Tfap2b acts in GABAergic neurons to control sleep in mice

<jats:title>Abstract</jats:title><jats:p>Sleep is a universal state of behavioral quiescence in both vertebrates and invertebrates that is controlled by conserved genes. We previously found that AP2 transcription factors control sleep in <jats:italic>C. elegans, Drosophila,</jats:italic> and mice. Heterozygous deletion of <jats:italic>Tfap2b</jats:italic>, one of the mammalian AP2 paralogs, reduces sleep in mice. The cell types and mechanisms through which <jats:italic>Tfap2b</jats:italic> controls sleep in mammals are, however, not known. In mice, <jats:italic>Tfap2b</jats:italic> acts during early embryonic stages. In this study, we used RNA-seq to measure the gene expression changes in brains of <jats:italic>Tfap2b</jats:italic><jats:sup>−/−</jats:sup> embryos. Our results indicated that genes related to brain development and patterning were differentially regulated. As many sleep-promoting neurons are known to be GABAergic, we measured the expression of GAD1, GAD2 and Vgat genes in different brain areas of adult <jats:italic>Tfap2b</jats:italic><jats:sup>+/−</jats:sup> mice using qPCR. These experiments suggested that GABAergic genes are downregulated in the cortex, brainstem and cerebellum areas, but upregulated in the striatum. To investigate whether <jats:italic>Tfap2b</jats:italic> controls sleep through GABAergic neurons, we specifically deleted <jats:italic>Tfap2b</jats:italic> in GABAergic neurons. We recorded the EEG and EMG before and after a 6-h period of sleep deprivation and extracted the time spent in NREM and in REM sleep as well as delta and theta power to assess NREM and REM sleep, respectively. During baseline conditions, <jats:italic>Vgat-tfap2b</jats:italic><jats:sup>−/−</jats:sup> mice exhibited both shortened NREM and REM sleep time and reduced delta and theta power. Consistently, weaker delta and theta power were observed during rebound sleep in the <jats:italic>Vgat-tfap2b</jats:italic><jats:sup>−/−</jats:sup> mice after sleep deprivation. Taken together, the results indicate that <jats:italic>Tfap2b</jats:italic> in GABAergic neurons is required for normal sleep.</jats:p>

section_content: Tfap2b controls the expression of genes related to embryonic brain development. Tfap2b is expressed in early embryo stages, starting from E8, and is enriched in midbrain and hindbrain areas 32, 33. Complete deletion of Tfap2b is lethal at perinatal stages around P1-P2 34. Here, we investigated how Tfap2b regulates gene expression during embryonic development. We collected Tfap2b -/-embryos at stage E14. 5 for RNAsequencing. For this experiment, we dissected the secondary prosencephalon (SP) and diencephalon/midbrain/ hindbrain (DMH) areas of female and male E14. 5 embryos and compared homozygote deletion mutants with wild-type littermate controls. Overall, there were fewer differentially expressed (DE) genes in SP (Fig. 1A, B ) than in DMH samples (Fig. 1C, D ), and more genes were downregulated in DMH than upregulated (Fig. 1C, D ). Among the highlighted genes, Tfap2b was significantly down regulated in both female and male DMH but not in SP (Fig. 1 ). The highest number of DE genes (133 genes) was found in female DMH. These genes were hence further analyzed for functionally grouped gene ontology and pathway annotation networks using ClueGO 35 (Fig. 2 ). The downregulated genes were classified into two major networks, related to embryonic development and neural activity, respectively (Fig. 2A, B ). All of the DE genes that are related to embryonic brain development belong to the Hox gene family except for Pax8, which is from the Pax family that can be recruited by AP-2 paralogs during target gene activation 36. The enriched group of DE genes related to neurotransmission belong mostly to the solute carrier (Slc) family, except for Aqp1. Upregulated genes (31 genes) were categorized into one network consisting of neuronal generation and glial cell differentiation (Fig. 2C ). 

