BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells

Research Article BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells Cristina Ameneiro 1 , Tiago Moreira 1 , Alejandro Fuentes-Iglesias 1,2 , Alba Coego 1 , Vera Garcia-Outeiral 1,2 , Adriana Escudero 1,2 , Daniel Torrecilla 1 , Sonia Mulero-Navarro 3 , Jose Maria Carvajal-Gonzalez 3 , Diana Guallar 1,4 , Miguel Fidalgo 1,2 BMAL1 is essential for the regulation of circadian rhythms in differentiated cells and adult stem cells, but the molecular underpinnings of its function in pluripotent cells, which hold a great potential in regenerative medicine, remain to be addressed. Here, using transient and permanent loss-of-function approaches in mouse embryonic stem cells (ESCs), we reveal that although BMA L1 is dispensable for the maintenance of the pluripotent state, its depletion leads to deregulation of transcriptional programs linked to cell differentiation commitment. We further confirm that depletion of Bmal1 alters the differentiation potential of ESCs in vitro. Mechanistically, we demonstrate that BMAL1 participates in the regulation of energy metabolism maintaining a low mitochondrial function which is associated with pluripotency. Loss-offunction of Bmal1 leads to the deregulation of metabolic gene expression associated with a shift from glycolytic to oxidative metabolism. Our results highlight the important role that BMAL1 plays at the exit of pluripotency in vitro and provide evidence implicating a non-canonical circadian function of BMAL1 in the metabolic control for cell fate determination. DOI 10.26508/lsa.201900534 | Received 23 August 2019 | Revised 31 March 2020 | Accepted 1 April 2020 | Published online 13 April 2020 Introduction Circadian rhythms are necessary to coordinate key behavioural (e.g., sleep/wake cycle) and physiological (e.g., metabolism, hormone secretion, and stem cell homeostasis) processes in mammals (Bechtold & Loudon, 2013;Lopez-Minguez et al, 2016;McAlpine & Swirski, 2016;Weger et al, 2017;Dierickx et al, 2018). At the cellular level, the circadian clock is composed by transcriptional and translational feedback loops involving the clock master regulators BMAL1, CLOCK, PER, and CRY proteins, which ensure rhythmic gene expression to accommodate to the tissue and organ needs. Interestingly, although the proteins of the circadian clock are already present at early stages of embryonic development, circadian rhythms are not established until around the mid-gestation stage (Saxena et al, 2007;Umemura et al, 2017). In line with this, embryonic stem cells (ESCs), which are derived from the inner cell mass of the preimplantation blastocyst, are devoid of transcriptional circadian oscillations (Kowalska et al, 2010;Yagita et al, 2010;Umemura et al, 2014,2017;Dierickx et al, 2017). Given the lack of a compensating homologue in vivo, BMAL1 has been defined as the only essential component of the molecular circadian clock in mammals (Bunger et al, 2000). Bmal1 KO mice have impaired circadian behaviour and absence of rhythmicity in circadian target genes (Bunger et al, 2000). Moreover, they show infertility (Alvarez et al, 2008;Boden et al, 2010), show impaired glucose homeostasis (Rudic et al, 2004), and have been reported to have reduced life span and higher prevalence of age-related pathologies (Kondratov et al, 2006). Unexpectedly, many metabolic and age-related pathologies caused by Bmal1 depletion were not observed when using an inducible KO mouse model where Bmal1 depletion was performed in the adult age (Yang et al, 2016), suggesting importantfunctionsforthis master regulator during embryogenesis. Given that BMAL1 is readily expressed in ESCs, even in the absence of a functional circadian clock, we hypothesized that additional roles of this factor in pluripotency remain to be discovered and could yield insights into its function during early stages of embryonic development. To investigate the function of BMAL1 in pluripotent cells, which present a great therapeutic potential given their ability to generate cells of any adult tissue, we used transient and genetic models of Bmal1 loss-of-function in ESCs. We discovered that BMAL1 is dispensable for ESC maintenance, as its depletion does not affect pluripotency marker expression or colony formation. Nevertheless, we observed that ablation of Bmal1 in ESCs resulted in deregulation of genes from the three embryonic germ layers, and an aberrant 1 Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade de Santiago de Compostela (USC)-Health Research Institute (IDIS), Santiago de Compostela, Spain 2 Department of Physiology, USC, Santiago de Compostela, Spain 3 Department of Biochemistry, Molecular Biology and Genetics, Facultad de Ciencias, Universidad de Extremadura, Badajoz, Spain 4 Department of Biochemistry and Molecular Biology, USC, Santiago de Compostela, Spain Correspondence: miguel.fi[email protected]; [email protected] ©2020Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 1of15 on 14 January, 2025life-science-alliance.org Downloaded from http://doi.org/10.26508/lsa.201900534Published Online: 13 April, 2020 | Supp Info: induction of differentiation gene expression in vitro. Importantly, using embryonic organoids, we discovered that