Abstract
In mammals, circadian clocks are strictly suppressed during early embryonic stages as well as pluripotent stem cells, by the lack of CLOCK/BMAL1 mediated circadian feedback loops. During ontogenesis, the innate circadian clocks emerge gradually at a late developmental stage, then, with which the circadian temporal order is invested in each cell level throughout a body. Meanwhile, in the early developmental stage, a segmented body plan is essential for an intact developmental process and somitogenesis is controlled by another cell-autonomous oscillator, the segmentation clock, in the posterior presomitic mesoderm (PSM). In the present study, focusing upon the interaction between circadian key components and the segmentation clock, we investigated the effect of the CLOCK/BMAL1 on the segmentation clock Hes7 oscillation, revealing that the expression of functional CLOCK/BMAL1 severely interferes with the ultradian rhythm of segmentation clock in induced PSM and gastruloids. RNA sequencing analysis showed that the premature expression of CLOCK/BMAL1 affects the Hes7 transcription and its regulatory pathways. These results suggest that the suppression of CLOCK/BMAL1-mediated transcriptional regulation during the somitogenesis may be inevitable for intact mammalian development.
Introduction
The circadian clock is the cell-autonomous time-keeping system generating the orderly regulated various physiological functions, which enables cells, organs, and systems to adapt to the cyclic environment of the rotating Earth (1-5). The core architecture of the circadian molecular clock consists of negative transcriptional/translational feedback loops (TTFLs) composed of a set of circadian clock genes, including Bmal1, Clock, Period (Per1, 2, 3), and Cryptochrome (Cry1, 2), functioning under the control of E-box elements (2, 6). The kernel of TTFLs is composed of heterodimerized CLOCK/BMAL1 key transcriptional factors that positively regulate the circadian output genes, as well as Per and Cry genes via E-box. PERs and CRYs inhibit CLOCK/BMAL1 transcriptional activity, and the negative feedback loops between these genes generate oscillations of approximately 24 hr.
In mammalian development, it has been demonstrated that early embryos and pluripotent stem cells have no apparent circadian molecular oscillations (7-11), whereas the innate circadian clock develops during ontogenesis and is established at a late developmental stage (12-15). Regarding the mechanisms regulating circadian clock development, using an in vitro model of embryonic stem cell (ESC) differentiation and mouse embryos, it was shown that prolonged posttranscriptional mechanisms, such as suppressed translation of CLOCK protein and predominant cytoplasmic localization of PER proteins, inhibit the establishment of the circadian TTFL cycle (16-18). Although it was revealed that the multiple mechanisms strictly suppress the circadian molecular clock in the undifferentiated cells and early stage embryos, the biological and physiological significance of the delayed emergence of circadian clock oscillation in mammalian embryos has been unknown.
In the early developmental stages, a segmented body plan is essential for an intact developmental process. Somitogenesis is related to another cell-autonomous oscillator, the segmentation clock, in the posterior presomitic mesoderm (PSM) (19, 20). The mouse segmentation clock is underlain by a negative feedback loop involving Hes7 oscillation (21, 22). HES7 is a key transcriptional factor that represses its own expression and oscillates through a negative feedback loop in a period of 2–3 hr in mouse and 4–5 hr in humans. The NOTCH, WNT, and fibroblast growth factor signaling pathways are involved in the regulation of the Hes7 oscillator and its intercellular synchronization (20). In mammals, two different types of rhythm sequentially emerge during the developmental process, however, there is a lack of knowledge about the biological significance of the rhythm conversion during development.
In this study, focusing on the relationship between circadian clock and segmentation clock, we investigated the effect of the premature expression of CLOCK/BMAL1 on the segmentation clock oscillation, and revealed severe interference with the ultradian rhythm of segmentation clock in iPSM and gastruloids. RNA sequencing analysis showed that CLOCK/BMAL1 affects the Hes7 transcription and its regulatory pathways. These findings highlight that the suppression of functional CLOCK/BMAL1, which leads to arrest the circadian clock oscillation, during the early to mid-developmental stage may be inevitable for the intact process of mammalian embryogenesis.
