Rhythmic Expression of Cryptochrome Induces the Circadian Clock of Arrhythmic Suprachiasmatic Nuclei Through Arginine Vasopressin Signaling

Rhythmic expression of cryptochrome induces the circadian clock of arrhythmic suprachiasmatic nuclei through arginine vasopressin signaling

Mathew 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 suprachiasmatic behavioral rhythms under constant conditions whereas Cry2-null nucleus (SCN). SCN neurons define circadian time using transcriptional/ mice have longer periods (2). These differences are also seen at posttranslational feedback loops (TTFL) in which expression of the level of the SCN in vitro and are magnified in the presence of Afh Cryptochrome (Cry)andPeriod (Per) genes is inhibited by their protein the Fbxl3 mutation that stabilizes CRY proteins (4). Whether products. Loss of Cry1 and Cry2 stops the SCN clock, whereas individual these differences reflect intrinsic properties of the protein, differ- deletions accelerate and decelerate it, respectively. At the circuit level, ences in their phase of expression, or other features is unknown. neuronal interactions synchronize cellular TTFLs, creating a spatiotem- Indeed, it is unclear whether rhythmic expression of the Cry genes Cry1/2 poral wave of gene expression across the SCN that is lost in - is necessary for a fully functioning TTFL. Molecular rhythms can deficient SCN. To interrogate the properties of CRY proteins required be induced in Cry1/2-null mouse embryonic fibroblasts (MEFs) when for circadian function, we expressed CRY in SCN of Cry-deficient mice Cry is expressed rhythmically under a promoter containing E/E′-box, using adeno-associated virus (AAV). Expression of CRY1::EGFP or CRY2:: EGFP under a minimal Cry1 promoter was circadian and rapidly induced D-box, and RRE sequences (5, 6). On the other hand, constitutively PER2-dependent bioluminescence rhythms in previously arrhythmic available, cell-permeant CRY proteins can also induce rhythms in Cry1/2 Cry1/2-deficient SCN, with periods appropriate to each isoform. -null fibroblasts (7), and overexpression of CRY1 in WT MEFs CRY1::EGFP appropriately lengthened the behavioral period in does not abolish circadian rhythmicity (8), whereas constitutive Cry1-deficient mice. Thus, determination of specific circadian overexpression of PER2 does, suggesting that circadian oscillations periods reflects properties of the respective proteins, independently require rhythmic expression of Per2, but not Cry. The present study of their phase of expression. Phase of CRY1::EGFP expression was aimed to define critical properties of CRY proteins that sustain critical, however, because constitutive or phase-delayed promoters TTFL function in the SCN, the central circadian pacemaker. In failed to sustain coherent rhythms. At the circuit level, CRY1::EGFP contrast to mere loss-of-function studies, our approach focused on induced the spatiotemporal wave of PER2 expression in Cry1/2- the specific circadian properties that are conferred on the SCN circuit deficient SCN. This was dependent on the neuropeptide arginine va- by CRY proteins. We therefore developed a genetic restoration sopressin (AVP) because it was prevented by pharmacological block- approach using adeno-associated virus (AAV) gene delivery to SCN ade of AVP receptors. Thus, our genetic complementation assay of CRY-deficient mice in organotypic culture and in vivo. Restorative reveals acute, protein-specific induction of cell-autonomous and net- assays have been performed in fibroblast culture, but such cells lack work-level circadian rhythmicity in SCN never previously exposed to the intercellular coupling present in the SCN and do not control CRY. Specifically, Cry expression must be circadian and appropriately behavior. Our approach would allow us to investigate the circadian phased to support rhythms, and AVP receptor signaling is required to impose circuit-level circadian function. Significance period | bioluminescence | arginine vasopressin | clock | oscillation Daily cycles of behavior adapt us to the 24-h rhythm of day and he suprachiasmatic nucleus (SCN) is a precise biological clock night. These rhythms are coordinated by a central clock, the Tthat coordinates circadian (∼24 h) rhythms in mammals. suprachiasmatic nucleus (SCN), in which self-sustaining transcrip- Composed of ∼10,000 neurons, the SCN sits atop the optic chiasm, tional/posttranslational feedback loops define approximately 24-h through which it receives retinal innervation that entrains its cellular (circadian) time. Cryptochrome1 (CRY1) and CRY2 are essential clock clockwork to the light/dark cycle. In turn, the SCN entrains periph- components, but how they sustain cell-autonomous and circuit- eral circadian clocks distributed across the organism through neural level circadian timing in the SCN is not clear. To explore this, we pathways incorporating neuropeptidergic and GABA-ergic signaling developed a genetic complementation assay, using virally expressed, fluorescently tagged CRY proteins to test whether molecular (1). At the molecular level, circadian timing in SCN neurons revolves Cry1/2 around transcriptional/posttranslational feedback loops (TTFL), in pacemaking can be induced in arrhythmic -deficient SCN. which the positive components BMAL1 and CLOCK activate E/E′- We demonstrate protein-specific and robust induction of molecular – Period Per1 pacemaking, the efficacy of which depends on the circadian pattern box mediated transcription of the negative components ( , Cry Per2)andCryptochrome (Cry1, Cry2). PER/CRY heterodimers then and phase of expression and functional signaling via neuro- inhibit BMAL1/CLOCK activity and thus prevent their own tran- peptide (AVP) receptors. scription. Subsequent proteasomal degradation of PER and Author contributions: M.D.E., M.B., E.S.M., and M.H.H. designed research; M.D.E., J.E.C., CRY proteins relieves this inhibition, allowing the molecular cycle to E.S.M., and M.H.H. performed research; M.B. contributed new reagents/analytic tools; begin again. Genetic studies have established that CRY1 and CRY2 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. wrote are essential for behavioral and molecular circadian rhythmicity, the paper. acting as core components of the TTFL (2, 3). The properties that The authors declare no conflict of interest. allow them to fulfill this role are, however, unclear. For example, a This article is a PNAS Direct Submission. surprising feature of the clock is the opposing circadian phenotypes 1To whom correspondence should be addressed. Email: [email protected]. Cry in single knockouts. Although sequence similarity between the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. CRY proteins is extremely high, Cry1-null mice have short period 1073/pnas.1519044113/-/DCSupplemental.

