The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells

Analysis of the role of the molecular clock protein Bmal1 in pluripotent embryonic stem cells reveals a novel function of circadian proteins in cell differentiation and early embryo development.


Introduction
The earth rotates around its own axis with a 24-h period that generates repetitive changes in the intensity of sunlight reaching our planet. Organisms that live on the surface of the earth have developed mechanisms to optimize their physiology to this light-dark cycle. Circadian pacemakers allow to carry out an approximate measurement of time, so their phase must be adjusted daily to keep the internal clock in perfect synchrony with external signals. The main circadian synchronizer in mammals is light that is received in specialized photoreceptor cells in the retina, and the information is transmitted directly to the suprachiasmatic nucleus in the brain that synchronizes peripheral clocks through humoral signals such as hormones . Most of cells of the adult organism have their own internal clock that needs to be synchronized to be in the same circadian phase as the rest of the body and, therefore, facilitate optimal physiological functioning (Mohawk et al, 2012). Circadian regulation relies on the activity of the molecular clock that mediates the establishment of an autoregulatory loop that generates daily oscillations in the expression of target genes (Takahashi, 2017). This machinery is composed by the core Clock and Bmal1 (also known as Arntl, aryl hydrocarbon receptor nuclear translocator-like) heterodimer that activates transcription of their own negative regulators Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) genes. The molecular clock can regulate up to 10% of cellular transcripts in a tissue-specific way (Storch et al, 2002;Masri & Sassone-Corsi, 2010).
The function of the molecular clock during mammalian embryonic development is poorly understood (Seron-Ferre et al, 2012;Landgraf et al, 2014). Some components of the molecular clock are expressed during embryo development, but they do not generate consistent circadian fluctuations in embryo tissues until late stages of development when the suprachiasmatic nucleus is formed and the embryo is exposed to sunlight (Seron-Ferre et al, 2012;Landgraf et al, 2014;Umemura et al, 2017). In agreement, germline cells, zygotes, preimplantation embryos, and mouse embryonic stem cells (mESCs) derived from the developing blastocyst express components of the molecular clock but do not display circadian oscillations (Alvarez et al, 2003;Morse et al, 2003;Amano et al, 2009;Yagita et al, 2010). Importantly, despite mutant embryos lacking Bmal1 or other components of the molecular clock proceed through embryogenesis with no apparent phenotype at birth (van der Horst et al, 1999;Zheng et al, 2001;Kondratov et al, 2006;DeBruyne et al, 2007), recent evidence highlights that the lack of Bmal1 during embryo development is responsible for reduced life span, body weight, and fertility observed during the adult life in Bmal1−/− mice .
In this study, we have used naïve mESCs as a model system to analyse the molecular function of Bmal1 during early mouse development. We found that Bmal1−/− mESCs are viable and express pluripotency-associated markers. Bmal1−/− mESCs display increased expression of the pluripotency factor Nanog coupled to the mis-regulation of pluripotency signalling pathways. Importantly, the lack of Bmal1 protein in mESCs does not block pluripotency exit but perturbs the execution of multi-lineage cell differentiation as assayed using in vivo and in vitro cell differentiation systems. Our observations reveal a novel role of Bmal1−/− in the regulation of pluripotent cell differentiation and suggest that components of the molecular clock regulate mammalian development through molecular mechanisms that remain to be characterized.