Table 1 shows all of the genes found under each GO term for the functionally grouped gene ontology and pathway annotation networks for the genes that were differentially expressed in the Tfap2b -/-female DMH. The figure number refers to the panels where the corresponding network can be found.
section_name: Results

section_content: +/-mice. The analysis of the role of Tfab2b deletion in embryos suggested a role of this transcription factor in controlling brain development. GABA is a key neurotransmitter that is required for sleep initiation and regulation, especially for the control of NREMS 22, 37, but the transcriptome analysis could not directly detect a role for Tfap2b in GABAergic function at the early embryonic stage. We thus asked whether Tfab2b might be required for the formation of GABAergic circuits that control sleep at stages that follow after the embryonic development. We hence investigated whether Tfap2b regulates GABA-related gene expression in the brain of adult mice. As homozygous deletion of Tfab2b is perinatally lethal, we used Tfap2b +/-mice and compared their GABA gene expression to their wild-type littermate controls. We dissected the brain cortex (CTX); the hippocampus (HP); the hypothalamus (HY); the striatum (STR); the brainstem (BS); and the cerebellum (CB), and isolated separately the RNA of each of these parts. In each of these RNA samples we quantified the expression of the glutamate decarboxylase genes GAD67 and GAD65, and the GABA transporter gene Vgat using qPCR. We observed altered expression changes in several brain areas of Tfap2b +/-mice compared to their controls (Fig. 3 ). Specifically, there was an overall down regulation of all three genes in CTX, BS and CB areas. Interestingly, an up-regulation of Vgat was observed in the STR. These results suggest that Tfap2b plays a role regulating the expression of GABA-related genes.
section_name: GABA-related gene expression is impaired in the brains of adult Tfap2b

section_content: Vgat-tfap2b -/-mice. Our previous results showed that heterozygous deletion of Tfap2b in all cells of the mice shortened sleep length and reduced Figure 1. Genes differentially expressed in Tfap2b -/-developing mouse brains. We compared gene expression between homozygous mutant embryos (E14. 5) and their wild-type littermates. Volcano plots showing the significantly up-regulated (red) and down-regulated genes (blue) in female SP (A) and DMH (B). Up regulated genes are presented in orange for male (C) and DMH (D). We compared gene expression between homozygous mutant embryos (E14. 5) and their wild-type littermates. Significantly changed genes as defined by an FDR (False Discovery Rate) < 0. 2 and a logFC > 0. 5 were highlighted and the most robustly regulated genes were labeled (FDR < 0. 01; logFC > 2). Female E14. 5 embryos tfap2b +/+, n = 3; female E14. 5 embryos tfap2b -/-, n = 2 (based on PCA analysis shown in Fig. S1, the sample named "mut3" was excluded from the DE genes analysis); male E14. 5 embryos tfap2b +/+, n = 3; male E14. 5 embryos tfap2b, n = 3; SP, secondary prosencephalon; DMH, diencephalon, midbrain and hindbrain.
section_name: Shortened sleep and reduced sleep intensity in female

section_content: Functionally grouped gene ontology and pathway annotation networks for the genes that were differentially expressed in the Tfap2b -/-female DMH. We classified downregulated genes by ClueGO into networks of embryonic brain development (A) and neurotransmission (B). We classified upregulated genes into major groups that are called "negative regulation of glial cell differentiation" and "forebrain generation of neurons" (C). We categorized all genes by biological function with GO terms as nodes where the most significant terms were highlighted. Poorly grouped terms (< 10 nodes) are not shown. Table 1 shows all of the genes found under each GO term, which are represented by different colors as specified in Table 1. Table 1. Differentially expressed genes from GO enrichment analysis.
section_name: Figure 2.

section_content: Embryonic brain development (shown in light blue in Figure 2A )

Hoxa3, Hoxa4, Hoxa5, Hoxa6, Hoxb2, Hoxb3, Hoxb4, Hoxb5, Hoxb6, Hoxb7, Hoxb8, Hoxc4, Hoxd3, Pax8 Figure 2A Embryonic hindbrain patterning (shown in brown in Figure 2A )