BMAL1 is necessary for in vitro gastruloid formation and proper expression of lineage specification markers. Mechanistically, we discovered that depletion of Bmal1 produced a change in metabolism-related genes and pathways, which are now considered to be drivers in the differentiation process. In particular, we observed a reduction in basal glycolysis and a concomitant increase in respiration, which was accompanied by an increase in mitochondrial reactive oxygen species (mtROS) production. Thus, our results uncover an unexpected function of BMAL1 in ESCs in metabolic regulation, where the clock is not yet “ticking,” but BMAL1 function is already relevant for proper embryonic specification. Results Transient loss-of-function of BMAL1 is dispensable for ESC selfrenewal To define the role of BMAL1 in pluripotent cells, which have been previously reported to lack circadian rhythms (Kowalska et al, 2010; Yagita et al, 2010;Umemura et al, 2014,2017;Dierickx et al, 2017), we first determined the expression level of this core clock regulator in MEFs and pluripotent ESCs. Notably, when we analysed BMAL1 RNA and protein levels by RT quantitative PCR (RT-qPCR) and Western blot, respectively, we detected that its abundance was higher in ESCs compared with MEFs (Figs 1A and S1A). In contrast, expression of Clock, another circadian regulator, was higher in MEFs, which have been reported to possess a functional circadian clock (Yagita et al, 2001)(Fig S1A). These data prompted us to consider whether BMAL1 may be important for pluripotency. To understand the role of BMAL1 in ESCs, we first performed lossof-function experiments using two independent shRNAs against Bmal1 to reduce the likelihood of off-target effects. We confirmed the efficiency in silencing of Bmal1 compared with Luciferase control (shLuci) knockdown by analysing its protein levels by Western blot (Fig 1B). Importantly, we did not observe major differences in typical ESC morphology and AP staining in cells transduced with shRNAs against Bmal1 compared with shLuci (Fig 1C). Consistently, reduction of BMAL1 protein levels did not greatly impact pluripotency markers (i.e., Pou5f1 and Nanog) at the mRNA level (Fig S1B) and/or the protein level (Fig 1B). Indeed, immunofluorescence assays revealed that silencing of Bmal1 does not alter expression variability and population heterogeneity of pluripotency markers (i.e., SSEA1, POU5F1, and ZFP281) in ESCs (Figs 1D and S1C). We next examined whether self-renewal properties of pluripotent cells were affected by the loss-of-function of Bmal1 in the presence Figure 1. Silencing of Bmal1 does not compromise the maintenance of embryonic stem cells (ESCs). (A) (Left) Western blot of BMAL1 and the pluripotency markers NANOG and POU5F1 in MEFs and ESCs. βTUBULI N was used as loading control. Biological triplicates are shown. (Right) Quantification of the blots. (B) (Left) Western blot of BMAL1 and POU5F1 in ESCs transfected with shRNAs against Bmal1 or Luciferase as control. ACTIN (ACTB) was used as a loading control. One representative experiment is shown. (Right) Quantification of three independent experiments. (C) Bright-field (BF) and AP-staining images of ESCs transfected with shRNA against Bmal1 or Luciferase as a control. The scale bar represents 200 μm. (D) Immunofluorescence of the pluripotent marker SSEA1 in ESCs transfected with shRNAs against Bmal1 or Luciferase as control. Nuclei were stained with DAPI. The scale bar represents 50 μm. (E) Schematic representation for the colony formation assay. ESCs transfected with shRNA against Bmal1 or Luciferase as control are seeded at low confluence in standard medium with or without LIF, and colonies are counted and classified into the three indicated categories according to their AP-staining intensity. (F) Graphic representation for the counting and classification of colonies. (n = 3). In (A, B, F), bars represent mean ± SD. Two-tailed unpaired ttest analysis was performed. ****P< 0.0001, ***P< 0.001, **P< 0.01; ns, not significant. Source data are available for this figure. BMAL1 links metabolism & differentiation Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 2of15 or absence of leukaemia inhibitory factor (LIF). For this purpose, a reduced number of cells transduced with either the control shLuci or each of the two shRNAs against Bmal1 were plated and cultured at clonal density with or without LIF for 4 d, followed by AP staining (Fig 1E). Consistent with the effect observed in bulk-grown ESCs, silencing of Bmal1 did not impact the proportion of undifferentiated, partially or fully differentiated colonies both in pluripotencysustaining (+LIF) or differentiation-promoting (−LIF) culture conditions (Fig 1F). Taken together, our results show that BMAL1 is dispensable for ESC self-renewal, despite being abundantly expressed in pluripotent cells. Absence of BMAL1 protein is compatible with pluripotency maintenance Given that knockdown with RNA interference can result in variable amounts of mRNA reduction, we aimed at generating ESC lines completely lacking Bmal1 expression to systematically dissect BMA L1 function in pluripotent cells. To this end, we used the CRISPR-C as9 nuclease system (Jinek et al, 2012;Cong et al, 2013) to generate