Results
Bmal1 is expressed in pluripotent cells mESCs do not show circadian oscillation of clock genes, but these can be induced by in vitro differentiation towards mouse neural stem cells (mNSCs) (Yagita et al, 2010). Reciprocally, circadian oscillations in mNSCs are extinguished when they are artificially reprogrammed to pluripotency (Yagita et al, 2010). We asked whether the onset of circadian oscillations during mESCs differentiation was associated with differences in Bmal1 expression levels. Colonies of mESCs growing in FBS and leukemia inhibitory factor (LIF) (mESCs FBS + LIF or primed serum mESCs) were differentiated into mNSCs that showed the typical fusiform shape morphology and grew as a reticular network ( Fig 1A). mNSC cultures showed expected down-regulation of pluripotency genes Nanog, Oct4, and Rex1 coupled to up-regulation of mNSC genes Fabp7, Slc1a3, and Nestin ( Fig 1B). Immunofluorescence analysis confirmed that NSC cultures do not express the nuclear pluripotencyassociated transcription factor Oct4 and showed homogeneous staining of the NSC protein marker Nestin in their cytoplasms ( Fig  1C). Comparison of mRNA expression level in primed serum mESCs and NSCs showed that Bmal1 is expressed at a similar level in both cell types ( Fig 1D). Expression of Bmal1 was similar to the transcriptionally active housekeeping gene Hmbs, indicating that Bmal1 is expressed at functional levels in mESCs ( Fig 1D). Western blot analysis using anti-Bmal1 antibodies revealed two bands at 71 kD that correspond to previously described phosphorylated and unphosphorylated isoforms of Bmal1 (Yoshitane et al, 2009;Sahar et al, 2010) in mNSCs ( Fig 1E). Importantly, primed serum mESCs and mNSCs expressed similar levels of Bmal1 protein (Figs 1E and S1A), suggesting that Bmal1 might be functional in pluripotent cells.
To discard that expression of Bmal1 protein was associated to marginal spontaneous cell differentiation and cell heterogeneity typically found in primed serum mESC cultures (Li & Izpisua Belmonte, 2018), we analysed expression of Bmal1 in naïve pluripotent cells. Primed serum mESCs were transferred to defined media with LIF and inhibitors of MAPK and glycogen synthase kinase 3 (2i + LIF media), to establish cultures of naïve 2i pluripotent cells (Ying et al, 2008). Expression of Bmal1 was similar and slightly increased in naïve 2i mESCs as compared with primed serum mESCs, at the level of both mRNA and protein (Figs 1F and G and S1B), further indicating that Bmal1 is expressed at a functional level in pluripotent cells. To confirm that expression of Bmal1 in pluripotent mESCs is not an artefact of in vitro cell culture conditions, we analysed whether Bmal1 is also transcribed in pluripotent cells in vivo. We interrogated published single-cell messenger RNA high-throughput sequencing (mRNA-seq) datasets of developing preimplantation embryos (Deng et al, 2014) and found a heterogeneous pattern of Bmal1 expression at all stages from four cells to late blastocyst ( Fig 1H). Importantly, cells that express a higher level of the pluripotency gene Oct4 also tend to express higher levels of Bmal1 mRNA at the blastocyst stage (Fig 1I), demonstrating that Bmal1 mRNA is expressed in pluripotent cells in vivo.
Last, cell fractionation followed by Western blot experiments showed that distribution of Bmal1 protein in mESCs is similar to its distribution in NSCs (nucleus and cytoplasm) (Figs 1J and S1C), suggesting that the absence of circadian oscillations in ESCs is not a consequence of the exclusion of Bmal1 from the nucleus of pluripotent cells. Taken together, these results solidly demonstrate that albeit mESCs do now display circadian oscillations (Yagita et al, 2010), the molecular clock protein Bmal1 is present in pluripotent cells.