2732–2737 | PNAS | March 8, 2016 | vol. 113 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1519044113 Downloaded by guest on September 26, 2021 roles of CRY in both cell-autonomous and circuit-level pace- short (22.3 ± 0.4 h), and pCry1-Cry1::EGFP expression stably making in behaviorally relevant neurons. lengthened period (26.4 ± 0.1 h, P < 0.001) (Fig. 1E) from 3 d after transduction (Fig. S2C). In addition, these rhythms had a Results lower RAE, as CRY1::EGFP conferred stability to the endogenous Induction of Circadian Clock Function in Arrhythmic Cry1/2-Null SCN clockwork (Fig. S2D). In the converse test, long period Cry2-null pCry1-Cry2::EGFP E by AAV-Delivered Cry. Cry1/2-null SCN were transduced with AAVs SCN were transduced with (Fig. S2 ), and this encoding the CRY1::EGFP fusion protein controlled by a minimal decreased period (P < 0.01) (Fig. S2 F and G), although RAE did Cry1 promoter (pCry1) that exhibits circadian activation in the SCN not decrease following AAV transduction with Cry2 (Fig. S2H). Thus, (Fig. 1A)(9).Typically,∼5 × 109 viral particles (1 μLof∼5 × 1012 CRY2 did not enhance the definition of the molecular cycle. Trans- GC/mL) were dropped directly onto each slice 7 d after dissection genic CRY1 and CRY2 can, therefore, interact with and modulate the or after bioluminescence recordings, as previously reported (10). period of the endogenous clockwork in single Cry-null SCN. Confocal microscopy confirmed nuclear expression of CRY1 To demonstrate the behavioral relevance of the slice data and test (transduction efficiency of SCN cells, 88.0 ± 7.8%, n = 4) (Fig. 1A). the effect of AAV-mediated CRY expression in vivo, Cry1-null ani- Before AAV transduction, PER2::LUC bioluminescence was mals received bilateral stereotaxic injections of pCry1-Cry1::EGFP arrhythmic; however, ∼2 d after transduction with pCry1-Cry1:: AAV particles into the region of the SCN (Fig. S2I). Before treat- EGFP, rhythms emerged (Fig. 1B) with a long period (26.9 ± 0.2 h, ment the free-running period of Cry1-null animals over 10 d in determined after medium change) appropriate to the Cry2-null, continuous darkness (DD) (following entrainment to a 12-h light:12-h Cry1-proficient genotype of the transduced SCN (Fig. 1C). Thus, dark cycle) was 23.3 ± 0.1 h (as measured by wheel-running activity, arrhythmic SCN previously unexposed to CRY proteins can rapidly n = 8), consistent with the short period observed in Cry1-null SCN express circadian molecular rhythms after virally mediated CRY1 (Fig. 1 F and G). Following intracerebral injection of pCry1-Cry1:: expression. Cry1 cDNA was then replaced by Cry2 cDNA and AAV- EGFP, the free-running period of Cry1-null animals measured 2–4wk mediated nuclear expression of CRY2::EGFP in Cry1/2-null SCN post-administrationlengthenedto24.7± 0.1 h (P < 0.001) (Fig. 1 F confirmed by microscopy (83.2 ± 6.1%, n = 4) (Fig. S1A). CRY2:: and G), whereas injection of the AAV-EGFP control vector had EGFP induced strong bioluminescence rhythms in initially disorga- no effect on period (Fig. S2J). Virally expressed CRY1::EGFP nized Cry1/2-null SCN (Fig. S2A). Importantly, these rhythms had a in the SCN can therefore modulate the period of behavioral short period (22.0 ± 0.4 h) consistent with the Cry1-null, Cry2- rhythms in Cry1-null animals consistent with results seen in SCN proficient genotype. Both CRY1- and CRY2-induced rhythms slices ex vivo, validating the behavioral relevance of effects observed had periods significantly different from each other and from WT in SCN slice culture. SCN (24.5 ± 0.1 h, P < 0.0001 for each) (Fig. 1C), although The effect of virally expressed CRY1::EGFP on the TTFL of robustness of the oscillations, reported by the cycle-to-cycle individual SCN neurons was monitored by time-lapse CCD imaging variation of the relative amplitude error (RAE), was comparable (Fig. 1H and Movie S1). Nontransduced Cry1/2-null SCN neurons to WT SCN (Fig. S2B). Thus, not only can pCry1-driven CRY1 were arrhythmic, in contrast to SCN transduced with either Cry1 or and CRY2 induce coherent and stable molecular rhythms in Cry2 (Fig. 1I and Fig. S3A). These induced cellular oscillations had Cry1/2-null SCN, but also can do so with isoform-specific periods, stable periods: the mean SD of the period of individual oscillators even when driven by the same promoter. Therefore, CRY1 and was comparable to that of WT SCN (WT = 0.13 ± 0.03 h, CRY1- CRY2 are not simply permissive elements: the individual proteins induced = 0.27 ± 0.12 h, P = 0.43 vs. WT; CRY2-induced = 0.17 ± confer specific temporal properties to the clockwork. 0.07 h, P = 0.62 vs. WT) (Fig. S3 B and C). Moreover, single os- To test whether virally expressed CRY can intermesh with cillators were highly synchronized (Fig. S3D) as quantified by the ongoing molecular oscillations driven by endogenous CRY, length of the mean Rayleigh vector (r) of phase dispersal (Fig. S3E). rhythmic Cry1-null SCN were transduced with pCry1-Cry1::EGFP Thus, virally expressed CRY proteins initiated cellular circadian (Fig. 1D). The period of Cry1-null SCN before transduction was oscillations in Cry1/2-null SCN, and these oscillations were also

