BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells

Although pluripotent cells lack circadian oscillations, this study reveals a novel function of the master circadian regulator BMAL1 in metabolism and early differentiation of embryonic stem cells.

In this manuscript, the authors demonstrated that BMAL1 was dispensable for ESC self-renewal, but played a role in the exit of pluripotency and cell differentiation commitment. Mechanistically, the authors illustrated that BMAL1 participated in regulating energy metabolism and mitochondrial function. Overall, the part that BMAL1 was involved in the metabolic regulation is interesting. However, the other parts of this paper are not very conclusive due to either lack of statistical analysis or bad quality of the data. More experiments is needed to clarify these issues. Concerns: (1) In Fig1A, the authors examined the RNA level of Bmal1, Clock, Nanog and Pou5f1 in MEFs and ESCs. Since RNA level could not fully represent protein level, western blotting analysis of these proteins and statistical analysis is required.  (4) In the text, the authors claimed that the self-renewal of ESCs was not greatly affected by deletion of BMAL1 neither on colony number nor on the colony morphology (Fig 2F and 2G). However, in Fig2G, it seems that knockout of Bmal1 affects the differentiation. Convincing conclusion should be stated. In addition, statistical analysis is missing in Fig 1F. (5) ESC pluripotency analysis is required in in vivo experiment to check whether BMAL1 KO ESCs can form the three germ layers. (6) In Fig 5D, BMAL1 deficiency resulted in abnormal in vitro gastrulation, which lacks statistical analysis. More evidences are needed to confirm the relationship between aberrant upregulation of three-germ-layer markers and the deficiency of polarization or elongation during in vitro gastrulation. (7) The authors mentioned that close correlation of the transcriptional changes of regulators related with cellular differentiation and cellular metabolism including Zfx and Polycomb members (i.e. Eed and Suz12) were found upon depletion of Bmal1 and Pou5f1 (Fig EV3A) . Experiments including RT-qPCR or western blotting of these regulators in Bmal1 KO and Pou5f1 KO cells should performed to support the conclusion. The studies by Ameneiro and colleagues investigate the function of circadian gene BMAL1 in regulating embryonic stem cells (ESC) differentiation and metabolism. They first show that BMAL1 is dispensable for ESC pluripotency. They further show using both RNA interference and CRISPR-Cas9 that loss of BMAL1 does not significantly alter pluripotency in ESCs. Then through a set of experiments they propose that BMAL1 may be involved in early decisions during pluripotency exit. They further provide evidence that loss of BMAL1 impairs ESC differentiation and alters ESC gene expression (upregulation of ectoderm vs downregulation of mesoderm and endoderm genes). They also use in vitro gastruloid formation to show that BMAL1 KO cells do not elongate properly in vitro. Finally, they show that loss of BMAL1 is associated with metabolic alterations. This manuscript address an important question regarding BMAL1 regulation of ESC fate determination. The authors have performed enormous amount of work using a combination of approaches and have produced interesting data. However, their conclusion is some instances is premature or not supported by the data.
My comments are as follow: (1) Figure 6: please clarify how this figure supports BMAL1 control of transcriptional regulation of ESC differentiation. As the authors have noted loss of BMAL1 results in modulations of cellular status (pluripotency vs differentiation). The transcriptomic changes may be just a reflection of cellular differentiation and not the cause. The data does not support: "Thus, these results show that BMAL1 is involved in transcriptional regulation of developmental processes in ESCs" as stated by the authors. (2) Figure 3: the results in this figure are not clear. Experiments should be explained in more detail and results flushed out.
(3) Figure 7: BMAL1 KO results in modifications of metabolic gene expression (7A). The authors should clarify the identity of these genes (glycolytic? mitochondrial/TCA etc?). This is important in order to support their next finding. In addition, how do they reconcile reduced glycolysis in BMAL KO ESCs with their differentiation status? (4) Figure 4E: BMAL1 KO shows upregulation of ectoderm genes associated with downregulation of endoderm, mesoderm genes. Does loss of BMAL1 enhance the ESC differentiation to ectoderm (at the expense of other germ layers)? Minor: • The authors should quantify the bands in WB ( Figure 1B, 2B etc).
