Research Articles: Behavioral/Cognitive Neuronal Myocyte-specific enhancer factor 2D (MEF2D) is required for normal circadian and sleep behavior in mice https://doi.org/10.1523/JNEUROSCI.0411-19.2019
Cite as: J. Neurosci 2019; 10.1523/JNEUROSCI.0411-19.2019 Received: 20 February 2019 Revised: 8 July 2019 Accepted: 10 August 2019
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1 Title: Neuronal Myocyte-specific enhancer factor 2D (MEF2D) is required for 2 normal circadian and sleep behavior in mice 3 4 Abbreviated Title: Neuronal MEF2D and sleep behavior in mice 5 6 Jennifer A. Mohawk1,2, Kimberly H. Cox1, Makito Sato2,3, Seung-Hee Yoo1, Masashi 7 Yanagisawa2,3, Eric N. Olson4, Joseph S. Takahashi1,2* 8 9 Affiliations: 10 1 Department of Neuroscience, The University of Texas Southwestern Medical Center, Dallas, 11 TX 75390-9111, USA 12 13 2 Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, 14 TX 75390-9111, USA 15 16 3 Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, 17 TX 75390-9111, USA 18 19 4 Department of Molecular Biology, Hamon Center for Regenerative Science and Medicine, 20 University of Texas Southwestern Medical Center, Dallas, TX 75390, USA 21 22 23 * To whom correspondence should be addressed: [email protected] 24 25 Number of pages: 25 26 Number of figures: 6 27 Number of tables: 1 28 Number of extended data figures: 0 29 Number of words for abstract: 186 30 for introduction: 616 31 for discussion: 1141 32 33 Conflict of Interest Statement: The authors declare no competing financial interests. 34 35 Acknowledgements: This work was supported by the FIRST program from JSPS and 36 the WPI program from Japan's MEXT (M.Y.); NIH grants AR-067294 and HL-130253 37 and the Robert A. Welch Foundation grant 1-0025 (E.N.O.); and 38 NIH grants MH078024 and AG045795 (J.S.T.). M.Y. was, and J.S.T. is an Investigator 39 in the Howard Hughes Medical Institute. The authors would like to thank Dr. Rhonda 40 Bassel-Duby for contributing the Mef2d knockout and floxed mice, and Dr. Robert 41 Greene for suggesting the analysis in Figure 6g. 42 43 J.A. Mohawk’s current address: 2951 Gentle Creek Trail, Prosper, TX 75078. M. 44 Sato’s and M. Yanagisawa’s current address: International Institute for Integrative 45 Sleep Medicine (WPI-IIIS), University of Tsukuba, Tsukuba 305-8575, Japan. S-H. 46 Yoo’s current address: Department of Biochemistry and Molecular Biology, The 47 University of Texas Health Science Center at Houston, 6431 Fannin St., Houston, TX 48 77030, USA.
49 ABSTRACT 50 51 The transcription factor, myocyte enhancer factor-2 (MEF2), is required for
52 normal circadian behavior in Drosophila; however, its role in the mammalian circadian
53 system has not been established. Of the four mammalian Mef2 genes, Mef2d is highly
54 expressed in the suprachiasmatic nucleus (SCN) of the hypothalamus, a region critical
55 for coordinating peripheral circadian clocks. Utilizing both conventional and brain-
56 specific Mef2d knockout (Mef2d-/-) mouse lines, we demonstrate that MEF2D is
57 essential for maintaining the length of the circadian free-running period of locomotor
58 activity and normal sleep patterns in male mice. Crossing Mef2d-/- with Per2::luc
59 reporter mice, we show that these behavioral changes are achieved without altering the
60 endogenous period of the master circadian oscillator in the SCN. Together, our data
61 suggest that alterations in behavior in Mef2d-/- mice may be the result of an effect on
62 SCN output, rather than an effect on timekeeping within the SCN itself. These findings
63 add to the growing body of evidence that MEF2 proteins play important roles in the
64 brain.
65 66 67 SIGNIFICANCE STATEMENT 68
69 These studies are the first to show a role for MEF2 proteins in the brain outside
70 of the hippocampus, and our findings suggest that these proteins may play diverse roles
71 in the central nervous system. It is important to continue to build on our understanding
72 of the roles of proteins acting in the SCN, because SCN dysfunction underlies jet lag in
73 humans and influences the response to shift work schedules, which are now known as
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74 risk factors for the development of cancer. Our work on MEF2D could be the basis for
75 opening new lines of research in the development and regulation of circadian rhythms.
76 77 INTRODUCTION 78 79 The circadian clock is essential to the proper timing and regulation of behavior
80 and physiology. In mammals, a master pacemaker in the suprachiasmatic nucleus
81 (SCN) of the ventral hypothalamus coordinates circadian clocks throughout the
82 organism (Welsh et al., 2010; Albrecht, 2012; Herzog et al., 2017). The circadian time-
83 keeping mechanism is composed of a transcriptional autoregulatory feedback loop. At
84 the core of this loop, CLOCK and BMAL1 drive transcription of Period (Per1, Per2) and
85 Cryptochrome (Cry1, Cry2), whose protein products, in turn, dimerize, translocate back
86 into the nucleus, and repress the activity of CLOCK and BMAL1, thereby inhibiting their
87 own expression. In addition, accessory loops interact with the core clock loop, forming a
88 web of modulators contributing to the refinement of the oscillatory network and,
89 ultimately, circadian biological outputs (Shearman et al., 2000; Ueda et al., 2005;
90 Takahashi, 2017).
91 The transcription factor myocyte enhancer factor-2 (MEF2) has been linked to
92 the maintenance of normal circadian behavior in Drosophila (Blanchard et al., 2010;
93 Sivachenko et al., 2013). Overexpression of Mef2 in clock neurons leads to lengthening
94 of the free-running period of locomotor activity and weak behavioral rhythms in flies.
95 Repressing Mef2 activity has a similar effect, with flies displaying weakly rhythmic or
96 arrhythmic patterns of locomotor activity (Blanchard et al., 2010). However, the role of
97 MEF2 in the mammalian circadian system has not been established.
2
98 In vertebrates, there are four Mef2 genes: Mef2a, Mef2b, Mef2c, and Mef2d.
99 These genes display both differential and overlapping expression patterns throughout
100 the body, but are most abundantly expressed in muscle (both skeletal and cardiac)
101 (Black and Olson, 1998) and brain (Leifer et al., 1993; Ikeshima et al., 1995; Lyons et
102 al., 1995). Their protein products play vital roles in muscle development and stress- and
103 activity-dependent signaling (Shalizi and Bonni, 2005; Potthoff and Olson, 2007;
104 Dietrich, 2013). In neurons, MEF2 targets a diverse set of genes involved in synapse
105 development and remodeling (Flavell et al., 2006; Flavell et al., 2008), while loss of
106 MEF2 has been associated with neuronal apoptosis (Mao et al., 1999; Li et al., 2001;
107 Liu et al., 2003; Akhtar et al., 2012). Both in vitro and in vivo studies have shown that
108 MEF2 activation or overexpression can lead to the reduction of excitatory synapses in
109 hippocampal neurons (Barbosa et al., 2008; Pfeiffer et al., 2010). Interestingly, while
110 prenatal loss of Mef2c in the brain has been shown to impair hippocampal-dependent
111 learning in mice (Barbosa et al., 2008; Akhtar et al., 2012; Rashid et al., 2014),
112 postnatal loss of Mef2c had no such behavioral effect (Adachi et al., 2016), and
113 overexpression of MEF2 in discrete regions within the hippocampus disrupts memory
114 formation (Cole et al., 2012; Rashid et al., 2014). These findings suggest a complex role
115 for MEF2 proteins in the development and function of the hippocampus, but the impact
116 of loss of MEF2 on non-hippocampal dependent behaviors has not been thoroughly
117 investigated.