Fig. 1. Induction of molecular pacemaking in ar- A B 1500 C 28 rhythmic Cry1/2-null SCN. (A) EGFP signal from ITR pCry1 Cry::EGFP WPRE ITR * **** Cry1/2-null SCN transduced with AAV-pCry1-Cry1:: 26 EGFP.ITR:invertedterminalrepeats;WPRE:WHP 1000 24 posttranscriptional response element. (Scale bar: 500 μ **** 10 m.) (B) PER2::LUC bioluminescence from a (h) Period 22 Cry1/2-null SCN before (gray) and after (green) Biolum. (cps) 0 20 transduction (arrow). Asterisk indicates medium DAPI CRY1::EGFP Merge 0481216 20 24 AAV: none Cry1 Cry2 genotype: WT Cry1/2-null change. (C) The period of Cry1- and Cry2- induced Time (days) rhythms compared with WT (n = 6, 4, 6). All error D E SCN period FG0 Free-running 2000 28 + pCry1-Cry1 26 period bars represent mean ± SEM; ****P < 0.0001 vs. WT. *** (D) Bioluminescence from a Cry1-null SCN before 1500 26 10 25 **** (black) and after (green) transduction with pCry1- 24 1000 20 24 Cry1::EGFP.(E)PeriodofCry1-null (n = 5) SCN be- 22 Days Period (h) Period < 500 (h) Period 23 fore and after transduction with Cry AAVs; ***P Biolum. (cps) 20 30 0.001 vs. pretreatment, paired t-test. (F) Actogram 0 22 0 2 4 6 8 10 12 14 Pre- Post- 0 12 24 Pre- Post- of Cry1-null mouse before and after stereotaxic Time (days) Hours injection of Cry1 AAVs into the SCN. (G) The free- 0 h + 12 h Phase HICry1/2-null 1 J +Cry1 +Cry2 K running period of Cry1-null animals in continuous of GFP NEUROSCIENCE 1 darkness before and after Cry1 AAV injection. 0 Cry1/2-null + Cry1 CRY1:: ***P < 0.001 vs. pretreatment, paired t test (n = 8). Pre- EGFP (H) Time-lapse images of PER2::LUC biolumines- 0 n.s.

CT 0 CT 12 Cells cence from a Cry1/2-null SCN before (Top) and after Cry1/2-null + Cry2 CRY2:: EGFP (Bottom) transduction with pCry1-Cry1::EGFP.CT: -1 circadian time. (Scale bar: 100 μm.) (I) Raster plots Normalized signal Post- 062 4 02448720244872 02412 of bioluminescence from single cells in H.(J)Nor- Time (days) Time (h) CT malized PER2::LUC bioluminescence (magenta) and EGFP fluorescence (green/red) over 3 d from Cry1/2-null SCN + pCry1-Cry1::EGFP (Left)orpCry1-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).