Hoxa3, Hoxa4, Hoxa5, Hoxa6, Hoxb2, Hoxb3, Hoxb4, Hoxb5, Hoxb6, Hoxb7, Hoxb8, Hoxc4, Hoxc5, Hoxc6, Hoxd3 Figure 2A Neurotransmitter transmembrane transporter activity (shown in yellow in Figure 2B ) Aqp1, Slc18a3, Slc6a11, Slc6a15, Slc6a2 Figure 2B Solute:sodium symporter activity (shown in violet in Figure 2B ) Slc4a10, Slc4a5, Slc6a11, Slc6a15, Slc6a2 Figure 2B Forebrain generation of neurons (shown in dark blue in Figure 2C ) Dlx2, Dlx5, Nr2e1, Sox1 Figure 2C Negative regulation of glial cell differentiation (shown in pink in Figure 2C ) Dlx2, Hmga2, Nr2e1, Tmem98 Figure 2C NREM delta and REMS theta power, indicating lower NREM sleep intensity and impaired REMS 14. Our qPCR results above (Fig. 3 ) showed that the expression of GABAergic genes is impaired in the Tfap2b +/-mouse brain. GABAergic neurons are known to have sleep-promoting functions. For example, ablating GABAergic neurons in the parafacial zone (PZ) or ventrolateral preoptic area (VLPO) can reduce NREMS 22, 38, 39. To investigate whether Tfap2b acts in GABAergic neurons for sleep control, we deleted Tfap2b specifically in GABAergic neurons. For this experiment, we generated a conditional allele of Tfap2b, Tfap2b tm1c (see methods 40 ), and crossed it with a Cre recombinase driven by the Vgat promoter 41. To quantify sleep in these mice, we used electroencephalogram (EEG) analysis (see methods) and measured sleep over the 24-h Zeitgeber Time (ZT) course for 48 h. We compared the conditional knockdown animals with littermates that carried either the Cre recombinase or that were homozygous for the floxed allele. 

The EEG analysis of female Vgat-tfap2b -/-mice revealed that total sleep time was decreased by approximately 120 min per 24-h day (Fig. 4A, B ). NREMS and REMS time decreased most strongly within the first half of the dark phase (Fig. 4C-F ). In addition to the shortened sleep time, both delta and theta power were reduced in the female Vgat-tfap2b -/-mice (Fig. 5A-D ). Plotting power versus time of the day suggested that delta and theta power were most strongly reduced during the first half of the dark phase (Fig. 5B, D ). Hence, both sleep time as well as delta and theta power are mostly reduced during the first half of the dark phase. A small difference between the two control groups was observed in the spectrum power analysis of the wake state (Fig. 5E, F ). In the Tfap2b fl/fl control the EEG spectral power at slower frequency was lower than that in the Vgat-Cre control. Wake spectral power intensity of Vgat-tfap2b -/-knockouts fell in between the controls. The wake power intensity of wakefulness thus seems to change independently of the slow-wave intensity in sleep. The sleep and wake bout lengths in the mutant group were not affected by the altered sleep or wake spectral power (Fig. S2 ). In summary, the female Vgat-tfap2b -/-mice displayed reduced sleep time and sleep depth.
section_name: GO Term Associated Genes Found Figure Number

section_content: Vgat-tfap2b -/-mice. Sleep pressure, or sleep need, accumulates as wake time progresses and dissipates during sleep 42. The dynamics of sleep homeostasis is best characterized by spectral power changes during rebound sleep 43, 44. To determine the homeostatic sleep response, mice were deprived of sleep (SD) for 6 h after BSL recording and recovery sleep was measured for another 48 h. Following SD, a trend of increased sleep was observed during the first recovery day (R1) in both control and knockout groups with the main effect occurring during the dark phase (Fig. S3 ). The NREMS and REMS architecture was restored to baseline levels in all groups within the second day of recovery (R2) (Fig. S4 ). Spectral power analysis during R1 revealed a delta power increase compared to baseline sleep power within the equivalent ZT range in all groups tested. During R2, the delta power had returned to baseline levels. 