a Bmal1 KO ESC line. First, we designed a sgRNA specifically targeting the start codon site of Bmal1 gene, located at the Exon 5 (Fig 2A). PCR genotyping and Sanger sequencing identified a Bmal1 KO ESC line (Figs 2A and S2A–C). Western blot analysis confirmed complete depletion of BMAL1 protein in our selected ESC clone (Fig 2B). To establish whether depletion of Bmal1 influences the maintenance of the pluripotent state of ESCs, we first examined the morphology and AP-staining pattern in Bmal1 KO ESCs. Notably, consistent with the Bmal1 knockdown data, Bmal1 KO ESCs displayed normal pluripotent colony morphology and stained positive for AP compared with WT pluripotent cells over the course of multiple passages (>10) (Fig 2C). In addition, the expression of pluripotency markers (i.e., Nanog,Pou5f1, and Zfp42) at the RNA and protein levels was not significantly affected in the absence of BMAL1 Figure 2. Generation and characterization of a CRI SPR/Cas9 Bmal1 KO embryonic stem cell (ESC) line. (A) (Top) Schematic representation for the CRISPR/C as9 strategy used. The designed sgRNA is underlined and the PAM sequence is highlighted in red. (Bottom) Sequence deleted (22 bp) in Bmal1 KO alleles detected by Sanger sequencing. (B) (Top) Western blot of BMAL1 and the pluripotency factors NANOG and POU5F1 in Bmal1 WT and KO ESCs. βTUBULIN was used as a loading control. (Bottom) Quantification of three independent experiments. (C) Bright-field (BF) and AP-staining images of Bmal1 WT and KO ESCs. The scale bar represents 200 μm. (D) Immunofluorescence of POU5F1, SSEA1, and ZFP281 in Bmal1 WT and KO ESCs. Nuclei were stained with DAPI. The scale bar represents 50 μm. (E) (Left) Number of ESC colonies formed in a clonogenic assay with Bmal1 WT and KO ESCs. (Right) Representative images of AP-stained wells are shown. (F) Graphic representation for the counting and classification of colonies of a colonyformation assay. (n = 3) In (B, E, F), bars represent mean ± SD. Two-tailed unpaired ttest analysis was performed. ***P< 0.001, **P< 0.01, *P< 0.05; ns, not significant. Source data are available for this figure. BMAL1 links metabolism & differentiation Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 3of15 (Figs 2B and S2D and E). Moreover, immunofluorescence of key pluripotent markers (i.e., POU5F1, SSEA1, ZFP281, SOX2, and NANOG) (Figs 2D and S2F) suggested that pluripotency maintenance in Bmal1 KO ESCs was grossly unaffected. On the other hand, when we analysed the self-renewal capability of Bmal1-depleted ESCs, we did not observe changes in the number of colonies (Fig 2E) and only a mild reduction in the percentage of undifferentiated colonies (74.5 ± 3.3 versus 59.6 ± 3.9) in the presence of LIF (Fig 2F), which is in line with the acute silencing of Bmal1 using the shRNA approach (Fig 1F). Of note, Bmal1 KO ESCs showed a defect in differentiation upon LIF withdrawal as observed by the decrease in the percentage of fully differentiated colonies (65.6 ± 4.7 versus 34.7 ± 6.7) (Fig 2F), suggesting a possible role of BMAL1 during the exit of pluripotency. Collectively, taking together the knockdown and KO results, our data demonstrate that BMAL1 is dispensable for ESC maintenance. BMAL1 is required for exiting pluripotency in vitro Given that pluripotent cells can differentiate into all cell types from the three germ layers (i.e., ectoderm, endoderm, and mesoderm), we next interrogated the role of BMAL1 during the cellular differentiation process of ESCs. For this purpose, we first performed an in silico analysis using a genome-wide RNAi screen–published dataset for the identification of potential direct and indirect regulators of Nanog gene expression under mild retinoic acid (RA)–induced differentiation conditions, which could presumably be involved in the exit of pluripotency (Gingold et al, 2014). Remarkably, when we analysed theconsequencesofloss-of-functionin166genesinvolvedwith circadian rhythms (GO:0007623), we noticed the existence of eight positive, including BMAL1, and six negative potential regulators of the expression of the pluripotency marker Nanog (Z score > 2), measured by GFP fluorescent levels, which could play roles in the differentiation process (Fig 3A). Interestingly, we also observed the existence of opposing effects on Nanog promoter activity between the core circadian clock regulators BMAL1 and CLOCK (Fig 3B). Importantly, we further confirmed the regulatory effect of BMAL1 on Nanog promoter activity under the same RA-induced differentiation conditions, by flow cytometry analysis using the Nanog-GFP reporter ESC line NG4 (Schaniel et al, 2009) transduced with shRNAs against Bmal1 and shLuci as a control (Fig 3C and D). Future studies are warranted to investigate whether BMAL1 regulates Nanog promoter activity in a direct or indirect manner. Taken together, these results show that BMAL1 may be involved in early decisions during the exit of pluripotency and unveil the existence of specific functions of factors related with circadian processes in cell fate determination at the beginning of in vitro differentiation. Next, to investigate the function of BMAL1 in differentiation, we analysed the ability of Bmal1 KO and wild-type (WT) ESCs to form teratomas. When subcutaneously