, protein-specific induction of cell-autonomous and net-work-level circadian rhythmicity in SCN never previously exposed toCRY. Specifically, Cry expression must be circadian and appropriatelyphased to support rhythms, and AVP receptor signaling is requiredto impose circuit-level circadian function.

period | bioluminescence | arginine vasopressin | clock | oscillation

The suprachiasmatic nucleus (SCN) is a precise biological clockthat coordinates circadian (∼24 h) rhythms in mammals.

Composed of ∼10,000 neurons, the SCN sits atop the optic chiasm,through which it receives retinal innervation that entrains its cellularclockwork to the light/dark cycle. In turn, the SCN entrains periph-eral circadian clocks distributed across the organism through neuralpathways incorporating neuropeptidergic and GABA-ergic signaling(1). At the molecular level, circadian timing in SCN neurons revolvesaround transcriptional/posttranslational feedback loops (TTFL), inwhich the positive components BMAL1 and CLOCK activate E/E′-box–mediated transcription of the negative components Period (Per1,Per2) and Cryptochrome (Cry1, Cry2). PER/CRY heterodimers theninhibit BMAL1/CLOCK activity and thus prevent their own tran-scription. Subsequent proteasomal degradation of PER andCRY proteins relieves this inhibition, allowing the molecular cycle tobegin again. Genetic studies have established that CRY1 and CRY2are essential for behavioral and molecular circadian rhythmicity,acting as core components of the TTFL (2, 3). The properties thatallow them to fulfill this role are, however, unclear. For example, asurprising feature of the clock is the opposing circadian phenotypesin single Cry knockouts. Although sequence similarity between theCRY proteins is extremely high, Cry1-null mice have short period

behavioral rhythms under constant conditions whereas Cry2-nullmice have longer periods (2). These differences are also seen atthe level of the SCN in vitro and are magnified in the presence ofthe Fbxl3Afh mutation that stabilizes CRY proteins (4). Whetherthese differences reflect intrinsic properties of the protein, differ-ences in their phase of expression, or other features is unknown.Indeed, it is unclear whether rhythmic expression of the Cry genesis necessary for a fully functioning TTFL. Molecular rhythms canbe induced in Cry1/2-null mouse embryonic fibroblasts (MEFs) whenCry is expressed rhythmically under a promoter containing E/E′-box,D-box, and RRE sequences (5, 6). On the other hand, constitutivelyavailable, cell-permeant CRY proteins can also induce rhythms inCry1/2-null fibroblasts (7), and overexpression of CRY1 inWTMEFsdoes not abolish circadian rhythmicity (8), whereas constitutiveoverexpression of PER2 does, suggesting that circadian oscillationsrequire rhythmic expression of Per2, but not Cry. The present studyaimed to define critical properties of CRY proteins that sustainTTFL function in the SCN, the central circadian pacemaker. Incontrast to mere loss-of-function studies, our approach focused onthe specific circadian properties that are conferred on the SCN circuitby CRY proteins. We therefore developed a genetic restorationapproach using adeno-associated virus (AAV) gene delivery to SCNof CRY-deficient mice in organotypic culture and in vivo. Restorativeassays have been performed in fibroblast culture, but such cells lackthe intercellular coupling present in the SCN and do not controlbehavior. Our approach would allow us to investigate the circadian

Significance

Daily cycles of behavior adapt us to the 24-h rhythm of day andnight. These rhythms are coordinated by a central clock, thesuprachiasmatic nucleus (SCN), in which self-sustaining transcrip-tional/posttranslational feedback loops define approximately 24-h(circadian) time. Cryptochrome1 (CRY1) and CRY2 are essential clockcomponents, but how they sustain cell-autonomous and circuit-level circadian timing in the SCN is not clear. To explore this, wedeveloped a genetic complementation assay, using virally expressed,fluorescently tagged CRY proteins to test whether molecularpacemaking can be induced in arrhythmic Cry1/2-deficient SCN.We demonstrate protein-specific and robust induction of molecularpacemaking, the efficacy of which depends on the circadian patternand phase of Cry expression and functional signaling via neuro-peptide (AVP) receptors.

Author contributions: M.D.E., M.B., E.S.M., and M.H.H. designed research; M.D.E., J.E.C.,E.S.M., and M.H.H. performed research; M.B. contributed new reagents/analytic tools;M.D.E., J.E.C., and E.S.M. analyzed data; and M.D.E., M.B., E.S.M., and M.H.H. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. Email: mha@mrc-lmb.cam.ac.uk.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1519044113/-/DCSupplemental.

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roles of CRY in both cell-autonomous and circuit-level pace-making in behaviorally relevant neurons.