The theta power rebound during REMS showed a statistically significant increase in female Vgat-tfap2b -/-, but the changes in control groups did not reach statistical significance (Fig. S4 ). We therefore calculated the accumulative effect of the theta and delta power rebound for all genotypes. The cumulative delta and theta power was calculated for NREMS and REMS, respectively. Results of both delta and theta oscillations showed that the Figure 3. Expression of GAD67, GAD65 and Vgat genes was altered in the brains of adult Tfap2b +/-mice. Data were analyzed by two-way ANOVA followed by Sidak's multiple comparisons test and were shown as the mean ± SEM, *p < 0. 05, **p < 0. 01. Tfap2b +/+ (n = 3) and Tfap2b +/-(n = 3). CTX, cortex; HP, hippocampus; HY, hypothalamus; STR, striatum; BS, brainstem; CB, cerebellum. Asterisks (*) with short lines identify significance for pairwise comparisons for specific GABA-related genes in CTX and STR. In the BS and CB no significance could be found individual GABA-related genes. In the BS and CB, the asterisks with long lines identify significance in total variations of all three GABA-related genes combined. recovery sleep in female Vgat-tfap2b -/-mice was slower and weaker than their control littermates (Fig. 6 ). Taken together, the homeostatic sleep response was reduced in female Vgat-tfap2b -/-mice. 

Male Vgat-tfap2b -/-mice spent less time in NREM sleep. We showed here that knockdown of Tfap2b in GABAergic neurons reduces sleep in female mice. We also measured EEG/EMG in male mice. Consistent with our results in females, the total sleep time and NREMS were also reduced in male mutants (Fig. 7, S5 ). The effect of GABAergic neuron knockdown of Tfap2b extended to the rebound sleep that is observed following sleep deprivation, but less strongly than in females (Fig. S6 ). Hence Tfap2b in GABAergic neurons plays a role in both, baseline and rebound sleep following sleep deprivation. Thus, Tfap2b is required in GABAergic neurons also for sleep in males. 

The delta and theta power are, however, increased in Vgat-tfap2b -/-males (Fig. S7-S9 ). Male Vgat-cre control mice also displayed a stronger delta and theta power, suggesting that the CRE gene might have caused the power increase. The increased delta and theta power phenotype that is associated with the expression of CRE may have masked potential effects caused by Tfap2b knockdown. The effects of Tfap2b knockdown in GABAergic neurons on delta and theta power thus cannot be clearly interpreted in Vgat-tfap2b -/-males. In the power analysis of the male groups, the two controls, Vgat and tfap2b fl/fl, behaved differently albeit a similar genetic background and experimental conditions. Vgat-cre mice have been previously used in combinations with stereotaxic AAV injection [45] [46] [47]. In these conditions there is one control group involved in each experiment, whereas in the case of breeding conditional knockouts there are two parental control strains that need to be investigated, which increases the likelihood that one parental strain has altered sleep. It hence appears that CRE recombinase expression might only increase delta and theta power in males, but not in females. To clearly test the delta and theta power changes that are induced by knockdown of Tfap2b on sleep power, further studies would be required that, for example, employ a GABA-specific CRE driver that does not interfere with sleep power in males.
section_name: Reduced homeostatic sleep response in female

section_content: AP-2β is expressed in migrating neural crest cells and the expression continues in neural tissues, facial mesenchyme and peripheral system 48. Homozygous loss-of-function mutation in Tfap2b is perinatal lethal in mice, which is due to renal malfunction 34. In our study, a significant decrease of Tfap2b was detected in mutant DMH but not in SP. ISH data from other studies revealed a similar pattern that Tfap2b expression is more robust in mid-and hindbrain areas compared with forebrain 33. Thus, it is expected that more DEGs related to embryonic brain development identified in DMH than SP. This supports the view that Tfap2b plays a prominent role in neuronal development in the DMH. Consistent with our analysis, an in vivo study has demonstrated that Tfap2b is required for the differentiation of noradrenergic neurons 49. In addition, another study showed that Tfap2b mediates the differentiation of GABAergic interneurons in the cerebellum 29. Thus, consistently across species, Tfap2b homologs are required for brain development. The impaired GABAergic gene expression in multiple brain areas in our Tfap2b +/-model suggests that this transcription factor might control GABAergic function not only in the cerebellum but in multiple brain regions. 