injected in the flanks of immunodeficiency mice, both Bmal1 KO and WT ESCs were able to efficiently form teratomas of similar size (Fig 4A). Haematoxylin and eosin–stained sections of teratomas showed that a range of cell types and tissues from all three germ layers (i.e., mesoderm, ectoderm, and endoderm) was present in all teratomas regardless of BMAL1 presence (Fig 4B). These findings, together with our results showing that BMAL1 is dispensable for ESC maintenance, support that Bmal1 KO ESCs are pluripotent. It is possible that the role of BMAL1 in the exit of pluripotency observed in vitro (Fig 3) does not cause profound abnormalities which would be detectable during differentiation in vivo by teratoma assay. Thus, to determine if depletion of Bmal1 in ESCs can affect cellular differentiation in vitro, we performed embryoid bodies (EBs) assays, which mimic early embryonic development in a dish (Doetschman et al, 1985). After 6 d of culture in ESC medium without the LIF cytokine (Fig 4C), we observed that Bmal1 expression was down-regulated similarly to Nanog, concomitant with the up-regulation of markers from the three germ layers (i.e., Pax3,Gata6, and Mixl1)(Fig 4D). Consistent Figure 3. Absence of Bmal1 affects the exit from pluripotency. (A) (Left) Schematic representation for screening conditions. Nanog-GFP (NG4) ESCs are transfected with siRNAs and cultured for 1 d in standard pluripotency mediumfollowedby2dundermildretinoicacid (RA)–mediated differentiation. The cells are fixed, stained, and imaged. (Right) Scatter plot showing circadian clock core genes expression represented as FPKM and their effect in Nanog-GFP median fluorescence after screening conditions. (B) Summary table of Zscore median fluorescence values for circadian clock core genes after screening conditions highlighting Bmal1 as the gene with the highest effect among the core circadian regulators. (C) FACS analysis of Nanog-GFP intensity after RA-mediated differentiation of NG4 cells transfected with shRNA against Bmal1 or Luciferase as control. CCE parental ESC line is used as negative fluorescence control. The percentage represents the fraction of Nanog-GFP–positive cells in each sample in the indicated gate. One representative experiment is shown. (D) Percentage of Nanog-GFP–positive cells after transduction with the indicated shRNAs. (n = 3) Bars represent mean ± SD. Two-tailed unpaired ttest analysis was performed. **P<0.01,*P<0.05. Source data are available for this figure. BMAL1 links metabolism & differentiation Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 4of15 with their ability to generate teratomas, Bmal1 KO cells were able to form EBs (Fig 4E). However, we observed that Bmal1 KO EBs presented a significant increase in their size compared with the control (Fig 4E), suggesting intrinsic differences during the differentiation process through EB formation due to the absence of this master clock regulator. Moreover, when we analysed gene expression of several ectoderm, mesoderm, and endoderm markers, we found that they were differentially induced in Bmal1 KO at day 6, compared with WT EBs (Fig 4F). These results indicate that BMAL1 is required for ESC differentiation in vitro to properly establish germ layer–specific transcriptional programs. BMAL1 is important during in vitro gastrulation To further confirm our observation that loss of BMAL1 affects proper embryonic germ layer specification in vitro, we decided to use a recently reported gastruloid system (Beccari et al, 2018). Gastruloids are small aggregates of ESCs that undergo gastrulation-like events and elongation in vitro and mimic embryonic spatial and temporal gene expression (Beccari et al, 2018). Importantly, this gastrulation model can be used as an in vitro system to study early developmental events taking place in the mammalian embryo in vivo. Thus, we generated aggregates of WT or Bmal1 KO ESCs in N2B27 medium and subjected them to a pulse of a WNT agonist (i.e., CHIR99021) (Fig 5A). First, we analysed the expression pattern of WT gastruloids and observed that, similar to the pluripotency marker Nanog,Bmal1 was down-regulated at 120 h of gastruloid formationcomparedwithESCs(t=0h), concomitant with germ layer marker induction (i.e., Pax3,Gata6,and Mixl1)(Fig 5B). We then compared the efficiency in the generation of gastruloids in the presence or absence of BMAL1. Remarkably, we observed that after 120 h, the aggregates obtained from Bmal1 KO ESCs were significantly smaller (Fig 5C and D) and failed to elongate and polarize to give rise to gastruloid-like structures compared with wildtype cells (7.14% versus 41.07%, respectively) (Fig 5E). In addition, absence of BMAL1 during gastruloid formation was accompanied by altered expression of several lineage specification markers representative of three germ layers (Fig 5F). In particular, we observed deregulation, in the absence of BMAL1, of genes associated with in vivo gastrulation process such as Mixl1 and Eomes (Beccari et al, 2018). Likewise, we observed altered transcriptional dynamics of several members of the Hoxd gene cluster (Fig 5G), which is one of the hallmarks of axial gene regulatory systems whose sequential activation is associated with the patterning and