ResultsInduction of Circadian Clock Function in Arrhythmic Cry1/2-Null SCNby AAV-Delivered Cry. Cry1/2-null SCN were transduced with AAVsencoding the CRY1::EGFP fusion protein controlled by a minimalCry1 promoter (pCry1) that exhibits circadian activation in the SCN(Fig. 1A) (9). Typically, ∼5 × 109 viral particles (1 μL of ∼5 × 1012GC/mL) were dropped directly onto each slice 7 d after dissectionor after bioluminescence recordings, as previously reported (10).Confocal microscopy confirmed nuclear expression of CRY1(transduction efficiency of SCN cells, 88.0 ± 7.8%, n = 4) (Fig. 1A).Before AAV transduction, PER2::LUC bioluminescence wasarrhythmic; however, ∼2 d after transduction with pCry1-Cry1::EGFP, rhythms emerged (Fig. 1B) with a long period (26.9 ± 0.2 h,determined after medium change) appropriate to the Cry2-null,Cry1-proficient genotype of the transduced SCN (Fig. 1C). Thus,arrhythmic SCN previously unexposed to CRY proteins can rapidlyexpress circadian molecular rhythms after virally mediated CRY1expression. Cry1 cDNA was then replaced by Cry2 cDNA and AAV-mediated nuclear expression of CRY2::EGFP in Cry1/2-null SCNconfirmed by microscopy (83.2 ± 6.1%, n = 4) (Fig. S1A). CRY2::EGFP induced strong bioluminescence rhythms in initially disorga-nized Cry1/2-null SCN (Fig. S2A). Importantly, these rhythms had ashort period (22.0 ± 0.4 h) consistent with the Cry1-null, Cry2-proficient genotype. Both CRY1- and CRY2-induced rhythmshad periods significantly different from each other and from WTSCN (24.5 ± 0.1 h, P < 0.0001 for each) (Fig. 1C), althoughrobustness of the oscillations, reported by the cycle-to-cyclevariation of the relative amplitude error (RAE), was comparableto WT SCN (Fig. S2B). Thus, not only can pCry1-driven CRY1and CRY2 induce coherent and stable molecular rhythms inCry1/2-null SCN, but also can do so with isoform-specific periods,even when driven by the same promoter. Therefore, CRY1 andCRY2 are not simply permissive elements: the individual proteinsconfer specific temporal properties to the clockwork.To test whether virally expressed CRY can intermesh with

ongoing molecular oscillations driven by endogenous CRY,rhythmic Cry1-null SCN were transduced with pCry1-Cry1::EGFP(Fig. 1D). The period of Cry1-null SCN before transduction was

short (22.3 ± 0.4 h), and pCry1-Cry1::EGFP expression stablylengthened period (26.4 ± 0.1 h, P < 0.001) (Fig. 1E) from 3 dafter transduction (Fig. S2C). In addition, these rhythms had alower RAE, as CRY1::EGFP conferred stability to the endogenousclockwork (Fig. S2D). In the converse test, long period Cry2-nullSCN were transduced with pCry1-Cry2::EGFP (Fig. S2E), and thisdecreased period (P < 0.01) (Fig. S2 F and G), although RAE didnot decrease following AAV transduction with Cry2 (Fig. S2H). Thus,CRY2 did not enhance the definition of the molecular cycle. Trans-genic CRY1 and CRY2 can, therefore, interact with andmodulate theperiod of the endogenous clockwork in single Cry-null SCN.To demonstrate the behavioral relevance of the slice data and test

the effect of AAV-mediated CRY expression in vivo, Cry1-null ani-mals received bilateral stereotaxic injections of pCry1-Cry1::EGFPAAV particles into the region of the SCN (Fig. S2I). Before treat-ment the free-running period of Cry1-null animals over 10 d incontinuous darkness (DD) (following entrainment to a 12-h light:12-hdark cycle) was 23.3 ± 0.1 h (as measured by wheel-running activity,n = 8), consistent with the short period observed in Cry1-null SCN(Fig. 1 F and G). Following intracerebral injection of pCry1-Cry1::EGFP, the free-running period of Cry1-null animals measured 2–4 wkpost-administration lengthened to 24.7 ± 0.1 h (P < 0.001) (Fig. 1 Fand G), whereas injection of the AAV-EGFP control vector hadno effect on period (Fig. S2J). Virally expressed CRY1::EGFPin the SCN can therefore modulate the period of behavioralrhythms in Cry1-null animals consistent with results seen in SCNslices ex vivo, validating the behavioral relevance of effects observedin SCN slice culture.The effect of virally expressed CRY1::EGFP on the TTFL of

individual SCN neurons was monitored by time-lapse CCD imaging(Fig. 1H and Movie S1). Nontransduced Cry1/2-null SCN neuronswere arrhythmic, in contrast to SCN transduced with either Cry1 orCry2 (Fig. 1I and Fig. S3A). These induced cellular oscillations hadstable periods: the mean SD of the period of individual oscillatorswas comparable to that of WT SCN (WT = 0.13 ± 0.03 h, CRY1-induced = 0.27 ± 0.12 h, P = 0.43 vs. WT; CRY2-induced = 0.17 ±0.07 h, P = 0.62 vs. WT) (Fig. S3 B and C). Moreover, single os-cillators were highly synchronized (Fig. S3D) as quantified by thelength of the mean Rayleigh vector (r) of phase dispersal (Fig. S3E).Thus, virally expressed CRY proteins initiated cellular circadianoscillations in Cry1/2-null SCN, and these oscillations were also