Edwards et al. PNAS | March 8, 2016 | vol. 113 | no. 10 | 2733 Downloaded by guest on September 26, 2021 tightly synchronized. In the absence of any evident physical rear- and 2C). To characterize further the quality of the induced rhythms, rangement of the SCN, this suggests that the neural architecture to the period of individual cycles was measured over several days (Fig. support cellular synchrony preexists in the slice before transduction 2D). When either Cry1::EGFP or Cry2::EGFP were driven by the and thus does not require CRY proteins for its development. Cry1 promoter, molecular rhythms had a stable peak-to-peak in- To examine circadian expression of CRY1::EFGP and CRY2:: terval representative of the expected long (CRY1) or short (CRY2) EGFP, SCN were subjected to combined fluorescence and bio- period. Molecular rhythms induced by pSyn1-Cry1::EGFP, however, luminescence time-lapse imaging. The intensity of both CRY1:: varied greatly from <15 to >30 h over successive cycles in individual EGFP and CRY2::EGFP signals was rhythmic, peaking shortly af- SCN. Thus, constitutive expression of CRY1::EGFP could induce ter PER2::LUC, conventionally defined as circadian time (CT) 12 quasi-circadian cycles in Cry1/2-null SCN, but these were grossly (Fig. 1J). Following normalization to circadian time by correcting imprecise compared with circadian, pCry1-driven expression. The for period in solar time, no difference was observed in the phase of efficacy of CRY1 in driving the SCN TTFL, therefore, is dependent peak expression between the two CRY proteins (CRY1::EGFP = on its rhythmic availability. CT 17.0 ± 0.2 h vs. CRY2::EGFP = CT 17.3 ± 0.4 h, P = 0.52) (Fig. To explore further the temporal properties necessary for CRY1 1K). This is consistent with their expression from the same pro- to direct SCN circadian pacemaking, we tested the effects of in- moter and confirms that isoform-specific periods are determined by appropriately phased expression of Cry1. In vivo the positive TTFL intrinsic properties of the proteins, not by their phase of expression. component Bmal1 is expressed ∼8hafterPer and Cry (11). This phase difference was confirmed using the luciferase reporters SCN Circadian Clock Requires Expression of Cry1 to Be Circadian and pCry1-Luc and pBmal1-Luc, transiently transfected into NIH 3T3 Correctly Phased. The isoform-appropriate period difference of fibroblasts (12) (Fig. S4 B and C). The Cry1 promoter was replaced rhythms induced by Cry1 and Cry2, despite expression from the with the Bmal1 promoter (pBmal1) upstream of Cry1::EGFP. This same Cry1 promoter, led us to question whether the temporal directed nuclear CRY1::EGFP expression in Cry1/2-null SCN as pattern of promoter activity has any bearing on the effects of CRY effectively as pCry1 (transduction = 79.1 ± 5.6%, n = 3) (Fig. S1C). within the TTFL. We therefore tested the effects of constitutively Before transduction, the Cry1/2-null SCN were arrhythmic, but expressed Cry1 on Cry1/2-null SCN function by replacing the Cry1 pBmal1-driven expression of Cry1 was followed by cycles of promoter with the neuronally specific human synapsin 1 promoter PER2::LUC bioluminescence (Fig. 2E) with periods of 24.7 ± (pSyn1)(Fig. S1B).ThisisknowntobenoncircadianintheSCN 1.5 h (Fig. 2B). Although this was not significantly different from (10), and constitutive activity was confirmed using time-lapse fluo- that of rhythms induced by pCry1-Cry1::EGFP (P = 0.11 vs. rescence imaging of WT SCN transduced with an AAV encoding pBmal1), the peak-to-peak periods varied widely, within and be- an mCherry Cre recombinase fusion protein under the control of tween slices, by as much as 12 h (Fig. 2D). Moreover, cycle-to-cycle pSyn1 (Fig. S4A). Cry1/2-null SCN exhibited transduction with signal reproducibility of the molecular rhythms was less consistent pSyn1-Cry1::EGFP comparable to pCry1-Cry1::EGFP (86.6 ± 10.6%, (RAE, pBmal1 = 0.294 ± 0.150 AU, P < 0.05 vs. pCry1)(Fig.2C). n = 3). Constitutive expression of Cry1 rapidly suppressed PER2:: Heterochronic expression of CRY1::EGFP was therefore as in- LUC expression, accompanied by some weak rhythmicity in pre- effective as constitutively expressed CRY1::EGFP in its capacity to viously arrhythmic Cry1/2-null SCN (Fig. 2A). The period (24.3 ± induce circadian rhythms in Cry1/2-null SCN. 1.3 h) was not significantly different from that of rhythms induced by To test the effect of phase-delayed expression of CRY1::EGFP pCry1-Cry1::EGFP (P = 0.06) (Fig. 2B),butthisreflectsthelarge on an intact TTFL, rhythmic Cry1-null SCN were transduced with variance between individual SCN transduced with pSyn1-Cry1:: pBmal1-Cry1::EGFP (Fig. S4D). In contrast to the period-length- EGFP. Importantly, the cycle-to-cycle stability of the molecular ening effects of correctly phased CRY1::EGFP, the period did not rhythms within individual SCN was low, as indicated by significantly change significantly (Fig. S4E). The amplitude of the oscillation higher RAEs (pSyn1 = 0.087 ± 0.027 AU, P < 0.05 vs. pCry1)(Figs.1 was, however, markedly decreased, and RAE significantly increased

A B 30 C 0.5 2000 n.s. n.s. * * 28 0.4 1500 26 0.3 1000 24 0.2 * Period (h) Period 500 22 RAE (AU) 0.1 Fig. 2. Constitutive or phase-delayed expression of Biolum. (cps) 0 20 0.0 Cry1 compromises the SCN molecular clock. (A) Bio- 0481216 luminescence from a Cry1/2-null SCN before (gray) Time (days) pCry1 pCry1 and after (dark green) transduction (arrow) with + pSyn1 + pSyn1 D + +pBmal1E + +pBmal1 pSyn1-Cry1::EGFP. Asterisk indicates medium change. 35 + pCry1 + pSyn1- + pBmal1- 2500 (B) Period of molecular rhythms induced by pSyn1- Cry1 Cry1 Cry1 * 30 2000 Cry1::EGFP and pBmal1-Cry1::EGFP compared with = 1500 pCry1-Cry1::EGFP (n 5, 4, 6; data are from Fig. 1). 25 (C) RAE of induced molecular rhythms. (D) The peak- 20 1000 to-peak period of molecular rhythms in Cry1/2-null + pCry1 500 15 Cry2 Biolum. (cps) SCN induced by different Cry AAVs. Individual SCN

Period variation (h) 0 represented by different lines. (E)AsinA, with 024602468100246810 0481216 Cycle Time (days) pBmal1-Cry1::EGFP (blue). (F) Mean SD of period of single cells in Cry1/2-null SCN transduced with either FG+pBmal1-Cry1 H + pCry1-Cry1 + pBmal1-Cry1 = 7 1.0 pSyn1-Cry1::EGFP or pBmal1-Cry1::EGFP (n 4, 3 sli- 6 ** 1 ces; n = 660, 1,548 cells) compared with pCry1-Cry1:: 5 EGFP.(G) Normalized PER2::LUC bioluminescence 4 (magenta) and EGFP fluorescence (blue) traces over 3 0 0.0 2 * 3 d from SCN transduced with pBmal1-Cry1::EGFP. (H) Mean normalized EGFP fluorescence plotted Mean SD (h) SD Mean 1 -1

0 Normalized signal against normalized PER2::LUC bioluminescence through Normalized biolum. -1.0 0244872 -1.0 0.0 1.0 -1.0 0.0 1.0 time for Cry1/2-null SCN transduced with pCry1-Cry1:: pCry1 pSyn1 Time (h) Normalized fluorescence EGFP or pBmal1-Cry1::EGFP (n = 4, 3). *P < 0.05, **P < + + +pBmal1 0.01 vs. pCry1. All errors are SEM.