formation during in vitro gastruloid formation (Beccari et al, 2018). Collectively, these results show that BMAL1 is required for efficient gastruloid formation in vitro and confirm that BMAL1 deficiency abrogates the correct induction of ectoderm, mesoderm, and endoderm markers during the exit of pluripotency. BMAL1 regulates transcriptional networks related to cellular differentiation To gain further insight into the molecular underpinnings of BMAL1 function in pluripotent cells, we performed transcriptional profiling Figure 4. Bmal1 is required for embryonic stem cell in vitro differentiation. (A) (Left) Teratomas formed by wild-type (WT) and Bmal1 KO ES cells after 3–4 wk of injection. The bar represents 5 mm. (Right) Quantification of teratoma size (n = 8 tumours per genotype). (B) Histological analysis of teratomas of the indicated genotype by H&E staining. Structures representing the three embryonic germ layers in both genotypes were found. (*), connective tissue; (^), neural tissue; and (+), epithelial tissue. 4× magnifications for each tissue are shown. The bar represents 30 μm. (C) Schematic representation for the embryoid body (EB) differentiation assay. (D) Relative expression of genes at day 6 of EB differentiation assay compared with day 0 in WT embryonic stem cells determined by RT-qPCR. Ectoderm-specific (i.e., Pax3), endodermspecific (i.e., Gata6), and mesoderm-specific (i.e., Mixl1) markers are shown. (E) (Top) Representative images of EBs at day 6 of differentiation in the indicated cell lines. The scale bar represents 200 μm. (Bottom) Quantification of the diameter of day 6 EBs for Bmal1 WT and KO cells. The number of EBs analysed is indicated. Data are shown as mean ± SEM. (F) Relative expression of several germ layer–specific genes from day 0 and day 6 EBs determined by RT-qPCR. Expression fold changes are shown relative to the highest value. In (A, D), data are shown as mean ± SD. Two-tailed unpaired ttest was performed. **P< 0.01; ns, not significant. Source data are available for this figure. BMAL1 links metabolism & differentiation Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 5of15 of wild-type and Bmal1 KO ESCs. We identified 444 up-regulated and 197 down-regulated genes that were significantly changed with a difference in abundance greater than twofold in the absence of BMAL1 (Fig 6A and Table S1). Remarkably, Gene Ontology (GO) analysis of these misregulated genes upon Bmal1 depletion showed enrichment for differentiation processes (Fig 6B). To validate these results, we performed RT-qPCR analysis of a number of up-regulated (e.g., Tead4 and Mest) and down-regulated (e.g., Eomes and Snai3) early cell fate markers upon Bmal1 KO in ESCs (Fig 6C and D). Moreover, gene set enrichment analysis (GSEA) also identified the up-regulation of gene signatures related to stem cell differentiation in the absence of BMAL1 function when compared with their wild-type counterparts (Fig 6E). Thus, these results suggest that Bmal1 depletion leads to transcriptional changes in genes related to developmental processes by direct or indirect mechanisms, which is in line with our results showing the requirement of this circadian master regulator for proper cellular differentiation of ESCs. Indeed, we observed that depletion of Bmal1 led to a significant deregulation of genes related to the three embryo germ layers (i.e., endoderm, ectoderm, and mesoderm) in ESCs, whereas housekeeping genes remained unaltered (Fig 6F). Importantly, in spite of these global transcriptional changes, the GSEA analysis further confirmed the pluripotent cell identity of Bmal1 KO ESCs denoted by no significant changes in the expression of ESC-enriched genes as well as targets of the core pluripotency regulators POU5F1 (OCT4), SOX2, and NANOG (OSN) (Fig S3A). Taken together, our data suggest that BMAL1 loss may influence the differentiation potential of ESCs in vitro by altering the expression of early specification genes of the three germ layers in pluripotent cells. BMAL1 supports glycolytic metabolism in ESCs To understand how BMAL1 orchestrates transcriptional programs involved in proper cell differentiation in vitro, we next compared the gene expression changes after Bmal1 depletion in ESCs with those caused by loss-of-function of other transcription regulators using the Network2Canvas computational tool (Tan et al, 2013). Interestingly, we found a close correlation of the transcriptional changes in the absence of BMAL1 with those upon depletion of the master pluripotency regulator POU5F1 (Kim et al, 2015) as well as other pluripotent regulators related with differentiation and/or cellular metabolism, including ZFX (Galan-Caridad et al, 2007; Chen et al, 2008) and Polycomb members (i.e., EED and SUZ12) (Brookes et al, 2012;Di Croce & Helin, 2013)(Fig S3B). These results, together with the observation that the expression of these factors was not altered upon Bmal1 depletion in mouse ESCs (Figs 2B and S3C and D), suggest the existence of common pathways regulated Figure 5. Ablation of Bmal1 disrupts gastrulation in vitro. (A) Schematic depiction for the gastrulation assay performed (Beccari et al, 2018) for CHIR, GSK-3 inhibitor. (B) Relative expression of genes after 120 h of in vitro