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Fig. 1. Induction of molecular pacemaking in ar-rhythmic Cry1/2-null SCN. (A) EGFP signal fromCry1/2-null SCN transduced with AAV-pCry1-Cry1::EGFP. ITR: inverted terminal repeats; WPRE: WHPposttranscriptional response element. (Scale bar:10 μm.) (B) PER2::LUC bioluminescence from aCry1/2-null SCN before (gray) and after (green)transduction (arrow). Asterisk indicates mediumchange. (C ) The period of Cry1- and Cry2- inducedrhythms compared with WT (n = 6, 4, 6). All errorbars represent mean ± SEM; ****P < 0.0001 vs. WT.(D) Bioluminescence from a Cry1-null SCN before(black) and after (green) transduction with pCry1-Cry1::EGFP. (E ) Period of Cry1-null (n = 5) SCN be-fore and after transduction with Cry AAVs; ***P <0.001 vs. pretreatment, paired t-test. (F ) Actogramof Cry1-null mouse before and after stereotaxicinjection of Cry1 AAVs into the SCN. (G) The free-running period of Cry1-null animals in continuousdarkness before and after Cry1 AAV injection.***P < 0.001 vs. pretreatment, paired t test (n = 8).(H) Time-lapse images of PER2::LUC biolumines-cence from a Cry1/2-null SCN before (Top) and after(Bottom) transduction with pCry1-Cry1::EGFP. CT:circadian time. (Scale bar: 100 μm.) (I) Raster plotsof bioluminescence from single cells in H. (J) Nor-malized PER2::LUC bioluminescence (magenta) andEGFP fluorescence (green/red) over 3 d from Cry1/2-null SCN + pCry1-Cry1::EGFP (Left) or pCry1-Cry2::EGFP (Right). (K ) Peak phase of EGFP fluorescence(CT 12 defined as PER2::LUC peak) of CRY1::EGFP and CRY2::EGFP (n = 4, 3).

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tightly synchronized. In the absence of any evident physical rear-rangement of the SCN, this suggests that the neural architecture tosupport cellular synchrony preexists in the slice before transductionand thus does not require CRY proteins for its development.To examine circadian expression of CRY1::EFGP and CRY2::

EGFP, SCN were subjected to combined fluorescence and bio-luminescence time-lapse imaging. The intensity of both CRY1::EGFP and CRY2::EGFP signals was rhythmic, peaking shortly af-ter PER2::LUC, conventionally defined as circadian time (CT) 12(Fig. 1J). Following normalization to circadian time by correctingfor period in solar time, no difference was observed in the phase ofpeak expression between the two CRY proteins (CRY1::EGFP =CT 17.0 ± 0.2 h vs. CRY2::EGFP = CT 17.3 ± 0.4 h, P = 0.52) (Fig.1K). This is consistent with their expression from the same pro-moter and confirms that isoform-specific periods are determined byintrinsic properties of the proteins, not by their phase of expression.

SCN Circadian Clock Requires Expression of Cry1 to Be Circadian andCorrectly Phased. The isoform-appropriate period difference ofrhythms induced by Cry1 and Cry2, despite expression from thesame Cry1 promoter, led us to question whether the temporalpattern of promoter activity has any bearing on the effects of CRYwithin the TTFL. We therefore tested the effects of constitutivelyexpressed Cry1 on Cry1/2-null SCN function by replacing the Cry1promoter with the neuronally specific human synapsin 1 promoter(pSyn1) (Fig. S1B). This is known to be noncircadian in the SCN(10), and constitutive activity was confirmed using time-lapse fluo-rescence imaging of WT SCN transduced with an AAV encodingan mCherry Cre recombinase fusion protein under the control ofpSyn1 (Fig. S4A). Cry1/2-null SCN exhibited transduction withpSyn1-Cry1::EGFP comparable to pCry1-Cry1::EGFP (86.6 ± 10.6%,n = 3). Constitutive expression of Cry1 rapidly suppressed PER2::LUC expression, accompanied by some weak rhythmicity in pre-viously arrhythmic Cry1/2-null SCN (Fig. 2A). The period (24.3 ±1.3 h) was not significantly different from that of rhythms induced bypCry1-Cry1::EGFP (P = 0.06) (Fig. 2B), but this reflects the largevariance between individual SCN transduced with pSyn1-Cry1::EGFP. Importantly, the cycle-to-cycle stability of the molecularrhythms within individual SCN was low, as indicated by significantlyhigher RAEs (pSyn1= 0.087± 0.027 AU, P < 0.05 vs. pCry1) (Figs. 1