Bmal12/2 mESCs remain undifferentiated and express a higher level of Nanog protein To investigate the role of Bmal1 protein in pluripotent cells, we derived Bmal1−/− mESCs using CRISPR/Cas9 and grew them in the 2i + LIF medium. The amino terminus of Bmal1 was targeted (exon 5) (Fig 2A), and a panel of genetic clonal mESC lines were screened and genotyped. The frequency of Bmal1 mutants in one or both alleles was high (four heterozygous [Bmal1+/−] and four compound heterozygous [Bmal1−/−] mutants of 12 screened clonal colonies), suggesting that Bmal1 is not essential to establish undifferentiated mESC cultures. Of the four Bmal1−/− clonal cell lines, we focused on one in which both alleles contained DNA indel mutations (−22 and +61 nucleotides, respectively) at expected target sites ( Fig 2B) and resulted in frame shifts and premature stop codons at the very beginning of Bmal1 open reading frame, impeding the production of peptides that include any of the annotated functional domains included in Bmal1 protein (BHLH, PAS1, PAS2, and TAD). Bmal1−/− mESCs formed typically tight three-dimensional colonies similar to the ones generated by parental wild-type cells ( Fig 2C). As expected, expression of Bmal1 mRNA was not detected in Bmal1−/− mESCs compared with wild-type cells (Fig 2D), and Western blot analysis indicated that Bmal1−/− mESCs do not express detectable levels of Bmal1 protein (Fig 2E). Bmal1−/− mESC cultures remain undifferentiated because they retained expression of pluripotency-associated transcription factors Oct4, Nanog, and Rex1 ( Fig 2F). Interestingly, we found that Bmal1−/− mESCs express even higher levels of Nanog mRNA and protein than parental wild-type cells (Figs 2F-I and S1D). Taken together, these results demonstrate that Bmal1 protein is not essential for mESCs self-renewal, and that its depletion results in upregulation of Nanog expression in naïve 2i mESCs.
Bmal12/2 mESCs display altered expression of signalling pathway genes and cell differentiation potential To further understand the role of Bmal1 in pluripotent cells, we compared mRNA expression in Bmal1−/− and wild-type naïve 2i mESCs by mRNA-seq. 854 gene transcripts were differentially expressed (up-or down-regulated by more than twofold and adjusted P-value < 0.05) in cells that lack Bmal1 as compared with parental wild-type cells ( Fig 3A). Unexpectedly, 689 of these transcripts (80.6%) were up-regulated in Bmal1−/− cells, suggesting that, in contrast to the role of Bmal1 in promoting transcription of target genes in adult tissues (Takahashi, 2017), Bmal1 is not acting as a transcriptional activator in pluripotent mESCs. Most of the upregulated genes in Bmal1−/− mESCs were already transcriptionally active in wild-type cells (Fig 3B), suggesting that Bmal1 negatively modulates transcription of active genes. Functional categories analysis revealed that Bmal1−/− mESCs display mis-regulation of genes encoding for 180 glycoproteins and 59 developmental proteins, suggesting that Bmal1 is involved in the regulation of cell communication and development ( Fig 3C). In fitting, gene ontology analysis showed that depletion of Bmal1 results in deregulation of genes involved in cell differentiation and organism development (Fig 3D), and pathway enrichment analysis exposed that Bmal1−/− mESCs display altered regulation of pluripotency-associated signalling pathways including phosphoinositide 3-kinase (PI3K), MAPK, and Wnt ( Fig 3E). In agreement with the general trend towards   Fig S2A). In addition, analysis of components of signalling pathways deregulated in Bmal1−/− mESCs confirmed widespread abnormal expression patterns of mRNA transcripts involved in pluripotency signalling and cell differentiation including the MAPK pathway (Figs 3F and S2B). MAPK pathway, also known as extracellular signal-regulated kinases (Erk1/2) pathway, inhibits Nanog expression in mESCs (Wray et al, 2010), and thus, we wondered whether deregulation of this pathway underlie the up-regulation of Nanog protein found in Bmal1−/− mESCs (Fig 2F-I). However, expression of Erk1/2 protein was unaltered in Bmal1−/− mESCs in both primed serum and naïve 2i conditions (Fig S2C), and mRNA expression of Erk1/2 targets (Hamilton & Brickman, 2014) was not affected by depletion of Bmal1 protein (Fig S2D and E), suggesting that up-regulation of Nanog protein is not a consequence of reduced Erk1/2 signalling in Bmal1−/− mESCs.