gastrulation assay of wild-type embryonic stem cells (ESCs) determined by RT-qPCR and expressed relative to ESCs (0 h). Ectoderm-specific (i.e., Pax3), endoderm-specific (i.e., Gata6), and mesodermspecific (i.e., Mixl1) markers are shown. (C) Representative images of 120 h aggregates for Bmal1 WT and KO cells. The scale bar represents 200 μm. (D) Quantification of the size of the aggregates formed by Bmal1 WT and KO cells at 120 h of in vitro gastrulation. (E) Percentage of gastruloid-like structures formed by Bmal1 WT and KO aggregates at 120 h of in vitro gastrulation (n = 14 independent experiments with at least eight formed aggregates per experiment). (F) Relative expression of Nanog and several germ layer–specific genes at the indicated time points of the gastruloid differentiation assay of WT and Bmal1 KO ESCs determined by RT-qPCR. Expression of fold changes are shown relative to the highest value. (G) Expression of Hoxd1,Hoxd3,Hoxd4, and Hoxd10 dynamics during in vitro gastrulation of Bmal1 WT and KO ESCs at the indicated time points. (n = 3). In (B, D, E, G), data are represented as mean ± SD. Two-tailed unpaired ttest was performed. ****P< 0.0001, ***P< 0.001, **P< 0.01; a.u., arbitrary units; ns, not significant. Source data are available for this figure. BMAL1 links metabolism & differentiation Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 6of15 by this set of factors and BMAL1. Indeed, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the misregulated genes in Bmal1 KO ESCs revealed an enrichment in metabolicassociated processes (Fig S4A). Notably, among the most differentially expressed genes (false discovery rate [FDR] > 0.05 and fold change > 2), we found 19 down-regulated and 49 up-regulated metabolism-related genes in Bmal1-depleted cells (Fig S4B). In particular, we observed that BMAL1 loss influences the expression of several genes related with mitochondrial complex I–V and the tricarboxylic acid cycle or Krebs cycle (Figs 7A and S4C). Collectively, our results show that BMAL1 contributes to the proper transcriptional landscape regulation of pluripotent cells and that its depletion leads to the deregulation of metabolism-related transcriptional pathways. Changes in metabolic activity are closely linked to the exit of pluripotency, partly by influencing the epigenome during cell commitment (Cliff & Dalton, 2017;Dahan et al, 2019). Thus, we speculated that Bmal1 depletion can alter early cell differentiation potential through changes in the expression of metabolic gene networks that govern the balance between glycolytic and oxidative phosphorylation (OXPHOS) activity. In agreement with our hypothesis, we found that Bmal1 KO cells showed reduced basal glycolysis compared with WT cells (Fig 7B). Conversely, depletion of Bmal1 in ESC s led to an increase in the basal oxygen consumption rate (OCR) (Fig 7C). Thus, we observed a metabolic switch in ESCs caused by Bmal1 depletion, which was translated into a more oxidative versus glycolytic use of glucose in Bmal1 KO ESCs compared with WT ones (Fig 7D). Although there is usually a tight coupling between electron transport and ATP synthesis, under certain conditions, protons can Figure 6. Bmal1 participates in the regulation of cell differentiation transcriptional programs in embryonic stem cells (ESCs). (A) Scatter plot of gene expression (in log 2 FPKM) in Bmal1 KO versus wild-type (WT) control ESCs determined by RNA-seq. Biological duplicates were analysed. Genes significantly (false discovery rate [FDR] < 0.05) up-regulated and down-regulated more than twofold are shown in red and blue, respectively. (B) Gene Ontology (GO) analysis of genes misregulated in Bmal1 KO ESCs. The x-axis corresponds to the negative log 10 P-values. (C) (Left panel) Integrative genome viewer representation of RNA-seq reads for representative up-regulated genes in Bmal1 KO cells. (Right panel) RT-qPCR analysis validating the RNA-seq results (n = 3). (D) (Left panel) Integrative genome viewer representation of RNA-seq reads for representative down-regulated genes in Bmal1 KO cells. (Right panel) RT-qPCR analysis validating the RNAseq results (n = 3). (E) Gene set enrichment analysis from WT and Bmal1 KO RNA-seq data against a stem cell differentiation gene set. Normalized enrichment score (NES) and FDR are shown. (F) Fold RNA expression changes of the indicated gene categories in WT and Bmal1 KO ESCs. The number of genes included in each category is indicated. Whiskers extend to the 10 th –90 th percentile range. In (C, D), data are shown as mean ± SD. Two-tailed unpaired ttest was performed. ****P< 0.0001, **P< 0.01, *P< 0.05. Source data are available for this figure. BMAL1 links metabolism & differentiation Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 7of15 re-enter the mitochondrial matrix with no contribution to ATP generation, in a process known as mitochondrial uncoupling or proton leak (Cadenas, 2018). Indeed, we observed that the increase of basal oxidative metabolism in cells lacking BMAL1 (Fig S4D) was accompanied by an increase in proton leak (Fig 7E)anda reduction in the coupling efficiency of mitochondria (Fig S4E). Although the exact link between proton leak and mtROS production is controversial, it is now clear