and 2C). To characterize further the quality of the induced rhythms,the period of individual cycles was measured over several days (Fig.2D). When either Cry1::EGFP or Cry2::EGFP were driven by theCry1 promoter, molecular rhythms had a stable peak-to-peak in-terval representative of the expected long (CRY1) or short (CRY2)period. Molecular rhythms induced by pSyn1-Cry1::EGFP, however,varied greatly from <15 to >30 h over successive cycles in individualSCN. Thus, constitutive expression of CRY1::EGFP could inducequasi-circadian cycles in Cry1/2-null SCN, but these were grosslyimprecise compared with circadian, pCry1-driven expression. Theefficacy of CRY1 in driving the SCN TTFL, therefore, is dependenton its rhythmic availability.To explore further the temporal properties necessary for CRY1

to direct SCN circadian pacemaking, we tested the effects of in-appropriately phased expression of Cry1. In vivo the positive TTFLcomponent Bmal1 is expressed ∼8 h after Per and Cry (11). Thisphase difference was confirmed using the luciferase reporterspCry1-Luc and pBmal1-Luc, transiently transfected into NIH 3T3fibroblasts (12) (Fig. S4 B and C). The Cry1 promoter was replacedwith the Bmal1 promoter (pBmal1) upstream of Cry1::EGFP. Thisdirected nuclear CRY1::EGFP expression in Cry1/2-null SCN aseffectively as pCry1 (transduction = 79.1 ± 5.6%, n = 3) (Fig. S1C).Before transduction, the Cry1/2-null SCN were arrhythmic, butpBmal1-driven expression of Cry1 was followed by cycles ofPER2::LUC bioluminescence (Fig. 2E) with periods of 24.7 ±1.5 h (Fig. 2B). Although this was not significantly different fromthat of rhythms induced by pCry1-Cry1::EGFP (P = 0.11 vs.pBmal1), the peak-to-peak periods varied widely, within and be-tween slices, by as much as 12 h (Fig. 2D). Moreover, cycle-to-cyclesignal reproducibility of the molecular rhythms was less consistent(RAE, pBmal1 = 0.294 ± 0.150 AU, P < 0.05 vs. pCry1) (Fig. 2C).Heterochronic expression of CRY1::EGFP was therefore as in-effective as constitutively expressed CRY1::EGFP in its capacity toinduce circadian rhythms in Cry1/2-null SCN.To test the effect of phase-delayed expression of CRY1::EGFP

on an intact TTFL, rhythmic Cry1-null SCN were transduced withpBmal1-Cry1::EGFP (Fig. S4D). In contrast to the period-length-ening effects of correctly phased CRY1::EGFP, the period did notchange significantly (Fig. S4E). The amplitude of the oscillationwas, however, markedly decreased, and RAE significantly increased

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Fig. 2. Constitutive or phase-delayed expression ofCry1 compromises the SCN molecular clock. (A) Bio-luminescence from a Cry1/2-null SCN before (gray)and after (dark green) transduction (arrow) withpSyn1-Cry1::EGFP. Asterisk indicates medium change.(B) Period of molecular rhythms induced by pSyn1-Cry1::EGFP and pBmal1-Cry1::EGFP compared withpCry1-Cry1::EGFP (n = 5, 4, 6; data are from Fig. 1).(C) RAE of induced molecular rhythms. (D) The peak-to-peak period of molecular rhythms in Cry1/2-nullSCN induced by different Cry AAVs. Individual SCNrepresented by different lines. (E) As in A, withpBmal1-Cry1::EGFP (blue). (F) Mean SD of period ofsingle cells in Cry1/2-null SCN transduced with eitherpSyn1-Cry1::EGFP or pBmal1-Cry1::EGFP (n = 4, 3 sli-ces; n = 660, 1,548 cells) compared with pCry1-Cry1::EGFP. (G) Normalized PER2::LUC bioluminescence(magenta) and EGFP fluorescence (blue) traces over3 d from SCN transduced with pBmal1-Cry1::EGFP.(H) Mean normalized EGFP fluorescence plottedagainst normalized PER2::LUC bioluminescence throughtime for Cry1/2-null SCN transduced with pCry1-Cry1::EGFP or pBmal1-Cry1::EGFP (n = 4, 3). *P < 0.05, **P <0.01 vs. pCry1. All errors are SEM.