Results
A circadian clock gene, Per1, and a segmentation clock gene, Hes7, are adjacent genes in the mammalian genome
In mammals, the temporal relationship between the segmentation clock and circadian clock appears to be mutually exclusive (Fig. S1) (12-15, 19, 20). To explore the functional interaction between these two biological rhythms with different frequencies during the developmental process, we focused on the genomic architecture of genes comprising circadian and segmentation clocks, respectively. Intriguingly, one of the core circadian clock genes, Per1, is physically adjacent to an essential component of the segmentation clock, Hes7, in a genomic region conserved in higher vertebrates, including mice and humans. The Per1 homolog Per2 is adjacent to the Hes7 homolog Hes6 in the genome (Fig. 1A). Since Hes7 exhibits the essential characteristics of a segmentation clock (23) and neighboring genes can influence the expressions with each other during somitogenesis in zebrafish (24), we focused on the effect of the regulation mechanism of the circadian clock on segmentation clock oscillation. Therefore, we investigated the effect of the CLOCK/BMAL1-mediated activation of Per1 transcription on the segmentation clock oscillation in induced presomitic mesoderm (iPSM), an in vitro recapitulating model of a segmentation clock, using ESCs carrying the Hes7-promoter-driven luciferase reporter (pHes7-luc) (25) (Fig. 1B). In the iPSM, the pluripotent markers have not yet been down-regulated sufficiently as previously reported (25), and the iPSM differentiated from Per2Luc ESCs showed no apparent circadian clock oscillation (Fig. S2A and B). In addition, the immunostaining pattern of CLOCK, BMAL1, and PER1 in the iPSMs was quite similar to that in the undifferentiated ESCs (Fig. S2C) (16, 17), confirming that the circadian TTFL was not established and circadian clock oscillation was also strictly suppressed in the iPSM by the common inhibitory molecular mechanisms to the undifferentiated ESCs.
Because CLOCK/BMAL1 is key transcription regulator of circadian TTFL, and the expression of CLOCK protein is suppressed post-transcriptionally in iPSM as well as ESCs and early embryos (17), we established two ESC lines carrying both the doxycycline (dox)-inducible Clock and Bmal1 genes (Fig. 1C, Fig. S3). In iPSM differentiated from ESCs, the expression of both Clock/Bmal1 mRNA and CLOCK/BMAL1 proteins was confirmed after the addition of dox (Fig. 1 D and E), and we found that overexpression of both Clock and Bmal1 successfully activated the expression of core clock genes (Fig. 2A). As the dominant negative mutant of Bmal1 (Bmal1DN) (26) co-expressed with Clock did not activate the Per1/2 and Cry1/2 genes, we concluded that CLOCK/BMAL1 specifically activated the expression of these clock genes via an E-box (Fig. 1 C–E, 2A). We then examined the expression of genes in Hes7, which is proximal to Per1, and Hes6, which is proximal to Per2 in the genome. The expression of Clock/Bmal1 induced by dox in the iPSM induced significant upregulation of the expression of the Hes7, but not Hes6, gene (Fig. 2B). Similarly, we also observed the upregulation of Hes7 expression by Clock/Bmal1 induction in the undifferentiated ESCs (Fig. S4 A and B). These results indicate that the circadian components CLOCK/BMAL1 also affect the segmentation clock gene Hes7, as well as Per1.
Inhibition of Hes7 ultradian rhythm by CLOCK/BMAL1 in iPSM
We next performed a functional analysis using the in vitro recapitulation model of a segmentation clock oscillation in iPSM (25). The oscillations in bioluminescence from pHes7-luc reporters were observed using a photomultiplier tube device (PMT) and an EM-CCD camera (Fig. 3A). We confirmed an oscillation of Hes7-promoter-driven bioluminescence with a period of approximately 2.5–3 hr in control iPSM with or without dox using PMT and the EM-CCD camera (Fig. 3 B and C). Traveling waves of pHes7-luc bioluminescence were observed, indicating that the segmentation clock oscillation in iPSM was successfully recapitulated, consistent with a previous report (25). Using this iPSM-based segmentation clock system, we investigated the effect of Clock/Bmal1 expression on Hes7-promoter-driven oscillation. The expression of Clock/Bmal1 genes (Dox+) resulted in defects of the oscillation in Hes7 promoter activity, whereas pHes7-luc bioluminescence continued to oscillate under Dox– conditions. Oscillation of the segmentation clock was observed even during the induction of Clock/Bmal1DN (Fig. 3D), indicating that the CLOCK/BMAL1-mediated mechanism interfered with the transcriptional oscillation of Hes7. A traveling wave of Hes7 promoter activity disappeared with the expression of Clock/Bmal1 (Fig. 3 E and F), and dox-dependent arrest of pHes7-luc traveling wave (Fig. 3 G and H) clearly demonstrated the CLOCK/BMAL1-mediated interference with Hes7-driven segmentation clock oscillation in iPSM.