118 Global deletion of Mef2a or Mef2c is lethal in mice, while Mef2d null mice are
119 viable (Arnold et al., 2007; Kim et al., 2008; Omori et al., 2015) and Mef2d is highly
120 expressed in the SCN(Lein et al., 2007). Therefore, utilizing both a conventional and
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121 brain-specific Mef2d KO, we began our investigation of the role of MEF2 proteins in the
122 regulation of mammalian circadian behavior. We demonstrate that Mef2D is essential
123 for maintaining the length of the circadian free-running period of locomotor activity. Loss
124 of Mef2D leads to a lengthening of the behavioral period and to disruption and
125 fragmentation of sleep patterns without altering the endogenous period of the master
126 circadian oscillator. Importantly, these effects are specific to Mef2D, as loss of either
127 Mef2A or Mef2C in the brain had no effect on locomotor free-running period.
128 129 MATERIALS AND METHODS 130 131
132 Animals.
133 Mef2d-/- mice (Arnold et al., 2007; Kim et al., 2008) and wild-type littermates on a
134 (129SvEv/C57BL/6J) mixed genetic background were derived from heterozygote
135 crosses. Brain-specific Mef2a, Mef2c, and Mef2d heterozygote and knockout mice (and
136 floxed, cre-negative controls) were derived from crossing CamKIIa-iCre mice (Casanova
137 et al., 2001) with Mef2a/c/dfx/fx triple-floxed mice (Barbosa et al., 2008; Liu et al., 2014;
138 Adachi et al., 2016) and intercrossing to obtain the desired genotypes. For
139 bioluminescence recordings to assess circadian rhythms in the SCN and peripheral
140 tissues, Mef2d-/- mice were crossed with mice carrying the Per2::luc reporter (Yoo et al.,
141 2004). These reporter mice are used to study circadian gene expression in vivo as
142 the Period2 (mPer2) promoter drives luciferase (luc) expression. In all of our studies,
143 Zeitgeber time (ZT) 0 denotes the time of lights on in light/dark cycles (LD), whereas
144 circadian time (CT) 0, denotes start of subjective day and CT12 denotes start of
145 subjective night in constant darkness (DD).Food and water were provided ad libitum.
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146 All animal care and procedures were performed in strict accordance with the
147 recommendations of the Guide for the Care and Uses of Laboratory Animals of the
148 National Institutes of Health and approved by the UT Southwestern Medical Center
149 Institutional Animal Care and Use Committee.
150
151 Circadian Behavioral Analyses.
152 Adult male mice (8-12 weeks, n > 12/group) were housed individually in running
153 wheel cages and their activity was recorded continuously as described previously
154 (Shimomura et al., 2001) using ClockLab hardware and software (Actimetrics, Wilmette,
155 IL; RRID:SCR_014309). Phase angle, activity bouts, and free-running period were
156 calculated using ClockLab. Activity onset (psi) was calculated during LD. Bout analyses
157 were calculated after approximately 8 weeks in DD, across 14 days of data, with bouts
158 defined by a 10 counts/min threshold and a 20min maximum gap. Free-running period
159 was calculated after approximately 8 weeks in DD, across 14 days of data using a χ2
160 periodogram. Animals whose data could not be scored for the full time for any measure
161 were excluded from analysis. For behavioral ethograms, mice (n=2/group) were housed
162 in LD and visualized with a Sony DCR-HC62 camcorder, recording 1 frame/minute.
163 Data were recorded over the active (dark) portion of the LD cycle, with recordings
164 extending into the morning hours (~ZT12-ZT3.5).
165
166 Bioluminescence Recordings of Circadian Rhythms.
167 To investigate circadian rhythms of clock gene expression in the SCN and
168 peripheral tissues of these mice, bioluminescence recordings were performed as
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169 previously described (Yoo et al., 2004; Liu et al., 2007). Briefly, adult male Per2::luc
170 (WT and Mef2d-/-) mice (n=7/group) were euthanized by rapid cervical dislocation
171 between approximately ZT11-12 for LD experiments or CT11-12 for DD experiments.
172 Tissues were harvested and kept in chilled Hanks’ balanced salt solution. SCN was
173 taken from a 300mm coronal brain section sliced on a vibratome. A heart sample was
174 taken from the exsanguinated atrium. Kidney, liver, and lung were hand sliced into thin
175 samples. Anterior pituitary was cultured whole. Explanted tissues were placed on
176 Millicell culture inserts (MilliporeSigma, Burlington, MA; RRID: SCR_015799) in 35-mm
177 culture dishes in tissue culture medium containing luciferin (Promega, Madison, WI).
178 Cultures were incubated for at least 6 days under constant conditions (constant
179 darkness, constant temperature of 35 °C, and no medium change). The light emitted
180 from each individual culture dish was recorded in real-time with photomultiplier tube
181 detectors using LumiCycle hardware and software (Actimetrics, Wilmette, IL).
182 Bioluminescence data were detrended by subtracting the 24h running average from the
183 raw data. The detrended data sets were smoothed by taking 2h running averages. Free-
184 running period was calculated over 5 cycles with LumiCycle software using Levenberg-
185 Marquardt (LM) sin fit, and mean periods.
186
187 RNA Expression.
188 Tissue was collected from male mice (n=2/3 per group) housed in either LD or
189 DD. Mice were euthanized at the indicated times (2 timepoints for LD and 6 timepoints
190 for DD) by rapid cervical dislocation. RNA isolation and qRT-PCR were performed as
191 previously described (Siepka et al., 2007). Primers for qRT-PCR are shown in Table 1.
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192 Relative quantification was calculated using the ∆∆ Ct method and Gapdh as
193 housekeeping gene.
194
195 Cell Culture and In Vitro Assays. 196 197 HEK293A cells were cultured in DMEM (#D5671; Sigma-Aldrich, St. Louis, MO)
198 supplemented with 10% fetal bovine serum (#F2442; Sigma-Aldrich, St. Louis, MO).