We have mapped a sleep-promoting effect of Tfap2b onto GABAergic neurons. While we have mapped an effect of Tfap2b to GABAergic neurons, the specific populations of GABAergic neurons in which Tfap2b acts to promote sleep remain unidentified. GABA is widely used in the nervous system. It would thus be interesting in future studies to further map the function of Tfap2b onto specific subpopulations of GABAergic neurons. It would be particularly interesting to test whether Tfap2b is required for the development or functioning of sleep-promoting neurons. It is equally possible that Tfap2b is required for sleep in neurons that are not sleep active. This is particularly relevant as GABAergic neurons control many functions such as motor control 50, thermoregulation 51, 52, pain 53, memory and anxiety 54. Hence, effects on sleep could potentially be indirect. Also, it is possible that Tfap2b acts not only in GABAergic neurons but also in other neuron types. A mapping of the sleep function of Tfap2b on specific populations of neurons would also facilitate the identification of the molecular function of Tfap2b in these specific neuronal populations. 

In the mammalian brain there are both, populations of GABAergic neurons that promote sleep as well as populations of GABAergic neurons that promote wakefulness. For example, chemogenetic activation of GABAergic neurons in the basal forebrain (BF) promotes wakefulness 55, 56, whereas chemogenetic activation of populations of GABAergic neurons in the VLPO and PZ promote sleep 22, 24. Also, brain areas that control sleep and wakefulness may contain mixed populations of both sleep-promoting neurons and wake-promoting GABAergic neurons. For example, the BF and the VLPO contain such mixed populations of GABAergic neurons, some of which are wake active and others are sleep active 55, 57. Some approaches have been established to specifically target subpopulations of such GABAergic neurons. For example, a specific subpopulation of VLPO GABA neurons, which have projections to the tuberomammillary nucleus (TMN), can be targeted by retrograde labeling, which demonstrated that this specific neuronal subpopulation promotes sleep 24. Hence, some populations of GABAergic neurons can promote sleep, whereas other populations of GABAergic neurons might function to inhibit sleep-promoting neurons 58, 59. Hence, the identification of the molecular function of Tfap2b in sleep will ultimately require more specific manipulations of Tfap2b. 

We did not detect a defect in GABAergic gene expression in the hypothalamus, which contains sleep-active neurons in the VLPO 58. As brain regions such as the hypothalamus contain many different populations of different neuron types, this does not however exclude the possibility that Tfap2b might still be playing a role in sleep-promoting areas within the hypothalamus and it does not exclude the possibility that Tfap2b controls GABA-related gene expression in subsets of neurons. Hence, the effects of Tfap2b knockdown in GABAergic neurons could be different for different populations of GABAergic neurons, and the effects on GABAergic gene expression could be restricted to a small subset of GABAergic neurons within the hypothalamus such as sleepactive neurons of the VLPO or other neurons and might thus be difficult to detect by analyzing gene expression at the level of the entire hypothalamus. It remains unclear why Tfap2b is required for sleep in GABAergic neurons. Tfap2b has been shown to control the expression of GABA-related genes, but it is not known whether this function also underlies its role in sleep. In C. elegans, the Tfap2b homolog aptf-1 does not seem to control sleep by controlling the expression of GABA-related genes, but through the control of expression of a neuropeptide gene 19. While the idea that Tfap2b is required for sleep by controlling GABA-related gene expression seems plausible, it is likely that Tfap2b is playing a role in sleep regulation through other genes. 