• Figure 2D, it is important to show protein expression.
• Figure 5C-D: please provide quantitative data measuring the size of the gastruloid structure and possibly the elongation/polarization. • How do gene expressions in Figure 5B relate to that in 5E? • The authors should explain in detail of how the results from Figure 7F-7G have been generated.

Appeal Request
October 25, 2019 Dear Dr. Leibfried, Thanks for your kind efforts in handling our manuscript and sending us your thoughtful editorial comments.
After carefully reading through your decision letter as well as reviewers' comments, we took time to reevaluate all our data-sets (including some new data-sets) and were able to put together the rebuttal document (see attached PDF file) with the following highlights: 1) We feel that the two reviewers recognized the significance and conceptual novelty of our findings to the field and that they have provided many constructive suggestions for us to improve our manuscript; 2) We are in a very strong position to address all their concerns within a reasonable time frame. In fact, we have many data-sets available already to address many of their concerns (please see the rebuttal PDF file). These data-sets are now ready to be incorporated into the revised manuscript.
3) We have a solid and realistic plan to address the remaining issues raised by both reviewers (please see our rebuttal PDF file). 4) We feel that some of the concerns raised by the reviewers are caused by misunderstandings with another manuscript (i.e. data quality not sufficient and conclusions not well supported).
I sincerely hope that you will also find our rebuttal reasonable and our experimental plan realistic and promising to improve our story towards a final product (i.e. a revised manuscript) that meets the publication standards of Life Science Alliance.
Thanks again for your time and effort. Looking forward to hearing from you soon,

Best regards, Miguel Fidalgo
We appreciate the constructive and in-depth comments of the two reviewers, which have helped us to design and perform additional experiments, thus further clarifying the presentation of our data and strengthening the conclusions of our manuscript. Here we have provide detailed point-by-point responses to reviewers' comments together with our additional information and new experimental data. For the reviewers' convenience, we have highlighted in red those new data figures that have been incorporated in the revised manuscript and are also mentioned in this rebuttal.

Reviewer #1 (Comments to the Authors (Required)):
In this manuscript, the authors demonstrated that BMAL1 was dispensable for ESC selfrenewal, but played a role in the exit of pluripotency and cell differentiation commitment.
Mechanistically, the authors illustrated that BMAL1 participated in regulating energy metabolism and mitochondrial function. Overall, the part that BMAL1 was involved in the metabolic regulation is interesting.
We thank the reviewer for his/her positive comments and appreciation of our work.
However, the other parts of this paper are not very conclusive due to either lack of statistical analysis or bad quality of the data. More experiments are needed to clarify these issues.
We apologize for having forgotten to perform the statistical analysis of some panels in the first version of the manuscript. We have now fixed this in the revised current version of the manuscript. On the other hand, we respectfully disagree with the reviewer on the comment that "bad quality of the data" and we think this may be due to a confusion (please see below point no. #1.2). Concerns: (1.1) In Fig1A, the authors examined the RNA level of Bmal1, Clock, Nanog and Pou5f1 in MEFs and ESCs. Since RNA level could not fully represent protein level, western blotting analysis of these proteins and statistical analysis is required. This is a good suggestion from the reviewer. Indeed, we have followed his/her indications and analysed the protein expression levels of BMAL1, NANOG and POU5F1 (Additional Figure   1A) ( Figure 1A). Importantly, consistent with the RNA expression ( Figure 1A in original manuscript version) we found that BMAL1 protein levels are also significantly higher in pluripotent ESCs than in somatic MEFs (Additional Figure 1B) (Figure 1A). We respectfully disagree with the reviewer and as stated above, we think that there is a confusion when he/she states that "the quality of western blotting in Fig2A and Fig 2B are bad" because: i) Figure 2A in the previous version of the manuscript was not a Western Blot (WB), but instead it showed a schematic representation for the CRISPR/Cas9 strategy used in our study.
ii) The WB showed in Figure 2B   We apologize that in our current version of the manuscript we mislabeled the deletion as remarked by the reviewer. Now we have fixed this issue (Additional Figure 3) (Figure 2A). (1.4) In the text, the authors claimed that the self-renewal of ESCs was not greatly affected by deletion of BMAL1 neither on colony number nor on the colony morphology (Fig 2F and 2G).