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(Fig. S4F). Before transduction the molecular rhythms had verystable peak-to-peak periods, but afterward they varied greatlywithin and between individual SCN by more than 15 h (Fig. S4G).Thus, incorrectly phased expression of Cry1 perturbed the ongoingrhythms in SCN with an otherwise competent clock.The molecular mechanism underpinning the TTFL is not fully

induced when CRY1 is expressed either constitutively or in an in-correct phase. To explore this further, single-cell rhythms were ex-amined by time-lapse imaging of Cry1/2-null SCN transduced witheither pSyn1-Cry1::EGFP or pBmal1-Cry1::EGFP. Compared withrhythms induced by pCry1-Cry1::EGFP, individual oscillators in Cry1/2-null SCN transduced with pSyn1-Cry1::EGFP or pBmal1-Cry1::EGFP displayed a much broader range of periods (Fig. S4 H and I)with a significantly higher mean SD of period across multiple slices(pSyn1 = 1.7 ± 0.5 h, P < 0.05 vs. pCry1, pBmal1 = 5.4 ± 0.8 h,P < 0.01 vs. pCry1) (Fig. 2F). In contrast to pCry1-Cry1::EGFP,constitutive or phase-delayed expression of Cry1 did not induce ac-curate (∼27 h) TTFL oscillations within individual SCN neurons. Toexamine the cause of this inadequacy, we examined the relativephases of expression of CRY1::EGFP and PER2::LUC using com-bined time-lapse imaging of Cry1/2-null SCN transduced withpBmal1-Cry1::EGFP. Although CRY1::EGFP fluorescence wasrhythmic, it was less well defined than in SCN transduced with pCry1-Cry1::EGFP, and a clear phase relationship between PER2::LUCbioluminescence and CRY1::EGFP fluorescence could not becalculated due to the high variability of both (Fig. 2G). To comparethe temporal relationship between PER2::LUC bioluminescenceand CRY1::EGFP fluorescence formally, the two measures fromindividual SCN were plotted against each other sequentiallythrough time. In the case of oscillations driven by pCry1-Cry1::EGFPand pCry1-Cry2::EGFP, a clear circular trajectory was observed,reminiscent of a limit-cycle plot (Fig. 2H and Fig. S4J). The re-lationship between PER2::LUC and CRY1::EGFP in Cry1/2-nullSCN transduced with pBmal1-Cry1::EGFP, however, failed to de-fine such a trajectory, emphasizing failure to sustain a stable os-cillation (Fig. 2H). Together, these data show that the molecularrhythms induced in Cry1/2-null SCN by constitutive or phase-delayed expression of CRY1::EGFP are less accurate and robustand that the internal structure of the TTFL is compromised,compared with when CRY1 and CRY2 are rhythmically driven inthe correct phase by the Cry1 promoter.

Circuit-Level Organization of CRY-Dependent Molecular Rhythms in theSCN. Phase-dependent, rhythmic expression of Cry1 in Cry1/2-nullSCN supports a functioning TTFL within SCN neurons. We thentested whether Cry1 affected higher-order circuit-level behavior—specifically, the spatiotemporal wave of circadian gene expressionthat progresses dorsomedially to ventrolaterally across the SCN. Thedynamics of bioluminescence can be represented by center-of-lumi-nescence analysis (CoL), which tracks the geometric center of mass ofthe distributed bioluminescence signal across the SCN in X-Y co-ordinates. In PER2::LUC slices, this presents a specific trajectory thatis sustained by, and is reflective of, interneuronal communicationacross the SCN (Fig. 3A). In contrast, bioluminescence in Cry1/2-nullSCN did not exhibit a coherent spatiotemporal wave. The perimeterof CoL (an index of the wave) was significantly greater in WT than inCry1/2-null slices (110 ± 13 μm vs. 21 ± 7 μm, P < 0.01) (Fig. 3B).Cry1/2-null SCN transduced with pCry1-Cry1::EGFP showed spatio-temporal dynamics comparable to WT SCN, rather than non-transduced Cry1/2-null SCN (Fig. 3A). The CoL perimeter of theseinduced waves was significantly higher than Cry1/2-null SCN (98 ±10 μm, P < 0.01) and no different from WT slices (P = 0.56), dem-onstrating that the AAV-delivered Cry induced and sustained ap-propriate circuit-level organization of the SCN. CoL analysis wasthen used to assess network dynamics in Cry1/2-null SCN transducedwith other Cry constructs. The CoL of SCN transduced with pCry1-Cry2::EGFP was 63 ± 5 μm: greater than nontransduced Cry1/2-nullSCN (P < 0.01) but significantly less than WT controls (P < 0.05)(Fig. 3 A and B). Similarly, oscillations induced by pSyn1-Cry1::EGFPhad CoL (57 ± 9 μm) intermediate between those of Cry1/2-null(P < 0.05) and WT (P < 0.05) SCN (Fig. 3 A and B). The network

dynamics of bioluminescence in Cry1/2-null SCN transduced withthe phase-delayed pBmal1-Cry1::EGFP construct were also disor-ganized, with a CoL of 23 ± 3 μm, not significantly different fromthat of nontransduced Cry1/2-null SCN (P = 0.76) and significantlylower than WT SCN (P < 0.01) (Fig. 3 A and B). Thus, to induceappropriate circuit-level SCN timekeeping, Cry expression has to becircadian and correctly phased. Finally, to explore the evolution ofcircuit-level organization of the induced rhythms, Cry1/2-null SCNwere imaged for at least 3 d before and 10 cycles after transductionwith pCry1-Cry1::EGFP. As expected, Cry1/2-null SCN werearrhythmic before transduction and lacked apparent spatiotem-poral dynamics (Fig. 3C). After transduction with pCry1-Cry1::EGFP,however, stereotypical spatiotemporal dynamics emerged (Fig.3C) with the perimeter of CoL over each circadian day increasingsteadily and reaching a plateau after 1 wk (P < 0.001, one-wayANOVA) (Fig. 3D). These results show that the circuit-level be-havior, stereotypical of WT SCN, progressively emerges in disor-ganized arrhythmic Cry1/2-null SCN following CRY1-mediatedrestoration of cellular TTFLs.