2734 | www.pnas.org/cgi/doi/10.1073/pnas.1519044113 Edwards et al. Downloaded by guest on September 26, 2021 F (Fig. S4 ). Before transduction the molecular rhythms had very A WT Cry1/2-null B stable peak-to-peak periods, but afterward they varied greatly Y Y 150 n.s. within and between individual SCN by more than 15 h (Fig. S4G). D †† Thus, incorrectly phased expression of Cry1 perturbed the ongoing L M V 100 * rhythms in SCN with an otherwise competent clock. ††* † X X The molecular mechanism underpinning the TTFL is not fully Cry1/2-null Cry1/2-null 50 inducedwhenCRY1isexpressedeitherconstitutivelyorinanin- + pCry1-Cry1 + pCry1-Cry2 ** **n.s. correct phase. To explore this further, single-cell rhythms were ex- Y Y Cry1/2 0 amined by time-lapse imaging of -null SCN transduced with (μm) CoL mean of Perimeter pCry1- pCry1- pSyn1- pBmal1- either pSyn1-Cry1::EGFP or pBmal1-Cry1::EGFP. Compared with AAV: none none Cry1 Cry2 Cry1 Cry1 Cry1/2-null rhythms induced by pCry1-Cry1::EGFP, individual oscillators in Cry1/ genotype: WT 2 pSyn1-Cry1::EGFP pBmal1-Cry1:: X X CD -null SCN transduced with or Cry1/2-null Cry1/2-null 150 *** EGFP displayed a much broader range of periods (Fig. S4 H and I) + pSyn1-Cry1 + pBmal1-Cry1 Y Y Y ** with a significantly higher mean SD of period across multiple slices 100 (pSyn1 = 1.7 ± 0.5 h, P < 0.05 vs. pCry1, pBmal1 = 5.4 ± 0.8 h, 50 CoL (μm)