199 Cells were plated 1 day before transfection and Effectene reagent (#301425; Qiagen,
200 Germantown, MD) was used to transfect DNA according to the manufacturer's protocol.
201 Approximately 36hr after transfection, cells were harvested for downstream
202 applications. For the luminescence assays, cells were lysed, and luminescence was
203 measured from 20 μl of lysate with the Luciferase Assay System (#E1500; Promega,
204 Madison, WI) using a luminometer (AutoLumat Plus; Berthold Technologies, Oak Ridge,
205 TN). Luminescence is reported relative to Renilla (#E2810; Renilla Luciferase Assay,
206 Promega, Madison, WI). For the immunoprecipitation assay, cells were lysed, and
207 immunoprecipitation and Western blotting was performed as previously described
208 (Siepka et al., 2007), using anti-FLAG M2 agarose (#A2220; Sigma-Aldrich, St. Louis,
209 MO). Western blot was performed using anti-HA (Roche, Branchburg, NJ;
210 RRID:AB_2687407), anti-CLOCK (generously given bDr. Seung-Hee YooȌǡ anti-Flag
211 (Sigma-Aldrich, St. Louis, MO; RRID:AB_796202), and anti-α-tubulin (Santa Cruz,
212 Dallas, TX). All assays were performed in duplicate and, except for the BMAL::Luc
213 assay, were repeated in 2-3 independent transfection experiments.
214
215
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216 Sleep Recording and Analyses.
217 Sleep recording was performed as previously described (Funato et al., 2010).
218 Briefly, four electrode pins (for EEG) and two flexible wires (for EMG recording) were
219 implanted under ketamine/xylazine anesthesia. EEG electrodes were positioned over
220 the frontal and occipital cortices [anteroposterior (AP): 0.5mm, mediolateral (ML):
221 1.3mm, dorsoventral (DV): -1.3mm; and AP: -4.5mm, ML: 1.3mm, DV: -1.3mm,
222 respectively]. For recordings, male mice (n=8/group) were housed individually in LD and
223 tethered to a counterbalance arm (#SMCLA; Instech Laboratories, Plymouth Meeting,
224 PA) allowing for free movement. All mice were allowed 14d of recovery prior to
225 recording. EEG/EMG signal was recorded continuously for two, consecutive 24h
226 periods. EEG/EMG signals were amplified using a Grass Model 78 (AstroNova, West
227 Warwick, RI), filtered, digitized at a sampling rate of 250Hz, and displayed using custom
228 polygraph software. The vigilance state in each 20 sec epoch was classified as NREM
229 sleep, REM sleep, or wakefulness by a custom, semi-automated sleep staging software
230 based in MATLAB (Mathworks, Natick, MA). For power density analyses, EEG power
231 density in each frequency bin was expressed as a percentage of the mean total EEG
232 power over all frequency bins and vigilance states.
233 234 Experimental Design and Statistical Analyses. 235 236 All statistical tests were performed using Graphpad Prism version 8.0 for for Mac
237 OS X (GraphPad Software, San Diego, CA; RRID:SCR_002798). For behavioral
238 experiments, sleep studies, and SCN explant experiments, means were calculated for
239 each group for each measure and compared with students t-tests (conventional KO) or
240 one-way ANOVA (brain-specific KO) to compare across genotypes. Realtime PCR data
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241 were analyzed via two-way ANOVA with genotype and time as between and within
242 variables, respectively. In vitro assays were analyzed with one-way ANOVAs with
243 Bonferroni correction for multiple comparisons or unpaired t-tests with Welch’s
244 correction for differences in variance.
245 246 247 RESULTS 248 249 Mef2d knockout mice have altered circadian behavior. 250 251 Loss of functional MEF2D had a profound impact on behavioral circadian
252 rhythms (Fig. 1). Mef2d-/- (KO) mice housed with running-wheels in 12:12 light/dark
253 cycles with entraining T cycle 24h (T24, LD 12:12; hereafter referred to as LD) exhibited
254 a delayed phase of activity onset (t(29) = 3.762, *p=0.0008; t-test), beginning their
255 wheel-running approximately 25 min later than wild-type (WT) mice (Fig. 1a-c). KO
256 mice also had a substantially lengthened (40 min longer than WT) free-running period
257 (t(38) = 8.340, *p < 0.0001; t-test) in constant darkness (DD) (Fig. 1a-b, d). Notably,
258 running-wheel activity was fragmented in KO mice. KO mice displayed a greater
259 number of activity bouts per day (t(29) = 4.257, *p =0.0002; t-test), with the individual
260 activity bouts being of shorter duration (t(29) = 4.844, *p < 0.0001; t-test) as compared
261 to WT mice (Fig. 1a-b, e). These findings suggested that MEF2D could play a larger
262 role in the general control of behavior. Therefore, we performed an in-depth analysis of
263 the behavior of KO mice throughout the active portion of the 24h day. We compiled
264 ethograms from images of individual mice, which were recorded 1x/min throughout the
265 night. These analyses revealed that KO mice spent less consolidated time on the
266 running-wheel than WT mice, and wheel-running activity of KO mice was frequently
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267 interrupted by intermittent rest events (Fig. 1f-h). No other gross behavioral alterations
268 were observed.
269 Neuronal Mef2D is Required for Normal Circadian Behavior 270 271 To further investigate whether loss of MEF2D in the brain was responsible for the
272 behavioral phenotype in the global Mef2d KO, we crossed Mef2a/c/d floxed mice with a
273 CamKIIa-iCre line to generate mice with postnatal brain-specific loss of MEF2 function.
274 Intercrossing for subsequent generations resulted in mice with loss of varying
275 combinations of Mef2 genes in the brain. Only postnatal loss of MEF2D in the brain led
276 to a change in circadian free-running period during DD (F(3, 59) = 25.19, *p < 0.0001;
277 one-way ANOVA), as CamKIIa-iCre x Mef2afx/fx or Mef2cfx/fx mice were indistinguishable
278 from WT controls. In contrast, CamKIIa-iCre x Mef2dfx/fx mice had a long circadian
279 period of running-wheel activity similar to what was observed for the conventional KO
280 (Fig. 2a-c). However, these mice did not differ from controls in their phase-response
281 curves to light or dark pulses (data not shown).
282
283 Mef2d knockout in the SCN causes light-dependent effects on free-running 284 period. 285 286 To test the effects of Mef2d on circadian rhythms in the SCN, we crossed Mef2d-
287 /- mice with mice carrying the Per2::luc reporter. When SCNs were harvested from
288 animals housed in LD, the KO SCNs displayed a delayed phase of PER2
289 bioluminescence from the acute culture (Fig. 3a), recapitulating our behavioral findings.
290 Curiously, PER2 rhythms in the SCN of these animals were approximately 50 min
291 shorter in the presence of a T24 entraining cycle (Fig. 3a-b; t(12) = 3.443, *p=0.0049; t-
292 test). PER2::LUC rhythms in other tissues from KO mice were largely unaffected, with
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293 the exception of a lengthened period in lungs (Fig. 3c; t(17)=3.279, *p=0.0044; t-test).
294 The shorter free-running period in KO SCNs harvested from LD led us to hypothesize
295 that the SCNs of these mice may be exhibiting an after-effect of the LD cycle on the
296 master oscillator. To test this hypothesis, we harvested tissues from animals housed in
297 DD. Interestingly, despite the behavioral differences seen in DD (Fig. 1d), SCNs from
298 mice housed in DD had no observable difference in the rhythm of PER2::LUC (Fig. 3d-
299 e; t(6) = 0.598, p = 0.572; t-test). Likewise, no period difference was found in any of the
300 other tissues tested (Fig. 3f), and SCN amplitude did not differ between genotypes in
301 LD or DD (p > 0.05).
302 Therefore, to explore the impact of LD cycles on the PER2::LUC rhythms of
303 Mef2d-/- SCNs, we housed WT and KO mice in short (T22, LD11:11) and long (T26,
304 LD13:13) LD cycles. SCNs were harvested following at least three weeks of acclimation
305 to the altered cycle length. When analyzed by two-way ANOVA, neither a short (T22)
306 nor a long (T26) LD cycle produced a significant change in free-running period length
307 for either genotype (Fig. 3g-i); however, genotype effects were diminished under T26
308 conditions (Fig. 3i; F(1, 35) = 12.24, * p =0.0013 for T24; two-way ANOVA). Taken
309 together, the data from Mef2d KO SCNs suggests that the behavioral phenotype is not
310 a result of a gross change in Per2 expression in the SCN, but rather is a result of an
311 interaction between MEF2D and the photic environment.