We reasoned that up-regulation of Nanog protein and misregulation of signalling components might hinder the multilineage differentiation capacity of Bmal1−/− mESCs. We injected Bmal1−/− and wild-type mESCs into immunodeficient mice and assayed their ability to form teratomas and differentiate into the three germ layers. As expected, imaging analysis of teratomas using haematoxylin and eosin staining confirmed that wild-type mESCs can differentiate into tissues derived from the three germ layers. For example, we could detect skin (ectoderm), glandular tissue (endoderm), and smooth muscle (mesoderm) (Fig 3G). Bmal1−/− teratomas also contained tissues derived from the three germ layers, suggesting that the lack of Bmal1 does not fully block differentiation towards any the of three germ layers in vivo (Fig 3G). To complement this analysis using a quantitative approach, we performed immunohistochemistry using antibodies against glial fibrillary acidic protein (Gfap), cytokeratin, and smooth muscle actin that are well-established markers of ectoderm, endoderm, and mesoderm tissue respectively. Importantly, teratomas formed by Bmal1−/− cells displayed reduced labelling for Gfap, cytokeratin, and smooth muscle actin markers compared with wild-type cells ( Fig  3H and I), indicating that although Bmal1−/− cells can produce tissue derived from the three germ layers, their differentiation potential is altered compared with wild-type cells.

Bmal1 is required for the formation of gastrula-like organoids
To further examine the altered differentiation capacity of Bmal1−/− mESCs, we analysed their ability to differentiate and produce gastrula-like organoids (gastruloids) in vitro (Beccari et al, 2018). Bmal1−/− and wild-type mESCs were plated in low-attachment conditions to induce the formation of gastruloids, and their morphology and gene expression profile was compared during 5 d of differentiation. Compared with wild-type mESCs, Bmal1−/− cells formed smaller (average diameter on day 5 in wild-type is 371 ± 40 μm compared with Bmal1−/− 160 ± 16 μm) and less dense-looking wild-type mESCs of genes involved in pluripotency, PI3K, and MAPK signalling pathways using heat map clustering. (G) Representative images of haematoxylin and eosin staining of teratomas derived from wild-type and Bmal1−/− mESCs. Tissues corresponding to three germ layers are shown (black arrow): skin for ectoderm, glandular tissue for endoderm, and smooth muscle for mesoderm. Scale bar is 100 μm. (H) Representative images of immunohistochemistry analysis of teratomas derived from wild-type and Bmal1−/− mESCs. Antibodies against Glial fibrillary acidic protein (ectoderm marker), cytokeratin (endoderm marker), and smooth muscle actin (mesoderm marker) were used. Scale bar is 500 μm.  three-dimensional structures during the course of the experiment (Fig 5A, white arrows). Analysis of mRNA expression revealed that differentiating Bmal1−/− mESCs down-regulated pluripotency genes (Oct4 and Nanog) and induced the activation of ectoderm genes (Sox1, Gsx1, Wnt7, and Pax6) at a similar or even more accused rate than parental control cells (Figs 5B and C and S3C). In contrast, Bmal1−/− differentiating gastruloids showed minor induction of endoderm (Gata4, Gata6, and Foxa2) and mesoderm (Bmp2, Msx2, and Mixl1) genes compared with parental control cells (Figs 5D and E and S3C). Thus, we concluded that Bmal1−/− mESCs consistently fail to activate endoderm and mesoderm genes upon pluripotency exit in all cell differentiation systems tested.
To confirm that the phenotype of Bmal1−/− mESCs is a consequence of Bmal1 protein depletion and is not linked to a CRISPR/ Cas9 off-target effect, we derived an independent clonal population of Bmal1−/− mESCs (clone #G10). Genotyping confirmed that these cells are homozygous mutant for a deletion of four nucleotides in exon 5 in both alleles of the Bmal1 gene that result in a frameshift and premature stop codon at the beginning of the open reading frame (Fig S4A). Bmal1−/− (clone #G10) did not express detectable levels of Bmal1 protein (Fig 5F), and similarly to the previously characterized Bmal1−/− mutant (Fig 2), Bmal1−/− (clone #G10) grown in the 2i + LIF medium formed typical tight three-dimensional colonies ( Fig S4B) and expressed normal, or slightly increased, levels of the pluripotency-associated transcription factors Nanog, Oct4, and Rex1 ( Fig 5G). Importantly, Bmal1−/− (clone #G10) mESCs also displayed higher levels of Nanog protein than wild-type parental controls (Fig 5H) phenocopying the previous Bmal1−/− cells. In addition, analysis of the differentiation potential of Bmal1−/− (clone #G10) further confirmed that the lack of Bmal1 protein results in the formation of smaller gastruloids (Figs 5I and S4C) that can down-regulate pluripotency markers and up-regulate ectoderm genes but have compromised induction of endoderm and mesoderm markers (Figs 5J and S4D). Taken together, these analyses confirm that Bmal1 regulates Nanog expression and the multilineage potential of pluripotent cells and suggest that Bmal1 is required for formation of endoderm and mesoderm tissue during embryo development.