We have knocked down Tfap2b during development. It hence remains unclear whether Tfap2b is required specifically during development, i. e. for the normal creation of neuronal circuits that are involved in the control of sleep or whether Tfap2b is required for the functioning of mature neurons and circuits. Such adult-specific inhibition of Tfap2b could for example be tested by tamoxifen-inducible CRE 60, by injecting CRE-expressing AAV into specific brain areas in the adult 61, or by injecting AAV containing floxed shRNA against Tfap2b into specific brain regions such as the VLPO in animals expressing CRE in GABAergic neurons 62, 63. Such additional experiments will be instrumental also in identifying specific neuronal populations in which Tfap2b acts. Once specific populations of neurons are identified in which Tfap2b is required for sleep, transcriptomic and electrophysiological analysis of these specific neurons can be carried out with and without the knockdown of Tfap2b. www. nature. com/scientificreports/ Previous studies have investigated the function of other transcription factors in sleep regulation 7, 64, 65. For example, another transcription factor, Lhx6, exerts a mild sleep-reducing effect via GABA-releasing neurons in the zona incerta of the lateral hypothalamus 64, 66. Thus, Tfap2b might be one of the genetic factors that regulates sleep-initiating GABAergic neurons. Here, and consistent with our work in C. elegans, we find a role of Tfap2b in controlling sleep by acting in GABAergic neurons.
section_name: Discussion

section_content: Authorizations to carry out experiments with live mice. The LAVES (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) approved the experiments with live mice. All experiments were performed in accordance with relevant EU and German guidelines and regulations. The authors complied with the ARRIVE guidelines. 

Animals. Embryos carrying the Tfap2b tm1a allele (knock-out first tm1a allele) were purchased from EMMA. 

We first crossed Tfap2b tm1a with the FLP deleter strain 129S4/SvJaeSor-Gt(ROSA)26Sor tm1(FLP1)Dym /J (#003,946, Jackson laboratory). The FLP recombinase removed the trapping cassette from the Tfap2b tm1a allele, therefore converted the tm1a allele to the conditional (floxed) allele Tfap2b tm1c (here we call Tfap2b fl ). The deletion was verified by PCR. 

The Vgat-cre mouse line B6J. 129S6(FVB)-Slc32a1 tm2(cre)Lowl /MwarJ(#028,862, Jackson laboratory) was a gift from Professor Nils Brose from Max Planck Institute of Experimental Medicine. 

To conditionally delete Tfap2b from GABAergic neurons, we first crossed tfap2b fl/+ mice with Vgat-cre mice to obtain Vgat-tfap2b -/+ mice. Vgat-tfap2 +/-mice were then bred to Tfap2b fl/+ mice to generate homozygous knockouts (Vgat-tfap2b -/-) and the respective littermate controls. We used the Tfap2b fl/fl and heterozygous Vgatcre mice as the control groups in this experiment. Both male and female mice were used in the EEG recording. 

Tfap2b knockout (Tfap2b +/-) mice were provided by Markus Moser [Max Planck Institute (MPI) for Biochemistry] with PGK-neo cassette inserted into exon four of the Tfap2b gene 34. On embryonic day (E) 14. 5, mouse embryos were harvested from mother Tfap2b +/-mice, which had been mated with the male mice of Tfap2b +/-. The brains were extracted and dissected into secondary prosencephalon (SP) and diencephalon/midbrain/hindbrain (DMH) areas. All tissues were snap-frozen and stored at -80 °C before use. 

Genotyping. Ear biopsies of mice or tails from embryos were collected and genomic DNA was extracted as described previously 14. Briefly, samples were lysed in 50 µl of PBND buffer (50 mM KCl, 10 mM Tris HCl pH 8. 3, 0. 1 mg/mL MgCl 2 •6H 2 O, 0. 1 mg/mL Gelatin, 0. 45% NP-40, 0. 45% Tween20) with 2. 5 µl proteinase K (#P8107S, New England BioLabs) freshly added. The samples were incubated in a thermomixer at 55 °C overnight until samples were completely lysed. The proteinase K was then deactivated in the samples by incubation at 85 °C for 45 min. The lysate was then centrifuged at 6000 × g and the supernatant was used for PCR. Primers used for genotyping and PCR conditions are listed in Table S1. Tails from all embryos were measured for determining the genotype and the sex of the individuals.
section_name: Methods