that there is a mutual connection between these two processes (Nanayakkara et al, 2019). To interrogate the effect of Bmal1 depletion on the production of mtROS species, we stained WT and Bmal1 KO ESCs with MitoSOX Red. Interestingly, we observed that ESCs lacking BMAL1 displayed a significant increase in mtROS levels compared with WT cells (Figs 7F and G and S4F). Collectively, these findings show that BMAL1 is required for proper metabolic dynamics and mitochondrial function of pluripotent ESCs. Taking into account that the balance between glycolysis and OXPHOS is critical for modulating the differentiation potential of pluripotent cells (Wu et al, 2016;Cliff & Dalton, 2017;Zhang et al, 2018;Dahan et al, 2019), we tested whether reducing OXPHOS activity in Bmal1 KO ESCs could restore the proper expression of lineage specification markers during in vitro differentiation. For this purpose, we used an early differentiation in vitro assay to rapidly induce the expression of genes involved in mesendoderm (ME) lineage choice (Thomson et al, 2011). In line with our previous differentiation assays, absence of BMAL1 significantly alter the expression of lineage markers upon ME differentiation (Fig 7H). Importantly, presence of mitochondrial respiration inhibitors (i.e., antimycin A/rotenone [AA/Rotenone]) during ME differentiation of Bmal1 KO ESCs was sufficient to significantly rescue the expression of T,Mixl1,Fgf8, and Gpb2 lineage markers compared with Bmal1 WT ESCs (Fig 7H). These data further suggest that BMAL1 influences early cell fate specification during in vitro differentiation through the modulation of OXPHOS activity in ESCs. Figure 7. Depletion of Bmal1 alters metabolic gene pathways, causing an increase in respiration and mitochondrial ROS production in embryonic stem cells (ESCs). (A) Heat map of the expression of tricarboxylic acid cycle (TCA) and mitochondrial complex I–V genes from wild-type (Bmal1 WT) and Bmal1 KO ESCs RNA-seq. (B) Basal glycolysis levels determined as extracellular acidification rate (ECAR) in wild-type (WT) and Bmal1 KO ESCs (n = 3). (C) Bioenergetics assays in ESCs from WT and Bmal1 KO ESCs. The different drugs used in theassayareindicatedingrey(n=3).(D) OCR/ECAR under basal conditions for the indicated cell lines at three different time points (n = 3). (E) Proton leak shown as OCR in WT and Bmal1 KO ESCs (n = 3). (F) Fluorescence representative microscopy images showing MitoSOX fluorescence (red) in WT and Bmal1 KO ESCs. Nuclei were stained with DAPI and shown in blue. The scale bar represents 50 μm. (G) Box plots showing the mean fluorescence intensity of MitoSOX treated Bmal1 WT and KO cells. Center lines, medians; whiskers extend to the 10 th /90 th percentile. (H) Relative expression of the indicated genes in Bmal1 WT and KO cells with or without antimycin A/rotenone (AA/Rot) treatment. ttest was performed. (n = 3). In (B, C, D, E, H), data are represented as mean ± SD. Two-tailed unpaired ttest was performed. ****P< 0.0001, ***P<0.001, **P<0.01,*P< 0.05; AA, Antimycin A; FCCP, carbonyl cyanide4 phenylhydrazone; ns, not significant;OCR,oxygen consumption rate. Source data are available for this figure. BMAL1 links metabolism & differentiation Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 8of15 Discussion Here, we demonstrate that BMAL1 is not required for ESC maintenance. In contrast, we show that this core component of the circadian clock plays critical roles during ESC in vitro differentiation. Indeed, depletion of Bmal1 caused the deregulation of genes from the three germ layers (i.e., ectoderm, mesoderm, and endoderm). In line with this, we show that the absence of BMAL1 affects EB formation and gastrulation, using two independent differentiation in vitro organoid systems. Similar conclusions were drawn from an independently performed study, which recently came to our attention (Gallardo et al, 2020). Mechanistically, we find that depletion of Bmal1 induces a shift in the basal metabolism of pluripotent cells involving an increase OXPHOS activity accompanied by augmented mtROS species production. Importantly, decreasing OXPHOS activity is sufficient to restore proper induction of lineage-specific marker expression during mesendoderm differentiation in the absence of BMAL1. For circadian oscillation to occur, CLOCK and BMAL1 need to heterodimerize to activate their downstream transcriptional targets, which include Per1/2/3 and Cry1/2 (Takahashi, 2017). In agreement with the observation that circadian transcriptional oscillations are not functional in ESCs (Kowalska et al, 2010;Yagita et al, 2010;Umemura et al, 2014,2017;Dierickx et al, 2017), when we analysed Cry1/2 and Per1/ 2/3 expression in Bmal1 KO cells, we did not observe any significant change in their transcriptional levels (Table S1), further supporting a clock-independent function for BMAL1 in pluripotent stem cells. Several reports have demonstrated that circadian oscillations are absent from pluripotent cells by using bioluminescent reporter systems and analysing of clock gene expression (Kowalska et al, 2010; Yagita et al, 2010;Umemura et al, 2014,2017;Dierickx et al, 2017). Nevertheless, both BMAL1 and CLOCK proteins have been reported to be present in ESCs by us (this