Discussion
The most important discovery in this study is that the molecular clock protein Bmal1 is required for correct multi-lineage cell differentiation in pluripotent cells. This finding is interesting for the fields of circadian biology and pluripotency. It is currently not clear whether circadian proteins are expressed or not in pluripotent cells (Lu et al, 2016;Umemura et al, 2017), and therefore, our study is important to establish that the core component of the molecular clock Bmal1 is expressed in mESCs and that it has a functional role. Complementarily, our study show that circadian proteins are novel regulators of pluripotent biology and cell differentiation.
We show that Bmal1 regulates cell differentiation in mESCs where circadian rhythms cannot be detected using standard approaches (Kowalska et al, 2010;Yagita et al, 2010). Thus, our study support that during early development, the molecular clock carries out a novel and uncharacterized molecular function that does not rely in the canonical oscillatory production of gene transcripts. Therefore, an important question that arises from our study is what is the molecular function of Bmal1 in mESCs and whether it is carried out in coordination with the rest of the machinery of the molecular clock. Although there is evidence supporting the absence of circadian oscillations in pluripotent cells (Kowalska et al, 2010;Yagita et al, 2010), it is not clear whether the lack of circadian rhythms is a consequence of the absence of Clock protein; conflicting reports show that Clock protein is present (Lu et al, 2016) or degraded by posttranslational mechanisms (Umemura et al, 2017) in mESCs. Although this study cannot solve this discrepancy, our results indicate that the function of Bmal1 in mESCs might be different from the typical transcriptional regulation exerted in coordination with Clock in other tissues because depletion of Bmal1 in mESCs leads to up-regulation of gene transcripts, suggesting Bmal1 is unexpectedly acting as a transcriptional repressor. Further studies will be needed to address this important aspect and clarify what is the molecular function of Bmal1 in the regulation of pluripotency.
Mice lacking Bmal1 from conception are viable and show a pleiotropic phenotype including loss of circadian rhythms, an acceleration of aging, and shortened life span (Bunger et al, 2000;Kondratov et al, 2006). Strikingly, a current report by the FitzGerald Lab shows that most of this phenotype is a consequence of the function of Bmal1 during embryo development . Our findings might help explain these interesting observations. Bmal1−/− mESCs do not show a severe block in cell differentiation, but instead they show milder defects that result in unbalanced regulation of lineage choice during cell differentiation. This type of defects during early embryo development might allow the formation of viable newborns in Bmal1−/− mice that accumulate widespread minor developmental defects that will cause tissue misfunctioning during adult life, in agreement with reported phenotypes in life span, fertility, body weight, blood glucose and arthropathy, atherosclerosis, and hair growth (Kondratov et al, 2006;Yang et al, 2016). In this context, it is important to note that given that the differentiation phenotype of Bmal1−/− mESCs is associated to altered cell signalling, it is likely that developmental defects observed in our in vitro differentiation systems are partially ameliorated during embryo development because of compensatory mechanism that take place in vivo and, thus, facilitate full gestation of Bmal1−/− mice.
In summary, our study provides solid proof that the core molecular clock component Bmal1 is expressed in pluripotent cells and that it regulates pluripotent cell differentiation. This suggests the existence of an uncharacterized function of the molecular clock that is essential for pluripotency execution and proper embryo development in mammals. Similar conclusions were drawn from an independently performed study, which recently came to our attention (Ameneiro et al, 2020).