Interference with somitogenesis-like segmentation by induction of CLOCK/BMAL1 in gastruloids
In addition, to explore the effect of CLOCK/BMAL1 expression on somitogenesis, we established the ESC-derived embryonic organoids, gastruloids, recapitulating an embryo-like organization, including somitogenesis-like process in vitro (27) (Fig. 4A). The pHes7-luc bioluminescence represented a traveling wave accompanied by the formation of segment-like structures with anteroposterior polarity, in which the gastruloids were stained with stripes of a somite marker, Uncx4.1, by in situ hybridization (Fig. 4 B–D). Only dox treatment in control gastruloids induced no change in the pHes7-luc bioluminescence oscillation and somitogenesis-like process (Fig. 4 E–G). The dox-inducible Clock/Bmal1 ESC line carrying pHes7-luc was differentiated in vitro into gastruloids and produced the somitogenesis-like process without dox (Fig. 4 H–J). In contrast, the dox-dependent induction of Clock/Bmal1 expression in the gastruloids interrupted the pHes7-luc oscillation and disrupted the somitogenesis-like structures (Fig. 4 K–M). In gastruloids, the expression of both Clock/Bmal1 mRNA was confirmed after the addition of dox (Fig. S5). These results suggest that the premature expression of the circadian key transcriptional regulator CLOCK/BMAL1 critically interferes with not only Hes7 oscillation, but also somitogenesis.
CLOCK/BMAL1-mediated interference in Hes7 regulatory network
Next, to examine the perturbation mechanisms of the segmentation clock oscillation by the circadian components CLOCK/BMAL1, we analyzed the RNA sequencing (RNA-seq) data obtained from the total RNA of iPSM colonies. We extracted 509 upregulated and 88 downregulated differentially expressed genes (DEGs) after the induction of Clock/Bma11 gene expression in iPSM colonies (Fig. 5A). A KEGG pathway enrichment analysis for the DEGs revealed enrichment of the WNT, MAPK, and NOTCH signaling pathways related to Hes7 oscillation (28) (Fig. 5B). Almost all other ranked pathways also included the WNT, MAPK, and NOTCH signaling pathway-related genes (Fig. 5B). Similarly, enrichment of the WNT, MAPK, and NOTCH signaling pathways by Clock/Bmal1 induction was also observed in the undifferentiated ESCs (Fig. S6 A and B). These findings indicate that the expression of CLOCK/BMAL1 affects the Hes7-related signaling pathways, which interferes with the feedback loop regulating Hes7 oscillation. Intriguingly, in addition to Hes7 gene expression, the expressions of Aloxe3 in iPSM and Aloxe3 and Vamp2 in ESCs, the other contiguous genes with Per1, were upregulated with the induction of Clock/Bmal1 expression, and this result was confirmed by quantitative RT-PCR (qPCR) (Fig. 5 C–E, Fig. S6 C–E) suggesting that forced expression of CLOCK/BMAL1 also affects a wide region around the Hes7 gene locus on the same chromosome. These results suggest that the premature expression of the circadian components CLOCK/BMAL1 interfered with Hes7 oscillation and somitogenesis by perturbing the Hes7 expressions through indirect regulatory pathways (Fig. 5F). Because the loss of the Hes7 ultradian expression rhythm in the mouse cause segmentation defects (22, 29), the oscillatory expression of Hes7 is essential for mammalian development. Therefore, the results in this study suggest that it may be imperative that CLOCK/BMAL1 function is suppressed until the completion of segmentation and other related developmental events.
Discussion
Our present study showed that premature expression of circadian key components CLOCK/BMAL1 severely interferes with the ultradian rhythm of the segmentation clock in iPSM and gastruloids.
We have previously reported that during the early to mid-developmental stage, there are multiple molecular mechanisms that underlie the strict suppression of circadian TTFLs, such as the post-transcriptional suppression of CLOCK protein (17, 18) and the exclusive cytoplasmic localization of PER proteins (16). Furthermore, we have also reported that the maternal circadian clock cannot entrain the fetus until the establishment of the fetal circadian clock itself (17). These results suggest that the circadian rhythm in mammalian embryos is rigorously suppressed by the multilayered inhibitory mechanisms during the early to mid-developmental stage. During the multilayered suppression of circadian clock oscillation, the ultradian temporal oscillation of Hes7 expression, segmentation clock, proceeds and forms the spatial repetitive structure of somites.