P < 0.01 vs. pCry1)(Fig.2F). In contrast to pCry1-Cry1::EGFP, Perimeter of 0 Cry1 X constitutive or phase-delayed expression of did not induce ac- X X -4048 curate (∼27 h) TTFL oscillations within individual SCN neurons. To Cycle pre-/post- AAV transduction examine the cause of this inadequacy, we examined the relative Fig. 3. Circuit-level organization of molecular rhythms requires appropri- phases of expression of CRY1::EGFP and PER2::LUC using com- ately phased, circadian Cry1 expression. (A) Poincaré plots depicting pro- bined time-lapse imaging of Cry1/2-null SCN transduced with pBmal1-Cry1::EGFP gression of CoL in SCN over three circadian cycles from single SCN, with each . Although CRY1::EGFP fluorescence was cycle represented by a different color. (B) Comparison of the mean perimeter rhythmic, it was less well defined than in SCN transduced with pCry1- of CoL in different SCN; n = 5, 3, 4, 3, 4, 3 from left to right. *P < 0.05, **P < † †† Cry1::EGFP, and a clear phase relationship between PER2::LUC 0.01 vs. WT, P < 0.05, P < 0.01 vs. Cry1/2-null. (C) Poincaré plot depicting bioluminescence and CRY1::EGFP fluorescence could not be CoL over representative cycles in a Cry1/2-null SCN before (gray) and after calculated due to the high variability of both (Fig. 2G). To compare (green) transduction with pCry1-Cry1::EGFP.(D) Perimeter of CoL over each the temporal relationship between PER2::LUC bioluminescence circadian day before and after transduction (day 0) (n = 4). **P < 0.01, ***P < and CRY1::EGFP fluorescence formally, the two measures from 0.001 vs. pretransduction mean at cycle−1 (Bonferroni correction). individual SCN were plotted against each other sequentially through time. In the case of oscillations driven by pCry1-Cry1::EGFP and pCry1-Cry2::EGFP, a clear circular trajectory was observed, dynamics of bioluminescence in Cry1/2-null SCN transduced with reminiscentofalimit-cycleplot(Fig.2H and Fig. S4J). The re- the phase-delayed pBmal1-Cry1::EGFP construct were also disor- lationship between PER2::LUC and CRY1::EGFP in Cry1/2-null ganized, with a CoL of 23 ± 3 μm, not significantly different from SCN transduced with pBmal1-Cry1::EGFP,however,failedtode- that of nontransduced Cry1/2-null SCN (P = 0.76) and significantly fine such a trajectory, emphasizing failure to sustain a stable os- lower than WT SCN (P < 0.01) (Fig. 3 A and B). Thus, to induce cillation (Fig. 2H). Together, these data show that the molecular appropriate circuit-level SCN timekeeping, Cry expression has to be rhythms induced in Cry1/2-null SCN by constitutive or phase- circadian and correctly phased. Finally, to explore the evolution of delayed expression of CRY1::EGFP are less accurate and robust circuit-level organization of the induced rhythms, Cry1/2-null SCN and that the internal structure of the TTFL is compromised, were imaged for at least 3 d before and 10 cycles after transduction compared with when CRY1 and CRY2 are rhythmically driven in with pCry1-Cry1::EGFP.Asexpected,Cry1/2-null SCN were the correct phase by the Cry1 promoter. arrhythmic before transduction and lacked apparent spatiotemporal dynamics (Fig. 3C). After transduction with pCry1-Cry1::EGFP, Circuit-Level Organization of CRY-Dependent Molecular Rhythms in the however, stereotypical spatiotemporal dynamics emerged (Fig. SCN. Phase-dependent, rhythmic expression of Cry1 in Cry1/2-null 3C) with the perimeter of CoL over each circadian day increasing SCN supports a functioning TTFL within SCN neurons. We then steadily and reaching a plateau after 1 wk (P < 0.001, one-way tested whether Cry1 affected higher-order circuit-level behavior— ANOVA) (Fig. 3D). These results show that the circuit-level be- specifically, the spatiotemporal wave of circadian gene expression havior, stereotypical of WT SCN, progressively emerges in disor- that progresses dorsomedially to ventrolaterally across the SCN. The ganized arrhythmic Cry1/2-null SCN following CRY1-mediated dynamics of bioluminescence can be represented by center-of-lumi- restoration of cellular TTFLs. nescence analysis (CoL), which tracks the geometric center of mass of the distributed bioluminescence signal across the SCN in X-Y co- Arginine Vasopressin Signaling Is Required for CRY-Dependent ordinates. In PER2::LUC slices, this presents a specific trajectory that Induction of Circadian Timing in SCN. Induction of the spatiotem- is sustained by, and is reflective of, interneuronal communication poral wave in Cry1/2-null SCN transduced with pCry1-Cry1::EGFP across the SCN (Fig. 3A). In contrast, bioluminescence in Cry1/2-null is indicative of interneuronal signaling. Arginine vasopressin SCN did not exhibit a coherent spatiotemporal wave. The perimeter (AVP) is a neuropeptide expressed in the SCN shell region thought of CoL (an index of the wave) was significantly greater in WT than in to play a role in both SCN synchrony and output (13, 14). To test Cry1/2-null slices (110 ± 13 μmvs.21± 7 μm, P < 0.01) (Fig. 3B). the potential role of AVP as a mediator of CRY-dependent circuit- Cry1/2-null SCN transduced with pCry1-Cry1::EGFP showed spatio- level timekeeping, Cry1/2-null SCN were treated with a mixture of temporal dynamics comparable to WT SCN, rather than non- AVP-receptor antagonists (AVPx, OPC-21268 and SSR 149415: transduced Cry1/2-null SCN (Fig. 3A). The CoL perimeter of these V1a, V1b, respectively) (13). AVP-receptor blockade lengthened induced waves was significantly higher than Cry1/2-null SCN (98 ± the period of WT SCN, and this was reversed on washout (Fig. S5 10 μm, P < 0.01) and no different from WT slices (P = 0.56), dem- A–C). AVPx did not, however, affect the amplitude ratio or the NEUROSCIENCE onstrating that the AAV-delivered Cry induced and sustained ap- RAE (Fig. S5 D and E). This lack of disruption of the ongoing propriate circuit-level organization of the SCN. CoL analysis was circadian rhythm made it possible to test the effect of AVPx on then used to assess network dynamics in Cry1/2-null SCN transduced CRY-mediated induction of the molecular clock in Cry1/2-null with other Cry constructs. The CoL of SCN transduced with pCry1- SCN. We recorded bioluminescence from Cry1/2-null SCN before Cry2::EGFP was 63 ± 5 μm: greater than nontransduced Cry1/2-null application of AVPx or vehicle (Veh) in combination with pCry1- SCN (P < 0.01) but significantly less than WT controls (P < 0.05) Cry1::EGFP transduction. As anticipated, molecular rhythms (Fig. 3 A and B). Similarly, oscillations induced by pSyn1-Cry1::EGFP appeared in Veh-treated SCN after ∼2 d (Fig. 4A). In slices had CoL (57 ± 9 μm) intermediate between those of Cry1/2-null treated with AVPx, however, rhythms expressed following trans- (P < 0.05) and WT (P < 0.05) SCN (Fig. 3 A and B). The network duction were of significantly lower amplitude (amplitude ratio vs.

Edwards et al. PNAS | March 8, 2016 | vol. 113 | no. 10 | 2735 Downloaded by guest on September 26, 2021 A AVPx B Veh need for CRY to direct structural changes in postmitotic SCN 2000 Veh 2000 AVPx neurons and circuits. Adult animals and SCN require CRY proteins for the expression of circadian rhythms of behavior and gene 1500 1500 expression. Very early in postnatal development, however, well- 1000 1000 defined, short period oscillations of Per gene expression are observed 500 500 in ∼50% of Cry1/2-nullSCN(15,16),andsointhecurrentstudy Biolum. (cps) Biolum. (cps) Cry1/2-null SCN were prescreened to confirm arrhythmicity before 0 0 048121620 0 4 8 121620 transduction. Given that cellular circadian rhythms may involve Time (days) Time (days) TTFLs acting in combination with intrinsically oscillatory cytosolic pathways (17), the rhythms in Cry1/2-null SCN may arise from such C 0.4 DE120 * Y Pre n.s. ** cytosolic mechanisms, sustained by neuropeptidergic and/or meta- 0.3 AVPx Cry1/2 m) 80 bolic signaling. Early in development, therefore, the -null

w/o μ 0.2 SCN is formed as a competent circadian oscillator, but CRY pro- * D 40 teins later become necessary for expression of this latent capacity. 0.1 L M CoL ( Cry1 Cry1/ Perimeter of Perimeter When expressed under the same minimal promoter in Amplitude ratio ratio Amplitude V 0.0 0 2-null SCN, both CRY1 and CRY2 sustained robust molecular Veh AVPx X Pre- w/o rhythms with isoform-specific periods, and in single Cry mutant + pCry1-Cry1 + pCry1-Cry1 AVPx SCN they lengthened and shortened the circadian period in an F w/o w/o + AVPx + AVPx isoform-specific manner. The behavioral relevance of this effect 1 Cry1 AAV-Cry1 1 was confirmed in -deficient mice, in which -CRY1:: EGFP expression in the SCN lengthened the period of circadian