section_content: Brains from E14. 5 embryos were extracted and dissected into prosencephalon (SP) and diencephalon/midbrain/hindbrain (DMH) areas from three female and three male individuals per sex and genotype. Thus, in total, we used 12 embryos. We isolated RNA from each sample individually according to the manufacture's protocol (Qiagen RNeasy Mini plus). RNA sequencing was carried out by Bernd Timmermann and Stefan Börno at the sequencing facility of the Max Planck Institute for Molecular Genetics, Berlin, according to their protocol, which we have described previously in 14 and which is cited here: After quality control using Agilent's Bioanalyzer, sequencing libraries were prepared from 500 ng of total RNA per sample following Roche's stranded "KAPA RNA HyperPrep" library preparation protocol for single indexed Illumina libraries: First the polyA-RNA fraction was enriched using oligo-dT-probed paramagnetic beads. Enriched RNA was heat-fragmented and subjected to first strand synthesis using random priming. The second strand was synthesized incorporating dUTP instead of dTTP to preserve strand information. After A-tailing, Illumina sequencing compatible adapters were ligated. Following bead-based clean-up steps the libraries were amplified using 11 cycles of PCR. Library quality and size was checked with qBit, Agilent Bioanalyzer and qPCR. Sequencing was carried out on an Illumina NovaSeq2 system in PE100bp mode yielding around 50 million fragments per sample. For the analysis, we compared Tfap2b +/+ and Vgat-Tfap2b -/-. Quantitative PCR. Brains from adult Tfap2b +/-and their littermate controls (Tfap2b +/+ ) were extracted and dissected into different brain areas, which were: cortex, striatum, hypothalamus, hippocampus, brainstem, cerebellum and all of the remaining brain areas. Total RNA was extracted from each sample using TRIzol and was reverse transcribed into cDNA using the high-capacity cDNA RT kit (Applied Biosystems) according to the manufacturer's instructions. Reverse transcription was performed at 25 °C for 10 min, 37 °C for 120 min, 85 °C for 5 min and samples were kept in 4 °C or -20 °C until use. Subsequently, the cDNA levels were quantified using the Fast SYBR Green Master Mix (Applied Biosystems). mRNA expression was analyzed using the 2 -ΔΔCq method where the control was normalized to 100%, and the samples from the mutants were compared with their control. The primers used and qPCR conditions are listed in Table S2. 

Surgeries. Female and male mice were used for the EEG experiment and the surgeries were performed as described previously 14. Briefly, mice were anesthetized using isoflurane throughout the operation. Two EEG recording electrodes (Bilaney Consultants GmbH) were implanted bilaterally over frontal cortex (AP + 1. 5 mm from bregma; mediolateral ± 1. 7 mm), and one recording electrode was implanted over the right parietal lobe (AP + 1. 5 mm from lambda; mediolateral + 1. 7 mm). Two reference electrodes were placed bilaterally over the cerebellum (AP -1. 5 mm from lambda; mediolateral ± 1. 7 mm). One subcutaneous pad was inserted to the nuchal muscle to record the electromyogram (EMG). The wires attached to the electrodes and subcutaneous pad were assembled into a pedestal (PlasticsOne, Bilaney Consultants GmbH). The surgical exposure was covered with dental cement. At least 8 days were allowed for recovering before connecting to the recording cable. All mice were given 48 h to acclimate to the recording cylinder. 