study) and others (Lu et al, 2016), respectively, posing the question of how circadian oscillations are inhibited in pluripotent stem cells. One possible explanation was indicated by a study that demonstrated that in ESCs, PER is retained in the cytoplasm, therefore avoiding the proper nuclear function of the negative feedback loop required for cyclic circadian regulation (Umemura et al, 2014). Moreover, in contrast to Lu and colleagues (Lu et al, 2016), a recent study claimed that despite being expressed at the mRNA level, CLOCK protein was absent in ESCs because of microRNAmediated posttranscriptional repression (Umemura et al, 2017). Although the absence of transcriptional cycling in pluripotent cells seems faithfully demonstrated, the extent to which each of the proposed mechanisms contributes to inhibit the normal function of the clock remains to be clarified.Importantly,CLOCKandBMAL1seemto have opposite non-overlapping functions in terms of ESC differentiation (Gingold et al, 2014). This is in agreement with our observation that Bmal1 depletion facilitated the down-regulation of the pluripotency factor Nanog in conditions of mild differentiation, whereas Clock appeared to have the opposite effect, in agreement with what Lu and colleagues showed previously (Lu et al, 2016). Future studies are required to scrutinize common and specific roles of CLOCK and BMAL1 in ESCs, and additional levels of regulation that can shed light into the mechanisms by which pluripotent cells lack functional circadian rhythms. Previous reports on the effect of loss of Bmal1 expression on early embryo development are not consistent. Whereas Bradfield et al reported a normal mendelian ratio of Bmal1 −/− pups being born from the mating of two heterozygous Bmal1 +/− mice (Bunger et al, 2000), several recent reports have shown that Bmal1 KO zygotes show reduced blastocyst formation and post-implantation development (Xu et al, 2016,2017). Importantly, these defects could be directly associated with intrinsic developmental issues, given that they used females with at least one Bmal1 allele, which have been reported to possess normal reproduction physiology (Xu et al, 2017). Our observation that depletion of Bmal1 in ESCs, which are equivalent to the inner cell mass of the preimplantation blastocyst (around E3.5), show defects during in vitro differentiation to the three germ layers (i.e., ectoderm, endoderm, and mesoderm) would further support an embryo-autonomous role for BMAL1 in embryonic development. Taking into consideration the sterility issues described both in male and female Bmal1 KO mice (Alvarez et al, 2008;Boden et al, 2010;Ratajczak et al, 2012;Liu et al, 2014;Xu et al, 2016), the in vivo study of Bmal1 KO embryos is limited by the need of crossing heterozygous progenitors. We propose that ESC differentiation through gastruloid aggregation represents a powerful platform to investigate BMAL1 function in early stages of development where circadian rhythms have not yet been established. Moreover, this system allows to differentiate embryo-autonomous circadian mechanisms, excluding the influence from central and other periphery circadian clocks, which are also affected by the disruption of BMAL1 function. In this study, we demonstrate for the first time a non-canonical function of BMAL1 in cells lacking circadian oscillations. This is in line with the observation that conditional depletion of Bmal1 at the adult age does not recapitulate many of the metabolic and agerelated pathologies observed in Bmal1 KO mice (Yang et al, 2016). This observation implies that BMAL1 may have several important circadian-dependent and circadian-independent functions during embryogenesis. Importantly, the higher age-dependent accumulation of reactive oxygen species (ROS) observed in several tissues of Bmal1 KO animals (Kondratov et al, 2006) could explain the early onset of age-associated pathologies observed in these animals. In line with this, we observed an increase in the mtROS levels in ESCs lacking Bmal1, even when the clock is not yet active, pointing to a possible factor contributing to the aging phenotypes observed in these animals later during their lifetime. Moreover, previous studies have demonstrated that oxidative stress negatively impacts oocyte quality and fertilization, together with embryo development (Matsuzuka et al, 2005;Tamura et al, 2008). Thus, the decrease in these parameters observed in Bmal1 −/− in vivo may not only be caused by the reproductive female organs but also from excess of ROS at the embryo level. Finally, we observed that ablation of Bmal1 in ESCs affected their metabolism by reducing glycolysis and increasing oxidative phosphorylation (OXPHOS). In line with this observation, using genome-wide immunoprecipitation-based techniques, BMAL1 has been shown to target genes related to cellular metabolism in somatic cells (Hatanaka et al, 2010;Wu et al, 2017;Reinke & Asher, 2019), therefore pointing to a potential direct regulation of metabolic genes by BMAL1 through chromatin binding. Although until recently changes in metabolism were thought to be a consequence BMAL1 links metabolism & differentiation Ameneiro et al. https://doi.org/10.26508/lsa.201900534 vol 3 | no 5 | e201900534 9of15