312 313 Clock gene expression is normal in Mef2d knockout tissues. 314 315 To begin to elucidate the actions of MEF2D on core clock genes, we next
316 measured messenger RNA (mRNA) expression in cerebellum and heart samples from
317 WT and Mef2d KO mice housed in LD. There was no obvious effect of genotype on
11
318 clock gene expression in the cerebellum at two different time-points (Fig. 4a). However,
319 for Per1 mRNA expression there was an interaction between genotype and time-of-day,
320 with a less dramatic increase observed at ZT12 in the cerebellum of KO mice compared
321 to WT mice (interaction (F(1,7)= 8.502, p=0.0225) and main effect of time
322 (F(1,7)=21.19, p=0.0025); * p =0.0089; two-way ANOVA). In heart samples, only Rev-
323 erba expression differed between WT and KO mice, with KO mice having higher levels
324 of Rev-erba at ZT12 (Fig. 4b; interaction (F(1,7)=8.076, p=0.0250, main effect of time
325 (F(1,7)=43.10, p=0.0003, and main effect of genotype (F(1,7)=6.001, p=0.0441); *p =
326 0.0218; two-way ANOVA. Mef2d expression differed significantly with time-of-day in the
327 cerebellum where it was slightly higher at ZT12 than ZT0 (Fig. 4a; t(3)=3.296,
328 *p=0.0459; t-test). No differences were observed in clock gene expression or Mef2d
329 expression in either cerebellum or heart tissues from mice housed in constant darkness
330 (DD; data not shown).
331 Because such small differences in clock gene expression were observed, we
332 also tested for the ability of MEF2D to transcriptionally regulate clock genes in
333 fibroblasts using a luciferase reporter assay. MEF2D did not activate Per1, Per2, or
334 Bmal1 in our experiments (Fig. 5a-c), but did trans-activate a positive control (desMef2,
335 Fig. 5d; t(3.127)=8.472; *p=0.029; t-test). Further, we observed no physical interactions
336 between MEF2D and BMAL1 in co-immunoprecipitation experiments (Fig. 5e). Along
337 with our gene expression results, these findings suggest that the robust period
338 difference in the circadian behavior of Mef2d KO mice is not the result of a substantial,
339 direct impact on clock gene or protein expression.
340
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341 Mef2d KO mice have disrupted sleep patterns. 342 343 Our findings of disturbed circadian running-wheel behavior and increased rest
344 events in Mef2d-/- mice, without corresponding broad changes in the molecular circadian
345 clock, led us to hypothesize these behavioral changes were reflective of abnormal
346 sleep. Thus, as a final behavioral analysis, we recorded sleep patterns from WT and KO
347 mice. Wake and sleep episodes were phase delayed in KO mice (Fig. 6a-c), mirroring
348 what was observed for running-wheel behavior (Fig. 1a-c). There were no differences in
349 the EEG power density across sleep stages or the total time spent in any sleep stage
350 (data not shown); however, KO mice tended to have more transitions (Fig. 6d) from
351 wakefulness to NREM sleep (t(14)= 2.077; p = 0.0567; t-test) and from NREM sleep to
352 wakefulness (t(14)= 2.015; p = 0.0635; t-test) or REM sleep (t(14)= 2.002; p = 0.0651; t-
353 test). More in-depth analysis revealed that KO mice had extremely fragmented sleep
354 with significantly shorter REM latency (Fig. 6e; (Full day: t(14)=3.147, *p=0.0071),
355 (Light: t(14)=2.848, **p=0.0129), (Dark: t(14)=3.108, ***p=0.0077); t-tests) as well as
356 shorter NREM episode duration (Fig. 6f; Full day: t(14)=2.829, *p=0.0134), (Light:
357 t(14)=2.557, **p=0.0228), (Dark: t(14)=2.910, ***p=0.0114); t-tests). Plotting the number
358 of NREM episodes as a function of episode duration (Fig. 6g) revealed that the majority
359 of NREM episodes were shorter in KO mice as compared to WT mice. Taken together,
360 these results suggest that the circadian alterations in activity in Mef2d-/- mice are due, at
361 least in part, to disrupted sleep patterns.
362
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363 DISCUSSION 364 365 366 It is rare for a single-gene mutation/loss to cause a large change in the circadian
367 free-running period. When such changes do occur, they are typically the result of loss of
368 function for a gene directly involved in the core clock feedback loop or a gene that
369 strongly influences the expression or stability of core clock genes (Takahashi et al.,
370 2008). Here we demonstrate that loss of Mef2d results in a 40 min lengthening of the
371 free-running period of locomotor activity. This change in free-running period is not
372 associated with concomitant changes in the levels of core clock components, and thus
373 appears to result from a role of Mef2d downstream of the molecular clock.
374 In this study, we observed a lengthening of period both in conventional Mef2d-/-
375 and CamKIIa-iCre x Mef2dfx/fx mice, therefore it is likely that the loss of Mef2d impacts
376 the circadian system through its actions in the brain. The CamKIIa-iCre line used
377 expresses at postnatal day 3, with maximal expression occurring by postnatal day 15
378 (Casanova et al., 2001), thus our results suggest that the effects of Mef2d on the
379 circadian system are not due to developmental loss of Mef2d. The floxed Mef2d allele
380 does not cause a complete knockout, but rather it produces a non-functional truncated
381 protein product (Kim et al., 2008). However; these same mice have been crossed with
382 a different Cre-driver line shown to have reduced Mef2d gene expression in the
383 hippocampus (Akhtar et al., 2012). Importantly, the long free-running period observed in
384 Mef2d-/- mice is specific, as we did not observe a change in any circadian parameter in
385 brain-specific Mef2a or Mef2c floxed mice.
386 MEF2D has been shown to influence synapse elimination, dendritic spine
387 formation, and the expression of neurotrophic factors in hippocampal neurons (Lyons et
14
388 al., 1995; Akhtar et al., 2012; Dietrich, 2013; Rashid et al., 2014; Chang et al., 2017), so
389 it is possible that the postnatal loss of MEF2D causes reorganization of SCN circuitry.
390 While there is some evidence that alterations in synaptic connectivity lead to changes in
391 circadian outputs in the fly (Fernandez et al., 2008; Gilestro et al., 2009; Gorostiza et al.,
392 2014; Krzeptowski et al., 2018), it remains to be established whether this is the case in
393 mammals. Previous studies have concluded that MEF2 in the fly may be necessary for
394 a daily pattern of dendritic remodeling via inhibition of the cell-adhesion molecule
395 Fasciclin2 (Fas2) (Sivachenko et al., 2013), so it is possible that MEF2 may exert its
396 actions through similar mechanisms in mice.