Arginine Vasopressin Signaling Is Required for CRY-DependentInduction of Circadian Timing in SCN. Induction of the spatiotem-poral wave in Cry1/2-null SCN transduced with pCry1-Cry1::EGFPis indicative of interneuronal signaling. Arginine vasopressin(AVP) is a neuropeptide expressed in the SCN shell region thoughtto play a role in both SCN synchrony and output (13, 14). To testthe potential role of AVP as a mediator of CRY-dependent circuit-level timekeeping, Cry1/2-null SCN were treated with a mixture ofAVP-receptor antagonists (AVPx, OPC-21268 and SSR 149415:V1a, V1b, respectively) (13). AVP-receptor blockade lengthenedthe period of WT SCN, and this was reversed on washout (Fig. S5A–C). AVPx did not, however, affect the amplitude ratio or theRAE (Fig. S5 D and E). This lack of disruption of the ongoingcircadian rhythm made it possible to test the effect of AVPx onCRY-mediated induction of the molecular clock in Cry1/2-nullSCN. We recorded bioluminescence from Cry1/2-null SCN beforeapplication of AVPx or vehicle (Veh) in combination with pCry1-Cry1::EGFP transduction. As anticipated, molecular rhythmsappeared in Veh-treated SCN after ∼2 d (Fig. 4A). In slicestreated with AVPx, however, rhythms expressed following trans-duction were of significantly lower amplitude (amplitude ratio vs.

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Fig. 3. Circuit-level organization of molecular rhythms requires appropri-ately phased, circadian Cry1 expression. (A) Poincaré plots depicting pro-gression of CoL in SCN over three circadian cycles from single SCN, with eachcycle represented by a different color. (B) Comparison of the mean perimeterof CoL in different SCN; n = 5, 3, 4, 3, 4, 3 from left to right. *P < 0.05, **P <0.01 vs. WT, †P < 0.05, ††P < 0.01 vs. Cry1/2-null. (C) Poincaré plot depictingCoL over representative cycles in a Cry1/2-null SCN before (gray) and after(green) transduction with pCry1-Cry1::EGFP. (D) Perimeter of CoL over eachcircadian day before and after transduction (day 0) (n = 4). **P < 0.01, ***P <0.001 vs. pretransduction mean at cycle−1 (Bonferroni correction).

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pretreatment baseline, 0.25 ± 0.03 Veh vs. 0.08 ± 0.04 AVPx, P <0.05) (Fig. 4 B and C). When Veh-treated controls were treatedwith AVPx, following induction of rhythms, the ongoing rhythmspersisted (Fig. 4A), as in WT SCN treated with AVPx (Fig. S4). Inslices treated with AVPx at the time of transduction, washoutresulted in the emergence of molecular rhythms comparable to thoseof Veh-treated SCN. This demonstrates that competent AVP sig-naling is required for the induction, but not maintenance, of CRY-dependent circadian rhythms in Cry1/2-null SCN (Fig. 4B). Theseresults suggest that inhibition of AVP signaling prevents the circuit-level couplings between SCN neurons that are required to boostrhythm amplitude and initiate the spatiotemporal wave. Time-lapseimaging was performed on Cry1/2-null SCN transduced with pCry1-Cry1::EGFP in conjunction with AVPx. Pretreatment, Cry1/2-nullSCN had no clear wave and a correspondingly low perimeter of CoL(34± 10 μm) (Fig. 4D and E). Following coapplication of AVPx andpCry1-Cry1::EGFP, the perimeter of CoL was unchanged (34 ± 7μm, P = 0.95). Following washout of the AVPx, however, a stereo-typical wave emerged (102 ± 4 μm, P < 0.01 vs. AVPx) (Fig. 4D andE). Similarly, single cell rhythms failed to be induced during AVPxtreatment but did emerge on washout (Fig. 4F). Together, these datashow that CRY requires competent AVP signaling to initiate net-work-level organization and induce a fully functioning clock in Cry1/2-null SCN.