In the present study, we investigated the effect of the CLOCK/BMAL1-mediated activation of Per1 transcription on the segmentation clock oscillation by using the iPSM differentiated from ESCs. It was suggested that, similar to the undifferentiated ESCs, circadian clock oscillation is suppressed in the iPSM by the common mechanisms to the ESCs and early embryos (see Fig. S2). Recently, it was reported that hundreds of genes including Per1 also oscillates in the same phase as Hes7 ultradian rhythm in in vitro-PSM of both mouse and humans (30), suggesting that Per1 is deviated and free from the circadian gene regulatory mechanism of TTFL. These findings are consistent with the previously reported observations indicating that the multilayered inhibitory mechanisms including post-transcriptional inhibition of CLOCK and the predominant cytoplasmic accumulation of PER1 do not allow the oscillation of circadian TTFL (17, 18). Interestingly, although the expression of BMAL1 protein was observed even in ESCs (17), the dox-induced CLOCK sole expression in ESCs resulted in the only partial upregulation of E-box driven circadian clock genes (Fig. S3), raising the possibility that the endogenously expressed BMAL1 might be post-translationally modified to not function. Therefore, in this study, we used ESC lines carrying both the dox-inducible Clock and Bmal1 genes as a model system of premature expression of CLOCK/BMAL1 (see Fig. 1C).
We demonstrated that the expression of CLOCK/BMAL1 affected the WNT, MAPK, and NOTCH signaling pathways related to Hes7 oscillation in iPSM (see Fig. 5 A and B). In addition, the premature expression of CLOCK/BMAL1 resulted in not only the up-regulation of Per1 expression but also the expressions of Hes7, Aloxe3, and Vamp2, localized adjacently on the Per1 genomic locus (see Fig. 2 A and B, Fig. 5 C–E, Fig. S6 C–E). In the iPSM, the up-regulation of these gene expressions has already been induced after the 2-hour dox treatment (see Fig. 2 A and B, Fig. 5 C–E). Considering that the Per1 promoter harbors E-box elements with which CLOCK/BMAL1 heterodimer has a much higher affinity than the other genomic region (31), the immediate up-regulation of genes near Per1 gene locus after the induction of CLOCK/BMAL1 expressions could be caused by the ripple effect (32). On the other hand, the bioluminescence from Hes7-promoter driven luciferase reporters in the iPSMs not only lost cycling but also decreased signal intensity in approximately 2 hours after the dox addition (see Fig. 3D), indicating that the expression of CLOCK/BMAL1 in the iPSM has also inhibitory effects on the Hes7 gene expressions. Among components involved in the Hes7-regulatory signaling pathways, expression of CLOCK/BMAL1 induced some negative regulators, such as the Dusp phosphatase family (33) in the MAPK signaling pathway, Sfrp in the WNT signaling pathway (34), and Lfng in the NOTCH signaling pathway (35) (see Fig. 5B). Therefore, the premature expression of CLOCK/BMAL1 first may upregulate Hes7 transcription and induce subsequent downregulation of Hes7 gene expression by the induction of the negative regulators in addition to the HES7 autoinhibition. Consequently, the premature expression of the circadian components CLOCK/BMAL1 interfered with Hes7 oscillation by perturbing the Hes7 expression through various pathways.
In this study, we used a mouse embryonic organoid, gastruloids, as an in vitro recapitulation model of somitogenesis-like process (27). The premature expression of CLOCK/BMAL1 in the gastruloids disrupted not only the Hes7 oscillation but also the striped structure of the somite marker, Uncx4.1 (see Fig. 4M). Because the RNA-seq analysis data showed that hundreds of genes were affected by the induction of CLOCK/BMAL1 (see Fig. 5A), the possibility cannot be denied that the premature expression of CLOCK/BMAL1 affects cell fates or characters. However, the posterior structure in the gastruloids was held even after the induction of CLOCK/BMAL1 and then continued to extend, concomitant with the decrease of Hes7 bioluminescence signals and the arrest of the Hes7 oscillation (see Fig. 4K), suggesting that the premature expression of CLOCK/BMAL1 interfered with the somitogenesis process by perturbing Hes7 oscillation of the segmentation clock.
In vitro recapitulation of embryonic process using iPSM and gastruloids has differences such as no brain tissues comparing with in vivo process. However, key regulators of somitogenesis we focus on in this study are expressed similarly between embryos and gastruloids using single-cell RNA sequencing and spatial transcriptomics (27), and the in vitro recapitulation model enables to analyze the Hes7 oscillation in more detail using real-time imaging without maternal effects.