Cells 0

biolum. activity rhythms. The effectiveness of both CRY1 and CRY2 in the Relative 0 0 10 20 0 10 20 SCN contrasts with cell-based assays, in which CRY2 is insufficient Time (days) Time (days) to induce the molecular clock (6) and in dispersed SCN cultures, Cry1 Cry2 Fig. 4. AVP signaling is required for Cry-dependent induction of SCN circadian where loss of , but not loss of , compromises the clock- gene expression. (A) Bioluminescence from a Veh-treated (DMSO) Cry1/2-null work; i.e., CRY2 alone cannot sustain rhythms (18). The powerful SCN transduced with pCry1-Cry1::EGFP before application of AVPx. (B) Bio- coupling of SCN neurons, which is not present in fibroblast cell luminescence from a Cry1/2-null SCN transduced with pCry1-Cry1::EGFP in culture and SCN dispersals, may therefore permit self-sustaining combination with AVPx application before AVPx washout and treatment CRY2-mediated oscillations. Importantly, the sustained rhythms with vehicle (Veh). (C) Amplitude of induced rhythms under AVPx or Veh, were induced with long (CRY1: 26.9 h) and short (CRY2: 22.0 h) normalized to pretreatment baseline (n = 3, 4). (D) Poincaré plot depicting periods, respectively, comparable to the periods of single Cry CoL in Cry1/2-null SCN before (gray) and after (black) transduction with knockout SCN (26.2 and 22.3 h) (4). This protein-specific effect pCry1-Cry1::EGFP and application of AVPx. Subsequent CoL following AVPx was also observed in single Cry knockout SCN, although in these washout (w/o) is shown (green). (E) Comparison of the mean perimeter of cases the periods “over shot” the wild-type condition, likely due to CoL between conditions in D (n = 3). *P < 0.05, **P < 0.01, paired t tests. (F) an AAV copy number effect. This demonstrates that the presence Raster plot (Left) and single-cell trace (Right) from SCN in D. of CRY is not merely permissive: rather than being a passive component of the molecular clock, CRY proteins actually confer temporal information via endogenous properties, such as stability ± ± P < pretreatment baseline, 0.25 0.03 Veh vs. 0.08 0.04 AVPx, and transcriptional repression, possibly arising from differential B C 0.05) (Fig. 4 and ). When Veh-treated controls were treated posttranslational modifications (19, 20) independently of their with AVPx, following induction of rhythms, the ongoing rhythms phase of expression. A persisted (Fig. 4 ), as in WT SCN treated with AVPx (Fig. S4). In In cell-based assays, a delay in CRY-dependent feedback re- slices treated with AVPx at the time of transduction, washout pression mediated by an evening-phased RRE sequence in the first resulted in the emergence of molecular rhythms comparable to those intron of Cry1 (5) is necessary for accurate timekeeping. Limited of Veh-treated SCN. This demonstrates that competent AVP sig- AAV packaging capacity precluded inclusion of the RRE here, but naling is required for the induction, but not maintenance, of CRY- its absence did not compromise effective control of SCN rhythms. dependent circadian rhythms in Cry1/2-null SCN (Fig. 4B). These This suggests that RRE-dependent delay is not necessary in the SCN, results suggest that inhibition of AVP signaling prevents the circuit- consistent with the close phasing of Cry1 and Per2 mRNA expression level couplings between SCN neurons that are required to boost in the SCN, whereas in peripheral tissues and cells Cry1 is delayed rhythm amplitude and initiate the spatiotemporal wave. Time-lapse due to RRE activity (21). The view that RRE does not influence the imaging was performed on Cry1/2-null SCN transduced with pCry1- Cry1 phase in the SCN is supported by phase mapping of the minimal Cry1::EGFP in conjunction with AVPx. Pretreatment, Cry1/2-null Cry1 promoter in SCN of Cry1-luc transgenic mice (10). SCN had no clear wave and a correspondingly low perimeter of CoL Constitutive or phase-delayed expression of Cry1::EGFP using (34 ± 10 μm) (Fig. 4 D and E). Following coapplication of AVPx and the Syn1 or Bmal1 promoters, respectively, did induce molecular pCry1-Cry1::EGFP, the perimeter of CoL was unchanged (34 ± 7 rhythms, but they were unstable and less coherent than those in- μm, P = 0.95). Following washout of the AVPx, however, a stereo- duced by pCry1-Cry1::EGFP, demonstrating that phase-appropriate typical wave emerged (102 ± 4 μm, P < 0.01 vs. AVPx) (Fig. 4 D and Cry1 expression is necessary to generate accurate rhythms within E). Similarly, single cell rhythmsfailedtobeinducedduringAVPx the SCN. When expressed under the pCry1 promoter, the associ- treatment but did emerge on washout (Fig. 4F). Together, these data ation of CRY1::EGFP (and CRY2::EGFP) and PER2::LUC os- show that CRY requires competent AVP signaling to initiate net- cillations tracked a limit cycle, consistent with establishment of a work-level organization and induce a fully functioning clock in Cry1/ self-sustained oscillator (22). In contrast, phase-delayed expression 2-null SCN. of Cry1 using pBmal1 collapsed this trajectory. Thus, appropriately phased rhythmic feedback repression by CRY1 is required for full Discussion circadian clock function (5, 6). This may appear to run counter to Using a gain-of-function genetic complementation approach, we the detection of rhythms in Cry1/2-null fibroblasts treated with cell- show that both CRY1 and CRY2 can induce molecular circadian permeant CRY proteins (7) and WT MEFs overexpressing CRY1 rhythms in arrhythmic Cry-deficient SCN. Thus, despite developing (8), but the contrast between intrinsically sloppy cell-based rhythms in the absence of CRY proteins, Cry1/2-null SCN were nevertheless and the robust SCN oscillations makes comparison difficult. Sloppy able to express cell-autonomous and circuit-level circadian proper- rhythms were indeed also observed in SCN with constitutive CRY ties following appropriate virally mediated expression of Cry1 and expression, but they did not approach the quality of rhythms sup- Cry2. The rapidity of this effect (∼2d)arguesagainstanysignificant ported by circadian expression of CRY.