DiscussionUsing a gain-of-function genetic complementation approach, weshow that both CRY1 and CRY2 can induce molecular circadianrhythms in arrhythmic Cry-deficient SCN. Thus, despite developingin the absence of CRY proteins, Cry1/2-null SCN were neverthelessable to express cell-autonomous and circuit-level circadian proper-ties following appropriate virally mediated expression of Cry1 andCry2. The rapidity of this effect (∼2 d) argues against any significant

need for CRY to direct structural changes in postmitotic SCNneurons and circuits. Adult animals and SCN require CRY proteinsfor the expression of circadian rhythms of behavior and geneexpression. Very early in postnatal development, however, well-defined, short period oscillations of Per gene expression are observedin ∼50% of Cry1/2-null SCN (15, 16), and so in the current studyCry1/2-null SCN were prescreened to confirm arrhythmicity beforetransduction. Given that cellular circadian rhythms may involveTTFLs acting in combination with intrinsically oscillatory cytosolicpathways (17), the rhythms in Cry1/2-null SCN may arise from suchcytosolic mechanisms, sustained by neuropeptidergic and/or meta-bolic signaling. Early in development, therefore, the Cry1/2-nullSCN is formed as a competent circadian oscillator, but CRY pro-teins later become necessary for expression of this latent capacity.When expressed under the same minimal Cry1 promoter in Cry1/

2-null SCN, both CRY1 and CRY2 sustained robust molecularrhythms with isoform-specific periods, and in single Cry mutantSCN they lengthened and shortened the circadian period in anisoform-specific manner. The behavioral relevance of this effectwas confirmed in Cry1-deficient mice, in which AAV-Cry1-CRY1::EGFP expression in the SCN lengthened the period of circadianactivity rhythms. The effectiveness of both CRY1 and CRY2 in theSCN contrasts with cell-based assays, in which CRY2 is insufficientto induce the molecular clock (6) and in dispersed SCN cultures,where loss of Cry1, but not loss of Cry2, compromises the clock-work; i.e., CRY2 alone cannot sustain rhythms (18). The powerfulcoupling of SCN neurons, which is not present in fibroblast cellculture and SCN dispersals, may therefore permit self-sustainingCRY2-mediated oscillations. Importantly, the sustained rhythmswere induced with long (CRY1: 26.9 h) and short (CRY2: 22.0 h)periods, respectively, comparable to the periods of single Cryknockout SCN (26.2 and 22.3 h) (4). This protein-specific effectwas also observed in single Cry knockout SCN, although in thesecases the periods “over shot” the wild-type condition, likely due toan AAV copy number effect. This demonstrates that the presenceof CRY is not merely permissive: rather than being a passivecomponent of the molecular clock, CRY proteins actually confertemporal information via endogenous properties, such as stabilityand transcriptional repression, possibly arising from differentialposttranslational modifications (19, 20) independently of theirphase of expression.In cell-based assays, a delay in CRY-dependent feedback re-

pression mediated by an evening-phased RRE sequence in the firstintron of Cry1 (5) is necessary for accurate timekeeping. LimitedAAV packaging capacity precluded inclusion of the RRE here, butits absence did not compromise effective control of SCN rhythms.This suggests that RRE-dependent delay is not necessary in the SCN,consistent with the close phasing of Cry1 and Per2mRNA expressionin the SCN, whereas in peripheral tissues and cells Cry1 is delayeddue to RRE activity (21). The view that RRE does not influence theCry1 phase in the SCN is supported by phase mapping of the minimalCry1 promoter in SCN of Cry1-luc transgenic mice (10).Constitutive or phase-delayed expression of Cry1::EGFP using

the Syn1 or Bmal1 promoters, respectively, did induce molecularrhythms, but they were unstable and less coherent than those in-duced by pCry1-Cry1::EGFP, demonstrating that phase-appropriateCry1 expression is necessary to generate accurate rhythms withinthe SCN. When expressed under the pCry1 promoter, the associ-ation of CRY1::EGFP (and CRY2::EGFP) and PER2::LUC os-cillations tracked a limit cycle, consistent with establishment of aself-sustained oscillator (22). In contrast, phase-delayed expressionof Cry1 using pBmal1 collapsed this trajectory. Thus, appropriatelyphased rhythmic feedback repression by CRY1 is required for fullcircadian clock function (5, 6). This may appear to run counter tothe detection of rhythms in Cry1/2-null fibroblasts treated with cell-permeant CRY proteins (7) and WT MEFs overexpressing CRY1(8), but the contrast between intrinsically sloppy cell-based rhythmsand the robust SCN oscillations makes comparison difficult. Sloppyrhythms were indeed also observed in SCN with constitutive CRYexpression, but they did not approach the quality of rhythms sup-ported by circadian expression of CRY.

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Fig. 4. AVP signaling is required for Cry-dependent induction of SCN circadiangene expression. (A) Bioluminescence from a Veh-treated (DMSO) Cry1/2-nullSCN transduced with pCry1-Cry1::EGFP before application of AVPx. (B) Bio-luminescence from a Cry1/2-null SCN transduced with pCry1-Cry1::EGFP incombination with AVPx application before AVPx washout and treatmentwith vehicle (Veh). (C) Amplitude of induced rhythms under AVPx or Veh,normalized to pretreatment baseline (n = 3, 4). (D) Poincaré plot depictingCoL in Cry1/2-null SCN before (gray) and after (black) transduction withpCry1-Cry1::EGFP and application of AVPx. Subsequent CoL following AVPxwashout (w/o) is shown (green). (E) Comparison of the mean perimeter ofCoL between conditions in D (n = 3). *P < 0.05, **P < 0.01, paired t tests. (F)Raster plot (Left) and single-cell trace (Right) from SCN in D.