397 Interestingly, while the behavioral free-running period was lengthened in Mef2d-/-
398 mice, SCNs from animals previously entrained to LD cycles had shorter free-running
399 periods than WT mice. This effect was not observed when SCNs were harvested from
400 mice held in constant DD conditions, suggesting that the short period SCN in Mef2d-/-
401 mice is an after-effect of the LD cycle and not due to an intrinsic change in the period of
402 the SCN itself. After-effects of LD cycles on SCN rhythms have been reported
403 previously, although they are not well-understood (Aton et al., 2004; Mickman et al.,
404 2008; Molyneux et al., 2008). When SCNs were taken from mice housed in LD cycles
405 shorter than 24h, (T22; LD11:11), the periods of KO mice remained shorter than those
406 of WT mice, although this difference did not reach statistical significance. It should be
407 noted, however, that in this short LD cycle, the phase of locomotor activity onset was
408 very variable for both genotypes, suggesting that animals may not have been fully
409 entrained in this condition. It has been proposed that normal MEF2 levels may be
410 necessary to maintain synchrony between clock neurons in the fly brain (Blanchard et
15
411 al., 2010; Sivachenko et al., 2013). In mice, however, we did not observe any evidence
412 via gene expression data that the SCN is strongly affected by the loss of MEF2D. Based
413 on our data, we suggest that the long period of locomotor activity we observe in Mef2d-/-
414 mice may be due to their being pushed to conform to a 24-hour LD cycle, and could be
415 the result of an effect on SCN output, rather than an effect on timekeeping within the
416 SCN itself. While we did not directly assess the SCN anatomy or neuronal activity in
417 Mef2d-/- mice, nor did we measure core SCN-driven physiological outputs, these would
418 be important to measure in future studies to test this hypothesis.
419 Alternatively, it is possible that rather than affecting SCN outputs, the loss of
420 MEF2D alters SCN inputs from the retina. MEF2D binds to retinal-specific promoters to
421 drive photoreceptor development, and Mef2d-/- mice have progressive retinal
422 degeneration beginning at P30 (Andzelm et al., 2015; Omori et al., 2015). While these
423 studies examined gross changes to rods and cones, intrinsically photosensitive retinal
424 ganglion cells could also be disrupted. Changes in behavioral circadian rhythms have
425 been reported with retinal degeneration or bilateral enucleation (Lupi et al., 1999;
426 Yamazaki et al., 2002; Tosini et al., 2007), therefore it is possible that the Mef2d-/- mice
427 have altered behavioral rhythms due to decreased retinal signaling. Future studies
428 could examine light-induction in the SCN and light-induced phase shifts in behavior in
429 these mice. In addition, while we did not examine aging mice, it would be interesting for
430 future studies to examine the circadian phenotype over extended periods of time to
431 determine whether the progressive retinal degeneration seen in these mice correlates
432 with behavioral changes.
16
433 In addition to the circadian effects, loss of MEF2D had a profound effect on
434 sleep/wake patterning. Both activity and sleep were extremely fragmented in Mef2d-/-
435 mice. NREM sleep in particular was largely restricted to multiple, very short duration
436 bouts in KOs. This difference in the patterning of NREM sleep occurred without a
437 corresponding change in either total time spent in NREM sleep across the day or power
438 density during any sleep stage. While we did not measure melatonin levels or other
439 physiological markers of sleep patterning, future studies should examine the larger
440 implications of this disrupted sleep pattern on behavioral and physiological function in
441 these animals. In particular, it would be fascinating to determine whether loss of
442 MEF2D influences memory consolidation during sleep. The MEF2 family of
443 transcription factors have been implicated in memory formation and synaptic plasticity
444 (Barbosa et al., 2008; Akhtar et al., 2012; Dietrich, 2013; Rashid et al., 2014; Adachi et