Teratoma formation and analysis
One million cells of each cell line were resuspended in 250 μl of PBS and injected subcutaneously into the flanks of NOD/SCID IL-2Rγ−/− mice in the animal experimentation unit of the University of Granada. 5 wk later, the mice developed tumours that were removed, rinsed with PBS, fixed, and embedded in paraffin at the Biobank of Andalusia Public Health System. Tissue sections were cut and processed for haematoxylin and eosin staining or for immunohistochemistry staining with Anti-Human Smooth Muscle Actin (IR611; Dako), Ms Anti-Human Cytokeratin Clone AE1/AE3 (IR053; Dako), and Rb Anti-Human Glial Fibrillary Acidic Protein (IR 624; Dako). Species-specific secondary antibodies conjugated with peroxidase were used (EnVision Flex/HPR SM802; Dako). Immunohistochemistry staining images were quantified measuring the area that was positive for the signal of the antibody. For each staining, 10 images were quantified using Image J, averaged, and tested statistically using t test.

Derivation of Bmal12/2 mESCs
Single-guide RNA sequences (Oligo 1: 59-CACCGAACCGGA-GAGTAGGTCGGT-39, Oligo 2: 59-AAACACCGACCTACTCTCCGGTTC-39) were designed using CRISPR design tool and cloned into PX458 plasmid (Ran et al, 2013) upon testing with surveyor mutation detection kit (IDT). JM8 mESCs were transfected using lipofectamine 2000 (Thermo Fisher Scientific). Cells positive for GFP expression were sorted using flow cytometry and plated at serial dilutions to obtain isolated clonal colonies. DNA of 12 genetic clones was PCR-amplified (59-TCTGCCTGGCTTATTCTTCC-39 and 59-AGTGCTGCTGGCCATTTAAG-39) and sequenced upon pGEM-T (Promega) cloning. Four genetic clones showed mutations in both alleles and in the one containing mutation that produced a shorter version of Bmal1 protein was further characterized by RT-qPCR and Western blot.

RT-qPCR and immunofluorescence
RNA was isolated using Trizol reagent (Thermo Fisher Scientific), reverse transcribed using RevertAid Frist Strand cDNA Synthesis kit (Thermo Fisher Scientific), and analysed by SYBRG real-time PCR using GoTaq qPCR Master Mix (Promega). Primers used are provided in Table S1.

mRNA-seq and bioinformatic analysis
Total RNA of JM8 wild-type and Bmal1−/− mESCs growing in the 2i + LIF medium was isolated using Trizol (Thermo Fisher Scientific), and mRNA library preparation was carried out using TruSeq Stranded mRNA kit (Illumina). 30 million 75-bp paired end reads were analysed per sample. Two biological replicates were carried out for each cell line. Library preparation and Illumina sequencing was carried out at National Center for Genomic Analysis-Center for Genomic Regulation (CNAG-CRG). Raw FASTQ files were quality filtered and aligned to the mouse genome (version mm9) using STAR 2.5.2 (Dobin & Gingeras, 2015). Gene expression was normalized with trimmed mean of M values (Robinson & Oshlack, 2010) implemented in NOISeq R package (Tarazona et al, 2015). Differential expression analysis was performed with DESeq2 package (Love et al, 2014). MA plot was generated with edgeR (McCarthy et al, 2012) and heat maps were generated with pheatmap package. mRNA of deregulated genes (log2 fold change greater than 1 or smaller than −1 and adjusted P-value < 0.05) were used as input gene lists in DAVID bioinformatic gene enrichment analysis. List of differentially expressed genes is provided in Table S1.
Single-cell RNA expression of Bmal1 and Oct4 in developing blastocysts was extracted from published data (Deng et al, 2014). The cells were classified by the level of expression of Oct4 in four quartile groups (Oct4 low correspond to bottom quartile and Oct4 high corresponds to top quartile), and average expression of Bmal1 was plotted.