EEG recording setups and data analysis. As previously described, the recording was performed in condition under 12 h/12 h (Light/Dark) cycles with light-on at 6AM (ZT0) to 6PM (ZT12) every day. EEG/ EMG signals were recorded at a sampling rate of 2000 Hz (Multi Channel Systems MCS GmbH) starting from ZT0 for 48 h as baseline recording, followed by a 6 h-sleep-deprivation from ZT0 to ZT6 by gentle handling 67 and novel objects interaction 68, subsequently a recovery sleep of 48 h. EEG/EMG data were analyzed in a semiautomatic way that the signals were first trained by a human scorer using Sirenia Sleep Pro (Pinnacle Technologies, Lawrence, KS) at 10 s epoch and then analyzed by a MatLab-based custom-written system 69, followed by visual inspection by the same scorer. NREMS was scored based on the presence of the slow activity (0. 5-4 Hz) with high amplitude (delta waves) and quiet EMG activity. REMS was characterized as theta activity (6-10 Hz) with a slightly higher frequency than delta waves and EMG atonia. Time spent in each vigilance state was calculated and plotted over the 24 h course or as total hours during the 24 h/ light/ dark phase for each genotype. For 48 h of baseline recording, values were averaged and plotted as a 24 h scale. Spectral analysis was performed as described by Kent et al. 70 Raw power data of the vigilance states were presented based on the distribution of power in 0. 1 Hz windows or frequency classes. Accumulative spectral power was plotted to visualize the rebound power changes in NREMS and REMS. All power data were expressed as averaged values from frontal and temporal recordings. The scorer was blind to the genotypes of all mice during the analysis. 

Statistics. Statistics analysis and graph plotting were performed using SPSS or GraphPad Prism 8. Shapiro-Wilk Normality test was used to analyze the distribution of data. Levene's test was adopted to test whether variances were equal. Parametric tests were performed for the datasets showed a Gaussian distribution. Specifically, for comparisons between two groups, two-tailed paired/unpaired t-test was performed. As for multi-group comparisons, ANOVA followed by Sidak's multiple comparisons was performed. Nonparametric tests were used for the dataset that was not normally distributed or the variances were not equal. In such circumstances, Mann-Whitney test or Wilcoxon signed-rank were used for two-group comparison, or Friedman test followed by Dunn's multiple comparisons test was performed for multi-group comparisons. Linear regression analysis was performed to plot accumulative spectral power, and equality of slopes or intercepts was calculated to examine the difference between genotypes. ClueGO 35 was used in the network analysis of DE genes from RNA-analysis.
section_name: RNA-sequencing.

section_name: Acknowledgements

section_content: We thank Ulrike Teichmann, and Sara Kimmina for advice on electroencephalogram (EEG) and other animal experimentation, and help with obtaining permits. We are grateful to Gregor Eichele for providing laboratory space and resources. We thank Markus Moser and Nils Brose for mouse strains. Sequencing was carried out by Bernd Timmermann and Stefan Börno, Max Planck Institute for Molecular Genetics, Berlin. We thank Myrto Stylianou for helpful comments. This work was supported by the Max Planck Society ( Max Planck Research Group "Sleep and Waking "), the TU Dresden, and by a European Research Council Starting Grant (ID: 637860, SLEEPCONTROL).
section_name: Acknowledgements

section_name: Funding

section_content: Open Access funding enabled and organized by Projekt DEAL.
section_name: Funding

section_name: Data availability

section_content: The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article. All data are available at Dryad (https:// datad ryad. org/ stash/ share/ hzwhR Uy0u5 DTwk-CK4tc RFq1o 54oru MyWXp qJaPQ eUU; https:// doi. org/ 10. 5061/ dryad. 05qft tf6c). Raw RNA-seq data have been deposited in NCBI's Gene Expression Omnibus 71 and are available through GEO Series accession number GSE217724 (https:// www. ncbi. nlm. nih. gov/ geo/ query/ acc. cgi? acc= GSE21 7724).
section_name: Data availability

section_name: Author contributions

section_content: Y. H. carried out all experiments, analyzed the data, documented the data and prepared the figures, and wrote the manuscript. H. B. conceived the general outline of the project, acquired funding, supervised the project, and wrote the manuscript.
section_name: Author contributions

section_content: The authors declare no competing interests.
section_name: Competing interests