445 al., 2016). Further, a growing body of literature suggests that MEF2 proteins exert
446 neuroprotective effects in response to neuronal inflammation and oxidative stress (Gong
447 et al., 2003; Tang et al., 2005; Smith et al., 2006; She et al., 2011; Ryan et al., 2013;
448 Yang et al., 2015). The present study provides additional evidence for a specific role of
449 MEF2D in maintaining normal function of the mammalian circadian clock and sleep
450 homeostasis. Future studies should focus on understanding the influence of MEF2 on
451 sleep and circadian function and its impacts on neuronal health.
452 453
17
454 REFERENCES 455 456 ǡ ǡ ǡȋʹͲͳȌʹ 457 Ǥ 458 ͺͲǣͳͶͲǦͳͶͺǤ 459 ǡǡ ǡ ǡǡ ǡǦǡǡ 460 ǡȋʹͲͳʹȌ ʹ 461 Ǥǣ͵Ͷͺ͵Ǥ 462 ȋʹͲͳʹȌ ǣ 463 ǤͶǣʹͶǦʹͲǤ 464 ǡ ǡ ǡǡǡ ǡǡǡ 465 ǡǡǡ ȋʹͲͳͷȌ ʹ 466 Ǧ Ǧ 467 ǤͺǣʹͶǦʹ͵Ǥ 468 ǡ ǡǡǡ ǡǡ ǡ ǡǦ 469 ǡȋʹͲͲȌ ʹ 470 Ǥͳʹǣ͵Ǧ͵ͺͻǤ 471 ǡ ǡ ǡ ǡ ȋʹͲͲͶȌ 472 ǦʹͶǦ Ǥ 473 ͳͻǣͳͻͺǦʹͲǤ 474 ǡǡ ǡ ǡǡ ǡ ǡǡ 475 ǡǦǡȋʹͲͲͺȌ ʹǡ 476 477 Ǥ ͳͲͷǣͻ͵ͻͳǦͻ͵ͻǤ 478 ǡȋͳͻͻͺȌ 479 Ǧʹȋ ʹȌǤͳͶǣͳǦͳͻǤ 480 ǡǡǡ ǡǡ ȋʹͲͳͲȌ 481 ʹ Ǥ 482 ͵ͲǣͷͺͷͷǦͷͺͷǤ 483 ǡ ǡǡǡ ǡ ǡ 484 ȋʹͲͲͳȌ Ǧ Ǥ 485 ͵ͳǣ͵ǦͶʹǤ 486 ǡ ǡ ǡ ǡ ȋʹͲͳȌ 487 ͳ 488 ʹȀǤǤ 489 ǡ ǡǡ ǡ ǡ ǡ ǡǡ ǡ 490 ǡ ǡ ȋʹͲͳʹȌ ʹǦ 491 Ǥ ͳͷǣͳʹͷͷǦͳʹͶǤ 492 ȋʹͲͳ͵Ȍ ʹǣ Ǥ 493 ͵ͷʹǣͳͻǦͳͻͲǤ 494 ǡ ǡ ȋʹͲͲͺȌ 495 ǤǣͻǤ 496 ǡǡ ǡ ǡ ǡ ǡ Ǧ 497 ǡǡ ȋʹͲͲͺȌ Ǧ ʹ 498 Ǧ 499 ǤͲǣͳͲʹʹǦͳͲ͵ͺǤ
18
500 ǡǡǡ ǡ ǡǡ ǡ ǡǡ 501 ȋʹͲͲȌ Ǧ ʹ 502 Ǥ ͵ͳͳǣͳͲͲͺǦͳͲͳʹǤ 503 ǡǡǡ ǡǡ ǡ ǡ ǡ 504 ȋʹͲͳͲȌ Ǧ 505 ͳͶ 506 Ǥ ͳͲǣͳͺͳͷͷǦͳͺͳͲǤ 507 ǡ ǡȋʹͲͲͻȌ 508 Ǥ ͵ʹͶǣͳͲͻǦͳͳʹǤ 509 ǡǡǡǡ ǡ ǡǡ ǡ ȋʹͲͲ͵Ȍ 510 ͷǦ ʹ 511 Ǧ Ǥ͵ͺǣ͵͵ǦͶǤ 512 ǡǦǡ ǡǡ ȋʹͲͳͶȌ 513 ǤʹͶǣʹͳͳǦ 514 ʹͳǤ 515 ǡ ǡ ǡ ȋʹͲͳȌ 516 ȋȌ ǣ Ǧ 517 Ǧ Ǥ ͻǤ 518 ǡ ǡǡ ǡȋͳͻͻͷȌǡ 519 ʹǡ ǣ 520 Ǥ ʹͲͲǣͳͳǦͳʹͲǤ 521 ǡǡǡ ǡ ǡǡ ǡ ǡǦǡ 522 ȋʹͲͲͺȌ ʹ Ǧ 523 Ǥ ͳͳͺǣͳʹͶǦͳ͵ʹǤ 524 ǡ ǡȋʹͲͳͺȌ 525 Ǥ ͳʹǣ͵ʹǤ 526 ǡ ǡ ǡ ǡǡ ǡǡǡǦ 527 ǡȋͳͻͻ͵Ȍ ʹǡȀ ʹǦ 528 Ǥ 529 ͻͲǣͳͷͶǦͳͷͷͲǤ 530 ǤȋʹͲͲȌ ǦǤ 531 ͶͶͷǣͳͺǦͳǤ 532 ǡǡǡǡǡǡǡ 533 ȋʹͲͲͳȌ ʹʹ 534 Ǧ Ǥ 535 ʹͳǣͷͶͶǦͷͷʹǤ 536 ǡǡ ǡ ǡ ǡǡǡǡǡ 537 ǡ ǡ͵ǡ ǡȋʹͲͲȌ 538 ǤͳʹͻǣͲͷǦͳǤ 539 ǡ ǡ ǡ ǡ ǡ ȋʹͲͲ͵Ȍͷ ʹǦ 540 Ǧ 541 Ǥ ͳͲͲǣͺͷ͵ʹǦ 542 ͺͷ͵Ǥ 543 ǡǡǡ ǡ ǡǦǡ 544 ȋʹͲͳͶȌ ʹǡǡ Ǥ 545 ͳͳͳǣͶͳͲͻǦͶͳͳͶǤ
19
546 ǡ ǡ ǡǡ ǡ ȋͳͻͻͻȌ 547 Ǥ 548 ͺͻǣ͵͵Ǧ͵ͶǤ 549 ǡ ǡ ǡ ǡȋͳͻͻͷȌʹ 550 Ǥ 551 ͳͷǣͷʹǦͷ͵ͺǤ 552 ǡǡ ǡǦ ǡ ȋͳͻͻͻȌ Ǧ 553 ʹǤ ʹͺǣͺͷǦͻͲǤ 554 ǡ ǡ ǡȋʹͲͲͺȌ 555 ʹǣǣ Ǥ 556 ʹͻͷǣͳͺͺǦͳͻͶǤ 557 ǡǡ ȋʹͲͲͺȌ 558 ǤͳʹʹͺǣͳʹǦͳ͵ͶǤ 559 ǡǡ ǡǡǡ ǡ ȋʹͲͳͷȌʹ 560 Ǥ 561 ʹͲǣͶͲͺǦͶʹǤ 562 ǡ ǡ ǡ ǡǡǡǡ 563 ȋʹͲͳͲȌ 564 Ǧ ʹǤǣͳͻͳǦ 565 ͳͻǤ 566 ǡȋʹͲͲȌ ʹǣ 567 Ǥͳ͵ͶǣͶͳ͵ͳǦͶͳͶͲǤ 568 ǡ ǡ ȋʹͲͳͶȌ ʹ 569 Ǥ ͳ͵ǣͳͳͺǦͳʹͷǤ 570 ǤȋʹͲͳ͵Ȍ ̵Ǧ 571 ʹǦ ͳ Ǥͳͷͷǣͳ͵ͷͳǦͳ͵ͶǤ 572 ǡȋʹͲͲͷȌǣ ʹ 573 Ǥͻǣʹ͵ͻǦʹǤ 574 ǡ ǡǡ ǡ ǡǡ ȋʹͲͳͳȌ 575 ʹ 576 Ǥ ͳʹͳǣͻ͵ͲǦͻͶͲǤ 577 ǡǡǡǡ ǡ ǡǡǡ 578 ǡ ǡȋʹͲͲͲȌ 579 Ǥ ʹͺͺǣͳͲͳ͵ǦͳͲͳͻǤ 580 ǡǦ ǡǡǡǡ ǡ ǡ 581 ǡ ǡ ǡ ȋʹͲͲͳȌ Ǧ 582 583 Ǥ ͳͳǣͻͷͻǦͻͺͲǤ 584 ǡ ǡ ǡǡǡ ǡǡ ȋʹͲͲȌ 585 Ǧ ͵ 586 ǤͳʹͻǣͳͲͳͳǦͳͲʹ͵Ǥ 587 ǡ ǡǡȋʹͲͳ͵Ȍ ʹ 588 ʹǡǡ Ǥ 589 ͻǣʹͺͳǦʹͻʹǤ 590 ǡǡǡǡ ǡ ǡ ǡǡ ǡ 591 ȋʹͲͲȌǦ͵ͷȀ ͷ
20
592 593 ʹǤ ʹǣͶͶͲǦͶͶǤ 594 ȋʹͲͳȌ Ǥ 595 ͳͺǣͳͶǦͳͻǤ 596 ǡ ǡ ǡ ȋʹͲͲͺȌ 597 ǣ Ǥ ͻǣͶǦ 598 ͷǤ 599 ǡǡ ǡǡǡ ǡ ȋʹͲͲͷȌ Ǧͷ 600 Ǧ 601 ʹǤ ʹͷǣͶͺʹ͵ǦͶͺ͵ͶǤ 602 ǡ ǡ ǡǡȋʹͲͲȌ 603 Ǧ 604 ǤͳͳͶͺǣǦͺʹǤ 605 ǡ ǡǡǡ ǡ ǡ ǡ ȋʹͲͲͷȌ 606 Ǧ 607 Ǥ ͵ǣͳͺǦͳͻʹǤ 608 ǡ ǡȋʹͲͳͲȌ ǣ 609 ǤʹǣͷͷͳǦͷǤ 610 ǡǡȋʹͲͲʹȌ 611 Ǥ ͳǣ͵ͳͷǦ͵ʹͻǤ 612 ǡ ǡ ǡǡ ǡ ǡ ǡ ǡ ȋʹͲͳͷȌ 613 ʹǦͳͲ 614 Ǧ Ǥ 615 ͳʹǣ͵͵Ǥ 616 ǡ ǡǡǡ ǡǡǡ ǡ ǡ 617 ǡǡ ȋʹͲͲͶȌ ʹǣǣ Ǧ 618 619 Ǥ ͳͲͳǣͷ͵͵ͻǦͷ͵ͶǤ 620 621
21
622 FIGURE LEGENDS 623 624 625 Fig. 1: Global loss of Mef2d Lengthens Circadian Free-Running Period and Alters
626 Behavioral Activity Patterns. Representative actograms of WT (a) and KO (b) mice on
627 running-wheels. Actograms are double plotted, with each horizontal line representing 48h of
628 activity. Mice were initially housed in LD12:12, as indicated by the yellow shading (lights on) and
629 results analyzed by student’s t-tests. (c) Summary data (mean ± SEM) for activity onset in LD
630 (n= 13 WT, 18 KO; t(29) = 3.762, *p=0.0008); (d) Summary data (mean ± SEM) for free-running
631 period in DD (n = 18 WT, 22 KO; t(38) = 8.340, *p < 0.0001). (e) Mean ± SEM number (t(29) =
632 4.257, *p =0.0002) and duration (t(29) = 4.844, *p < 0.0001) of activity bouts as measured in DD
633 (n = 12 WT, 19 KO). (f, g) Representative ethograms from video analyses of WT (f) and KO (g)
634 mice (n=2/group). Ethograms are double plotted, with each horizontal line representing 48h of
635 activity. Mice were housed in LD12:12, as indicated by the yellow shading (lights on). (h).