However, in Fig2G, it seems that knockout of Bmal1 affects the differentiation. Convincing conclusion should be stated. In addition, statistical analysis is missing in Fig 1F. We would like to apologize if our choice of words when we said "was not greatly affected" could generate confusion. Following the reviewer's recommendation and to avoid any misinterpretation, we have now rephrased it indicating the specific percentages in the text as On the other hand, to address his/her suggestion regarding to lack of statistical analysis in Figure 1F in the previous version, we have now added that piece of information in the current revised manuscript (Additional We thank the reviewer for his/her suggestion. Following the reviewer´s suggestion we have now analysed the ability of Bmal1 KO ESCs to form the three layers in vivo using teratoma assays. For that purpose, eight female Swiss Nude mice at 5 weeks of age were injected subcutaneously in their flanks with wild type or Bmal1 KO ESCs. Teratomas were excised 4 weeks after injection, measured and processed for staining with hematoxylin and eosin (H&E).
Our results show that Bmal1 KO ESCs can form teratomas containing differentiated tissues from the three germinal layers (Additional Figure 5) (Figures 4A and 4B). Notably, this data is consistent with our results in the previous manuscript version, which show that Bmal1 KO ESCs are pluripotent given that: i) BMAL1 is dispensable for the maintenance of the pluripotent cellular state (Figures 1 and 2 in the previous version); ii) even though presence of BMAL1 in vitro is required for proper expression of the lineage markers of ectoderm, mesoderm and endoderm germ layers ( Figure 4E in the previous version), Bmal1 KO ESCs are capable of forming embryoid bodies (EBs) (Figures 4C and 4D in the previous version). Altogether, our data support that Bmal1 KO ESCs are pluripotent and that loss of Bmal1 leads to defects that interfere with normal expression patterns of lineage specification markers, which are detectable during in vitro differentiation. However, we cannot discard the possibility that during in vivo differentiation (i.e. by teratoma assays) there are also aberrant expression of specific markers of one or more germ layers even though Bmal1 KO teratomas seem grossly normal.
Additional Figure 4. 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 (n=3). Bars represent mean ± s.e.m. Significantly differences calculated using two-tailed unpaired Student's t-test analysis. ns: not significant. We apologize for not having included the statistical analysis in Figure 5D in the previous version of the manuscript. Following the reviewer´s suggestion we have now performed additional gastruloid formation assays and included statistical analysis (Additional Figure 6A) ( Figure 5E). Consistent with our previous data, we found that absence of BMAL1 significantly impact in gastruloid formation efficiency compared to wild-type (7.14% vs 41.07%, respectively). Additionally, to further characterize the effect of Bmal1 depletion in elongation during in vitro gastrulation, we have now analysed the size of Bmal1 WT or KO ESC-derived aggregates during gastruloid formation assay (Additional Figure 6B) ( Figure 5D). Our data supports that absence of BMAL1 impairs the elongation process in vitro, giving rise to cellular aggregates displaying reduced size at 120 hours. Likewise, we observed altered transcriptional dynamics of several members of the Hoxd gene cluster (Additional Figure 6C) ( Figure 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 by the seminal work of Beccari and collaborators [1]. Thus, these results are in line with the (1.8) Scale bar is missing in most of the staining data, such as in Fig1C, 1D, 5C, 7H, EV1B.