Our findings shown in this study indicated that the CLOCK/BMAL1, key components regulating the circadian TTFL, affected and interfered with the segmentation clock. Considering that transcriptional activation of CLOCK/BMAL1 is essential for the circadian regulatory networks, these results suggest that the strict suppression of circadian molecular oscillatory mechanisms during the early stage embryos is inevitable for the intact developmental process in mammals. Therefore, this may be the biological and physiological significance of the delayed emergence of circadian clock oscillation and the rhythm conversion observed in mammalian development.
Materials and Methods
Cell culture
KY1.1 ESCs (7), referred to as ESC in the text, and Per2Luc ESCs (5, 36) were maintained as described previously (17). E14TG2a ESCs carrying Hes7-promoter-driven luciferase reporters (25), referred to as pHes7-luc ESCs in the text, were maintained without feeder cells in DMEM (Nacalai) supplemented with 15% fetal bovine serum (Hyclone), 2 mM L-glutamine (Nacalai), 1 mM nonessential amino acids (Nacalai), 100 µM StemSure® 2-mercaptoethanol solution (Wako), 1 mM sodium pyruvate (Nacalai), 100 units/mL penicillin and streptomycin (Nacalai), 1000 units/mL leukemia inhibitory factor (Wako), 3 µM CHIRON99021 (Wako or Tocris Biosciences), and 1 µM PD0325901 (Wako) with 5% CO2 at 37°C.
Transfection and establishment of cell lines
ESCs stably expressing dox-inducible Clock/Bmal1 or Clock/Bmal1DN (I584X) were established as described previously (17). For TetO-Clock/Bmal1 or TetO-Clock/Bmal1DN ESCs, KY1.1 ESCs or pHes7-luc ESCs were transfected using 10.5 µl of FuGENE 6 mixed with 1 µg of pCAG-PBase, 1 µg of PB-TET-Clock (17), 1 µg of PB-TET-Bmal1 or PB-TET-Bmal1DN (I584X), 1 µg of PB-CAG-rtTA Adv, and 0.5 µg of puromycin selection vector. The transfected cells were grown in a culture medium supplemented with 2 µg/mL puromycin for two days. The ESC colonies were picked and checked by qPCR after treatment with 500 ng/mL dox. For PB-TET-Bmal1 and PB-TET-Bmal1DN (I584X), Bmal1 cDNA and Bmal1DN (I584X) cDNA (26) were cloned into a PB-TET vector (37). For the TetO-Clock Per2Luc ESCs, Per2Luc ESCs were established as described previously (17).
Bioluminescence imaging
The iPSM colonies were differentiated from the pHes7-luc ESCs and Per2Luc ESCs as described previously (25). The Per2Luc ESCs were cultured without feeder cells in the ES medium containing 3 µM CHIRON99021 and 1 µM PD0325901 before in vitro differentiation. Bioluminescence imaging of single pHes7-luc iPSM colonies and Per2Luc iPSM colonies was performed in gelatin-coated 24-well black plates or 35-mm dishes (26). DMEM was used that was supplemented with 15% Knock-out Serum Replacement (KSR), 2 mM L-glutamine, 1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 units/mL penicillin and streptomycin, 0.5% DMSO, 1 µM CHIRON99021, and 0.1 µM LDN193189 (Sigma) containing 1 mM luciferin and 10 mM HEPES. For live imaging of single iPSM colonies using an EM-CCD camera, each iPSM colony was cultured on a fibronectin-coated glass base dish for 6 h, and images were acquired every 5 min with an exposure time of 10 sec (control) or 2.5 sec (Clock/Bmal1 induction) under 5% CO2 using an LV200 Bioluminescence Imaging System (Olympus).
Gastruloids were generated as described in a previous report (27). In total, 200–250 live cells were plated in 40 µl of N2B27 medium into each well of a U-bottomed nontissue culture-treated 96-well plate (Greinier 650185). After a 96-hr cultivation, the gastruloids were embedded in 10% Matrigel (Corning 356231) containing 1 mM luciferin. For live imaging of single gastruloids, the images were acquired every 5 min with an exposure time of 3.5 sec (Clock/Bmal1 induction) or 10 sec (control) under 5% CO2 using the LV200 system. The Videos were analyzed using the ImageJ software (38). Kymographs of the averaged bioluminescence intensity along the straight or segmented line of 5-pixel width were generated using the plug-in KymoResliceWide.
In situ hybridization
Hybridization chain reaction (HCR) v3 was performed as described previously (27, 39) using reagents procured from Molecular Instruments. Uncx4.1 HCR probe (Accession NM_013702.3, hairpin B1) was labeled with Alexa Fluor 488.