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Limit-cycle behavior defines temporal relationships, but SCN geneexpression also follows a spatiotemporal wave that is dependent oninterneuronal signaling and is thought to reflect differences in cell-intrinsic periods (10, 23). Such dynamics were completely absent inCry1/2-null SCN, but transduction with pCry1-Cry1::EGFP initiatedand sustained waves of bioluminescence similar to those of WTSCN. Thus, the ability to express a spatiotemporal program of geneexpression is prespecified in the SCN by CRY-independent mech-anisms. The acute presence of CRY1 can induce its expression, asindividual cells become competent to oscillate and progressively,over about 1 wk, the wave is established with appropriate phaserelationships and an increasing trajectory, as an emergent property.This is consistent with reports based on quantitative spectral clus-tering of Cry1/2-null (24) where underlying spatial groupings can beidentified, even though ensemble circadian behavior is disrupted.Circuit-level organization was not induced in Cry1/2-null SCN

transduced with constitutive or phase-delayed Cry1, most likelybecause timing within the individual neurons was insufficientlystable for ensemble behavior to emerge. It remains to be seenhow manipulating the dynamics of gene expression across theSCN, using different promoters to drive Cry expression, relatesto circadian behavior in vivo, although proof-of-principle wasprovided by lengthening of behavioral rhythms in Cry1-null miceinjected with AAV-pCry1-CRY1::EGFP. Moreover, recent evi-dence has shown that the spatiotemporal wave is modified bylight exposure, with phase differences between SCN neuronsincreasing under longer day lengths (25). It is therefore likelythat the phase relationship between SCN neurons, which resultsin the specific spatiotemporal wave of gene expression charac-teristic of the SCN, encodes timekeeping information in vivo.Network dynamics of gene expression are dependent on intercel-

lular communication. For example, Vipr2-null SCN lacking VIP-mediated signaling also lack spatial organization (24). Further-more, rhythms of bioluminescence in Cry1/2-null SCN are inducedby paracrine signaling from a WT SCN graft (15). Of these para-crine-signaling molecules, AVP is thought to play a role in SCNsynchronization (26), with genetic loss of V1a and V1b receptorsloosening coupling within the SCN, facilitating re-entrainmentof molecular and behavioral rhythms (13). Additionally, when the

molecular clock is abolished in AVP-expressing cells, coupling be-tween SCN neurons is weakened, and the period of behavioralrhythms is lengthened (14). Here we show that the restoration ofmolecular rhythms using transgenic Cry1 is compromised by phar-macological blockade of the AVP receptor and that AVP signalingis therefore required for the circuit-level organization of the SCNclock. Transcription of the AVP gene is dependent on canonicalE-box sequences in the AVP promoter, subject to CLOCK/BMAL1activation and PER/CRY-mediated inhibition (27). This provides amolecular link to explain how recombinant CRY can rhythmicallyregulate AVP expression and hence direct network organization inCry1/2-null SCN. Further in vivo experiments in which SCN-specificCRY restoration with or without an AVP-signaling blockade wouldhelp determine the precise contribution of AVP signaling from theSCN neurons to overall behavioral rhythms. That is, can expressionof Cry in a small population of neurons induce behavioral circadianrhythms in an arrhythmic animal, and if so, is this AVP-mediated?In a similar vein, it would be interesting to investigate the role ofspecific SCN neurons in rhythm generation. The development ofCre-loxP technology, for example, would allow targeted expressionof Cry in SCN subpopulations both ex vivo and in vivo and wouldhelp address whether SCN neurons contribute equally and redun-dantly to circadian timekeeping or whether “pacemaker” cells existthat dictate time to the rest of the network.

Materials and MethodsAnimalworkwas licensedunder theUKAnimals (Scientific Procedures)Act 1986withLocal Ethical Review. SCN organotypic slices from 7- to 10-d-old pups were preparedas previously described (28). Bioluminescence emission was recorded using photonmultipliers (Hamamatsu), CCD cameras (Hamamatsu), or LV200 bioluminescenceimaging systems (Olympus). Graphs were plotted, data analyses were performed,and statistical tests were calculated using Prism 5 (GraphPad). Unless otherwisestated, unpaired t tests were used for statistical comparisons. All recombinant AAVswere manufactured by Penn Vector Core (University of Pennsylvania).

ACKNOWLEDGMENTS. We are grateful for excellent technical assistance pro-vided by biomedical staff at the Medical Research Council (MRC) Ares Facility. Thisworkwas supported by theMRC of the United Kingdom, and the ElectromagneticField Biological Research Trust.

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