636 Representative scored ethograms from WT (blue) and KO (red) mice, representing 15.5h of
637 data beginning at ZT12 (lights-off) on the day indicated by the red arrows in (f, g).
638
639 Fig. 2: Mef2d Activity in the Brain is Necessary for Normal Circadian Behavior. CamKIIα-
640 iCre mice were crossed with Mef2a, Mef2c, and Mef2d floxed lines to produce mice with a
641 varying combination of floxed Mef2 alleles. Representative actograms from (a) Mef2dfx/fx control
642 and (b) CamKIIα-iCre:Mef2dfx/fx mice. Actograms are double plotted, with each horizontal line
643 representing 48h of activity. Mice were initially housed in LD12:12, as indicated by the yellow
644 shading (lights on). (c) Free-running periods in DD (mean ± SEM) from floxed and control mice
645 (n = 14 Mef2a, 4 Mef2c, 13 Mef2d, 32 control; analyzed by one-way ANOVA, F(3, 59) = 25.19,
646 *p < 0.0001, different from all other conditions assessed by Tukey post hoc comparisons).
647
22
648 Fig. 3: Mef2d KO Causes a Short Free-Running Period in the SCN. (a) Detrended,
649 smoothed, bioluminescence traces from representative WT and KO mouse SCNs. Mice were
650 housed in T24 (LD12:12) prior to tissue harvest and results analyzed with student’s t-tests. (b)
651 Periods (mean ± SEM) of LD12:12 SCN explants (n= 7 WT, 7 KO; t(12) = 3.443, *p=0.0049).
652 (c) Periods (mean ± SEM) of LD12:12 peripheral explants (n= 3-10 mice/tissue/genotype; each
653 tissue analyzed separately, t(17)=3.279, *p=0.0044). (d) Detrended, smoothed,
654 bioluminescence traces from representative WT and KO mouse SCNs. Mice were housed in DD
655 prior to tissue harvest. (e) Periods (mean ± SEM) of DD SCN explants (n = 4 WT, 4 KO; t(6) =
656 0.598, p = 0.572). (f) Periods (mean ± SEM) of DD peripheral tissue explants (n= 2-4
657 mice/tissue/genotype). (g, h) Detrended, smoothed, bioluminescence traces from representative
658 WT and KO mouse SCNs. Mice in g were housed in T22 (LD 11:11) and mice in h were housed
659 in T26 (LD 13:13) prior to tissue harvest and results analyzed by two-way ANOVA. (i) Periods
660 (mean ± SEM) of SCN explants (T22: n = 6 WT, 9 KO; T24: n = 7 WT, 7 KO (also in b); T26: n =
661 6 WT, 6 KO; main effect of genotype F(1, 35) = 12.24, * p =0.0013 for T24; no main effect of T
662 length).
663
664 Fig. 4: Clock Gene Expression is Not Greatly Affected by loss of Mef2d. Quantitative RT-
665 PCR analysis of clock gene expression in WT and Mef2d KO cerebellum (a) and heart (b) taken
666 from mice housed in T24, LD12:12 (LD). Data are presented as relative quantification compared
667 to WT0; n = 2-3/group; and analyzed by two-way ANOVA. Per1: interaction (F(1,7)= 8.502,
668 p=0.0225) and main effect of time (F(1,7)=21.19, p=0.0025); * p =0.0089 WT12 different from
669 WT0; Reverba: interaction (F(1,7)=8.076, p=0.0250, main effect of time (F(1,7)=43.10,
670 p=0.0003, and main effect of genotype (F(1,7)=6.001, p=0.0441); *p = 0.0218 KO12 different
671 from WT12; Mef2d: t(3)=3.296, *p=0.0459 via student’s t-test.
672
23
673 Fig. 5: MEF2D Does not Directly Interact with Core Clock Components. Co-transfection of
674 Mef2d does not lead to activation of (a) Per1::Luc (average of 2 replicates across 3
675 experiments; 125 ng Mef2d transfected), (b) Per2::Luc (average of 2 replicates across 2
676 experiments; increasing doses of Mef2d from 5-50 ng), or (c) Bmal1:Luc (average of 2
677 replicates from 1 experiment; increasing doses of Mef2d from 5-50ng) in reporter assays. Both
678 Per1::Luc and Per2::Luc activation is observed in the presence of known transcriptional
679 activators, BMAL1 and CLOCK (Per1: f(3,20)= 10.80; ** p=0.0002; Per2: f(10,33)=3.42,
680 p=0.0037, ns after correction for multiple comparisons) (d) Co-transfection of Mef2d activates a
681 Mef2 binding domain fused to a TATA-box (average of 2 replicates across 2 experiments; 125
682 ng Mef2d transfected; t(3.127)=8.472; *p=0.029). (e) MEF2D is not pulled down with BMAL1 in
683 co-IP experiments, whereas the known binding partner, CLOCK, was successfully co-IP’d and
684 FLAG (BMAL1) enrichment is observed.
685
686 Fig. 6: Loss of Mef2d Leads to Fragmented Sleep. Patterns of wakefulness (a), NREM (b),
687 and REM sleep (c) are shifted towards a later phase in the KO mice, mirroring the delayed
688 phase observed from running-wheel behavior. (d) Mean ± SEM number of transitions over the
689 full day. Data are presented as mean ± SEM (n= 8 WT, 8 KO) and analyzed by student’s t-
690 tests; there were no significant differences between genotypes. However, KO mice tended to
691 have more transitions from wakefulness to NREM sleep (t(14)= 2.077; p = 0.0567) and from