Quantitative RT-PCR
The iPSM colonies, ESCs, and gastruloids were washed with ice-cold PBS, and total RNA was extracted using Isogen reagent (Nippon Gene) or miRNeasy Mini Kits (QIAGEN) according to the manufacturer’s instructions. To remove the feeder cells from ESCs cultured on a feeder layer, the cells were treated with trypsin, and then the mixed cell populations were seeded on gelatin-coated dishes and incubated for 25 min at 37°C three times in ES cell medium. Non-attached ESCs were seeded in a gelatin-coated dish overnight and then treated with or without 500 ng/mL doxycycline for 6 hr. The iPSM colonies and gastruloids were treated with or without 1000 ng/mL doxycycline for 2 hr. First-strand cDNAs were synthesized with 1000 or 280 ng of total RNA using M-MLV reverse transcriptase (Invitrogen) according to the manufacturer’s instructions. Quantitative PCR analysis was performed using the StepOnePlus™ Real-Time PCR system (Applied Biosystems) and iTaq™ Universal SYBR Green Supermix (Bio-Rad Laboratories). Standard PCR amplification protocols were applied, followed by dissociation-curve analysis to confirm specificity. Transcription levels were normalized to the level of β-actin. The following primer sequences were used:
RNA-seq
The iPSM colonies and ESCs were washed with ice-cold PBS, and total RNA was extracted using miRNeasy Mini Kits (QIAGEN) according to the manufacturer’s instructions. Total RNA sequencing was conducted by Macrogen Japan on an Illumina NovaSeq 6000 with 101-bp paired-end reads. After trimming the adaptor sequences using Trimmomatic (40), the reads that mapped to ribosomal DNA (GenBank: BK000964.1) (41) were filtered out and the sequence reads were mapped to the mouse genome (GRCm38/mm10) using STAR (42), as described previously (16). To obtain reliable alignments, reads with a mapping quality of less than ten were removed using SAM tools (43). The known canonical genes from GENCODE VM23 (44) were used for annotation, and the reads mapped to the gene bodies were quantified using Homer (45). The longest transcript for each gene was used for gene-level analysis. We assumed that a gene was expressed when there were more than 20 reads mapped on average to the gene body. Differential gene expression in the RNA-seq data was determined using DESeq2 with thresholds of FDR < 0.05, fold change > 1.5, and expression level cutoff > 0.1 FPKM (46). WebGestalt was used for KEGG pathway enrichment analysis (47). In the RNA-seq data using iPSM colonies, the reads mapped in the promoter (chr11:69115096-69120473) and 3′UTR (chr11:69122995-69123324) of Hes7 were filtered out to eliminate transcripts from the pHes7-luc reporter transgene. The heatmaps of gene expression and KEGG pathways were generated with R using the pheatmap and pathview packages, respectively.
Immunostaining
The iPSM colonies were fixed in cold methanol for 15 min at room temperature. The fixed iPSM was blocked with 1% BSA or 5% skim milk overnight at 4°C and then incubated with anti-CLOCK mouse antibody (CLSP4) (48), anti-BMAL1 mouse antibody (MBL, JAPAN), anti-BMAL1 guinea pig antibody (16), or anti-PER1 rabbit antibody (AB2201, Millipore) overnight at 4°C. After washing in 1% BSA, the iPSM colonies were incubated with a CF™488A-conjugated donkey anti-mouse IgG (Nacalai), Cy3-conjugated goat anti-guinea pig IgG (Jackson), DyLight™488-conjugated donkey anti-rabbit IgG (Jackson) for 2 hr at 4°C, and the nuclei were stained with TO-PRO®-3 1:1000 (Thermo Fisher Scientific, USA) for 10–20 min. The iPSM colonies were washed in 1% BSA and observed using an LSM510 or 900 confocal laser scanning microscope (Zeiss).
Data availability
RNA sequence data are available at the Gene Expression Omnibus. All other datasets generated in this study are available from the corresponding author upon reasonable request.
Supporting information
Acknowledgments
We thank the Yagita lab members for technical assistance. This work was supported in part by grants-in-aid for scientific research from the Japan Society for the Promotion of Science to Y.U. (19K06679) and K.Y. (18H02600), the Cooperative Research Program (Joint Usage/Research Center program) of the Institute for Frontier Life and Medical Sciences, Kyoto University (K.Y. and G.K.)
Footnotes
Competing Interest Statement: The authors declare no competing interests