We apologize as the scale bar size that we used in the previous manuscript version was too small and lead the confusion that they were not present. In order to improve the clarity of the

Reviewer #2 (Comments to the Authors (Required)):
The studies by Ameneiro and colleagues investigate the function of circadian gene BMAL1 in regulating embryonic stem cells (ESC) differentiation and metabolism. They first show that BMAL1 is dispensable for ESC pluripotency. They further show using both RNA interference and CRISPR-Cas9 that loss of BMAL1 does not significantly alter pluripotency in ESCs. Then through a set of experiments they propose that BMAL1 may be involved in early decisions during pluripotency exit. They further provide evidence that loss of BMAL1 impairs ESC differentiation and alters ESC gene expression (upregulation of ectoderm vs downregulation of mesoderm and endoderm genes). They also use in vitro gastruloid formation to show that BMAL1 KO cells do not elongate properly in vitro. Finally, they show that loss of BMAL1 is associated with metabolic alterations. This manuscript address an important question regarding BMAL1 regulation of ESC fate determination. The authors have performed enormous amount of work using a combination of approaches and have produced interesting data. However, their conclusion is some instances is premature or not supported by the data.
We thank the reviewer for these positive comments on our manuscript.
My comments are as follow: (2.1) Figure 6: please clarify how this figure supports BMAL1 control of transcriptional regulation of ESC differentiation. As the authors have noted loss of BMAL1 results in modulations of cellular status (pluripotency vs differentiation). The transcriptomic changes may be just a reflection of cellular differentiation and not the cause. The data does not support: "Thus, these results show that BMAL1 is involved in transcriptional regulation of developmental processes in ESCs" as stated by the authors.
We appreciate the reviewer´s comment and we apologize for the overstatement that "these results show that BMAL1 is involved in transcriptional regulation of developmental processes in ESCs". It is true that while BMAL1 has been widely reported to function as a transcriptional regulator in other cellular contexts (Reviewed in [2]), in this manuscript we have not directly Following the reviewer´s suggestion, we have now improved the section regarding Figure 3 (in the previous version) by providing more detailed information about the in silico analysis using our previously published genome-wide RNAi screening, which was designed to identify factors important in early stages of differentiation [6]. On the other hand, although how cellular metabolism at the molecular level directly influences cell fate decisions is an emerging area of study, accumulating evidence have shown active roles of metabolism influencing gene expression that is key for self-renewal and differentiation of pluripotent cells, including mouse and human ESCs [7][8][9][10][11]. In particular, it is well documented that increased OXPHOS and ROS levels accompany the differentiation process [8,10,11]. Indeed, the balance between glycolysis and OXPHOS is critical for modulating the differentiation potential of pluripotent cells [8,10,11]. Importantly, as the reviewer mentioned, This is an interesting remark from the reviewer. As we mentioned in the text of the previous version of the manuscript: "Indeed, 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 to WT EBs (Fig 4E).These results indicate that Bmal1 is required for ESC differentiation to properly establish germ layer specific transcriptional programs." As pointed by the reviewer, Bmal1 depletion induced ectoderm marker expression and reduced endoderm marker expression both in EB and gastruloid differentiation (Figures 4E and 5E in the previous version). Nevertheless, we cannot definitely conclude that Bmal1 loss-of-function skews differentiation towards ectoderm at the expense of mesoderm and endoderm germ layers, given that we observed a differentiation assay-dependent effect of BMAL1 on several ectoderm (i.e. Sox1) and mesoderm (i.e. Cxcl12, Mixl1 and T) markers. Altogether, from our data we can only conclude that BMAL1 loss influences the differentiation potential of ESCs in vitro, and future studies will be required to further characterize in detail the contribution of BMAL1 to the transcriptional regulation taking place during germ layers specification. Minor: (2.5) The authors should quantify the bands in WB ( Figure 1B, 2B etc).
Following the reviewer's suggestion, we have now quantified all WBs in the previous manuscript version: Figure 1B (Additional Figure 11) ( Figure 1B) and Figure 2B (Additional Figure 2) (Figure 2B).
(2.6) Figure 2D, it is important to show protein expression.
While the protein expression of both NANOG and POU5F1 was already shown in Figure 2B (previous version), following the reviewer´s suggestion we have now included the western blot analysis of ZFP42 protein abundance in wild type and Bmal1 KO ESCs (Additional Figure 2) ( Figure 2B). (2.8) How do gene expressions in Figure 5B relate to that in 5E?