692 NREM sleep to wakefulness (t(14)= 2.015; p = 0.0635) or REM sleep (t(14)= 2.002; p = 0.0651).
693 (e) Mean ± SEM REM latency is shorter in Mef2d KO mice (n’s same as above; results
694 analyzed with student’s t-tests). Data are presented over the full day (24; t(14)=3.147,
695 *p=0.0071), the light portion of the LD cycle (Light; t(14)=2.848, **p=0.0129), or the dark portion
696 of the LD cycle (Dark; t(14)=3.108, ***p=0.0077). (f) NREM episode duration is shorter in Mef2d
697 KO mice (n’s same as above; analyzed with student’s t-tests). Data are presented over the full
698 day (NREM24; t(14)=2.829, *p=0.0134), the light portion of the LD cycle (NREMLight;
24
699 t(14)=2.557, **p=0.0228), or the dark portion of the LD cycle (NREMDark: t(14)=2.910,
700 ***p=0.0114) (g) NREM episode duration as a percentage of all NREM episodes reveals
701 fragmented sleep patterns in KO mice.
702
703 TABLES
704
705 706 Table 1: Primers for Real-time PCR quantification of mRNA expression.
Gene Primer Set F 5' -CCA CCT CAG AGC CAT TGA TAC A- 3' Bmal1 R 5' -CGA AGA ACG TCA TGA TGC AG- 3' F 5' -CCC AGC TTT ACC TGC AGA AG- 3' Per1 R 5' -ATG GTC GAA AGG AAG CCT CT- 3' F 5' -TGT GCG ATG ATG ATT CGT GA- 3' Per2 R 5' -GGT GAA GGT ACG TTT GGT TTG C- 3' F 5' -CGA AGA ACG TCA TGA TGC AG- 3' Dbp R 5' -GGT TCC CCA ACA TGC TAA GA- 3' Nr1d1 F 5' -AGA CTT CCC GCT TCA CCA AG- 3' (Reverba) R 5' -AGC TTC TCG GAA TGC ATG TT- 3' F 5' -ACG AGA GCC GCA CCA ATG CT- 3' Mef2d R 5' -GGA ACA GAT GAC CCA TAG CGC CTG- 3' F 5' -CAA GGA GTA AGA AAC CCT GGA CC- 3' Gapdh R 5' -CGA GTT GGG ATA GGG CCT CT- 3' 707
708
25 Figure 1
abWT KO Day Day
0 8 16 0 8 16 0 0 8 16 0 8 16 0 Time (h) Time (h) c de
6 Ave Bout Length (min) 24.5 * * 250 0.0 24.0 200 4 -0.2 23.5 150
-0.4 2 * Period (h) 23.0 # Bouts/Day 100 WT -0.6 KO 22.5 0 50 Onset (h prior to lights off) * fgWT KO Day Day
18 2 10 18 2 10 18 18 2 10 18 2 10 18 Time (h) Time (h) h WT KO
Rest (sleep) 8 At end of cage (possible sleep) 7 General activity (far end of cage) 6 General activity (near wheel) 5 Near food hopper 4 At food hopper (eating) 3 At sipper (drinking) 2 On wheel 1
3 3 13 15 17 19 21 23 1 3 1 15 17 19 21 2 1 3 Time (h) Time (h) Figure 2
a Mef2dfx/fx
c 24.5 * Day 24.0
23.5 0 8 16 0 8 16 0
Time (h) Period (h) b CamKIIα-iCre x Mef2dfx/fx 23.0
22.5 Mef2c Mef2a Mef2d Day Control
0 8 16 0 8 16 0 Time (h) Figure 3 abc WT 200 LD 26 25 KO * 100 24 24 * (h)
0 Period Period (h) Period 23 22 -100
-200 22 20 Bioluminescence (detrended) Bioluminescence 1 2 3 4 5 6 7 LD SCN g ry Days in Culture Heart Liver Lun Kidney Pituita def
200 DD 25 26
100
24 (h) 24
0 Period (h) Period 23 Period 22 -100
-200 22 20 Bioluminescence (detrended) Bioluminescence 1 2 3 4 5 6 7 DD SCN ary Days in Culture Heart Liver Lung Kidney Pituit ghi
200 T22 200 T26 25 * 100 100 24 0 0
Period (h) Period 23 -100 -100
-200 -200 22 Bioluminescence (detrended) Bioluminescence 1234567 1234567 4 T22 T2 Days in Culture Days in Culture T26 Figure 4 WT a Cerebellum (LD) KO Bmal1 Per1 Per2 2.0 1.5 1.5 * 1.5 1.0 1.0
1.0
0.5 0.5 Relative 0.5
Quantification (RQ) 0.0 0.0 0.0 0 12 012 012 Dbp Rev-erbα Mef2d 1.5 1.5 1.5
* 1.0 1.0 1.0
0.5 0.5 0.5 Relative
Quantification (RQ) 0.0 0.0 0.0 012 012 012 Time (h)Time (h) Time (h)
b Heart (LD) WT KO Bmal1 Per1 Per2 10.0 1.5 1.5
8.0 1.0 1.0 6.0
4.0 0.5 0.5 Relative 2.0
Quantification (RQ) 0.0 0.0 0.0 012 012 012 Dbp Rev-erbα Mef2d 2.0 2.5 * 1.5 2.0 1.5 1.0 1.5 1.0 1.0 0.5 Relative 0.5 0.5
Quantification (RQ) 0.0 012012012 Time (h)Time (h) Time (h) Figure 5
abPer1::Luc Per2::Luc 200 12 ** 150 8
100
4 LUC/REN 50 LUC/REN
0 0 Mef2d-HA + + Mef2d-HA Flag-Bmal1 + + Flag-Bmal1 + + + + + + Flag-Clock + + Flag-Clock + + + + + + cde Bmal1::Luc desMef2::Luc Input IP: FLAG 200 10 * Mef2d-HA + + + + + + 8 Flag-Bmal1 + + + + + + 150 Clock + + 6 100 WB: HA 4 LUC/REN LUC/REN WB: CLOCK 50 2 WB: FLAG 0 0 WB: TUBULIN Mef2d-HA Mef2d-HA + Figure 6
abWake NREM c REM
60 60 10 WT KO 40 40 6
20 20 2 Time in state (mins) 0 0 0 0 2 4 6 8 6 0 2 4 6 8 10 12 14 16 18 20 22 10 12 14 16 18 20 22 0 2 4 6 8 10 12 14 1 18 20 22 Hour (ZT) Hour (ZT) Hour (ZT) de 400 WT 8 300 KO *** 6 200 * ** 100 4 40 30 # Transitions 2
20 REM latency (min) 10 0 0 M 24 Dark RE Light e to REM EM to Wake Wak R Wake to NREM NREM to WakeNREM to REM to NREM f g 20 100 15 10 80 8 60 6 *** 40 4 * ** 2 20 Episode Duration (min) 0 0 % of All NREM Episodes % of 0 5 10 15 20 25 30 35 40 45 5
REM24 NREM Episode Duration (epochs) Wake24NREM24 REMLightWakeDark REMDark WakeLightNREMLight NREMDark