2736 | www.pnas.org/cgi/doi/10.1073/pnas.1519044113 Edwards et al. Downloaded by guest on September 26, 2021 Limit-cycle behavior defines temporal relationships, but SCN gene molecular clock is abolished in AVP-expressing cells, coupling be- expression also follows a spatiotemporal wave that is dependent on tween SCN neurons is weakened, and the period of behavioral interneuronal signaling and is thought to reflect differences in cell- rhythms is lengthened (14). Here we show that the restoration of intrinsic periods (10, 23). Such dynamics were completely absent in molecular rhythms using transgenic Cry1 is compromised by phar- Cry1/2-null SCN, but transduction with pCry1-Cry1::EGFP initiated macological blockade of the AVP receptor and that AVP signaling and sustained waves of bioluminescence similar to those of WT is therefore required for the circuit-level organization of the SCN SCN. Thus, the ability to express a spatiotemporal program of gene clock. Transcription of the AVP gene is dependent on canonical expression is prespecified in the SCN by CRY-independent mech- E-box sequences in the AVP promoter, subject to CLOCK/BMAL1 anisms. The acute presence of CRY1 can induce its expression, as activation and PER/CRY-mediated inhibition (27). This provides a individual cells become competent to oscillate and progressively, molecular link to explain how recombinant CRY can rhythmically over about 1 wk, the wave is established with appropriate phase regulate AVP expression and hence direct network organization in relationships and an increasing trajectory, as an emergent property. Cry1/2-null SCN. Further in vivo experiments in which SCN-specific This is consistent with reports based on quantitative spectral clus- CRY restoration with or without an AVP-signaling blockade would Cry1/2 tering of -null (24) where underlying spatial groupings can be help determine the precise contribution of AVP signaling from the identified, even though ensemble circadian behavior is disrupted. SCN neurons to overall behavioral rhythms. That is, can expression Circuit-level organization was not induced in Cry1/2-null SCN Cry Cry1 of in a small population of neurons induce behavioral circadian transduced with constitutive or phase-delayed , most likely rhythms in an arrhythmic animal, and if so, is this AVP-mediated? because timing within the individual neurons was insufficiently In a similar vein, it would be interesting to investigate the role of stable for ensemble behavior to emerge. It remains to be seen specific SCN neurons in rhythm generation. The development of how manipulating the dynamics of gene expression across the Cry Cre-loxP technology, for example, would allow targeted expression SCN, using different promoters to drive expression, relates of Cry in SCN subpopulations both ex vivo and in vivo and would to circadian behavior in vivo, although proof-of-principle was help address whether SCN neurons contribute equally and redun- provided by lengthening of behavioral rhythms in Cry1-null mice dantly to circadian timekeeping or whether “pacemaker” cells exist injected with AAV-pCry1-CRY1::EGFP. Moreover, recent evi- that dictate time to the rest of the network. dence has shown that the spatiotemporal wave is modified by light exposure, with phase differences between SCN neurons Materials and Methods increasing under longer day lengths (25). It is therefore likely that the phase relationship between SCN neurons, which results Animal work was licensed under the UK Animals (Scientific Procedures) Act 1986 with in the specific spatiotemporal wave of gene expression charac- Local Ethical Review. SCN organotypic slices from 7- to 10-d-old pups were prepared teristic of the SCN, encodes timekeeping information in vivo. as previously described (28). Bioluminescence emission was recorded using photon Network dynamics of gene expression are dependent on intercel- multipliers (Hamamatsu), CCD cameras (Hamamatsu), or LV200 bioluminescence lular communication. For example, Vipr2-null SCN lacking VIP- imaging systems (Olympus). Graphs were plotted, data analyses were performed, mediated signaling also lack spatial organization (24). Further- and statistical tests were calculated using Prism 5 (GraphPad). Unless otherwise more, rhythms of bioluminescence in Cry1/2-null SCN are induced stated, unpaired t tests were used for statistical comparisons. All recombinant AAVs were manufactured by Penn Vector Core (University of Pennsylvania). by paracrine signaling from a WT SCN graft (15). Of these paracrine-signaling molecules, AVP is thought to play a role in SCN ACKNOWLEDGMENTS. We are grateful for excellent technical assistance pro- synchronization (26), with genetic loss of V1a and V1b receptors vided by biomedical staff at the Medical Research Council (MRC) Ares Facility. This loosening coupling within the SCN, facilitating re-entrainment work was supported by the MRC of the United Kingdom, and the Electromagnetic of molecular and behavioral rhythms (13). Additionally, when the Field Biological Research Trust.

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