In Figure 5B (previous version) we depicted the expression level of Bmal1, the pluripotency marker Nanog and the differentiation markers Pax3, Gata6 and Mixl1 after 120h of gastruloid formation using wild type ESCs (Relative expression is shown as relative to 0h). On the other hand, in Figure 5E  Thank you for your recent correspondence regarding our decision on your manuscript entitled "BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells". We appreciate the revision outline provided and will be happy to consider your work further here. Please note that we will need strong support on the revised version from experts. Not addressing pluripotency in vivo may be seen as impeding publication. It is difficult to predict such outcome at this pre-revision stage, but I wanted to be very open about this so that you consider your options carefully.

Reviewer #1 (Comments to the Authors (Required)):
In this manuscript, the authors demonstrated that BMAL1 was dispensable for ESC selfrenewal, but played a role in the exit of pluripotency and cell differentiation commitment.
Mechanistically, the authors illustrated that BMAL1 participated in regulating energy metabolism and mitochondrial function. Overall, the part that BMAL1 was involved in the metabolic regulation is interesting.
We thank the reviewer for his/her positive comments and appreciation of our work.
However, the other parts of this paper are not very conclusive due to either lack of statistical analysis or bad quality of the data. More experiments are needed to clarify these issues.
We apologize for having forgotten to perform the statistical analysis of some panels in the first version of the manuscript. We have now fixed this in the revised current version of the manuscript. On the other hand, we respectfully disagree with the reviewer on the comment that "bad quality of the data" and we think this may be due to a confusion (please see below point no. #1.2). Concerns: (1.1) In Fig1A, the authors examined the RNA level of Bmal1, Clock, Nanog and Pou5f1 in MEFs and ESCs. Since RNA level could not fully represent protein level, western blotting analysis of these proteins and statistical analysis is required. This is a good suggestion from the reviewer. Indeed, we have followed his/her indications and analysed the protein expression levels of BMAL1, NANOG and POU5F1 (Additional Figure   1A) (Figure 1A). Importantly, consistent with the RNA expression ( Figure 1A in original manuscript version) we found that BMAL1 protein levels are also significantly higher in pluripotent ESCs than in somatic MEFs (Additional Figure 1B) (Figure 1A). ii) The WB showed in Figure 2B  We apologize that in our current version of the manuscript we mislabeled the deletion as remarked by the reviewer. Now we have fixed this issue (Additional Figure 3) (Figure 2A).
(1.4) In the text, the authors claimed that the self-renewal of ESCs was not greatly affected by deletion of BMAL1 neither on colony number nor on the colony morphology (Fig 2F and 2G).
However, in Fig2G, it seems that knockout of Bmal1 affects the differentiation. Convincing conclusion should be stated. In addition, statistical analysis is missing in Fig 1F. We would like to apologize if our choice of words when we said "was not greatly affected" On the other hand, to address his/her suggestion regarding to lack of statistical analysis in (

1.5) ESC pluripotency analysis is required in in vivo experiment to check whether BMAL1 KO
ESCs can form the three germ layers.
We thank the reviewer for his/her suggestion. Following the reviewer´s suggestion we have now analysed the ability of Bmal1 KO ESCs to form the three layers in vivo using teratoma assays. For that purpose, eight female Swiss Nude mice at 5 weeks of age were injected subcutaneously in their flanks with wild type or Bmal1 KO ESCs. Teratomas were excised 4 weeks after injection, measured and processed for staining with hematoxylin and eosin (H&E).
Our results show that Bmal1 KO ESCs can form teratomas containing differentiated tissues from the three germinal layers (Additional Figure 5) (Figures 4A and 4B). Notably, this data is consistent with our results in the previous manuscript version, which show that Bmal1 KO ESCs are pluripotent given that: i) BMAL1 is dispensable for the maintenance of the pluripotent cellular state (Figures 1 and 2 in the previous version); ii) even though presence of BMAL1 in vitro is required for proper expression of the lineage markers of ectoderm, mesoderm and endoderm germ layers ( Figure 4E in the previous version), Bmal1 KO ESCs are capable of forming embryoid bodies (EBs) (Figures 4C and 4D in the previous version). Altogether, our data support that Bmal1 KO ESCs are pluripotent and that loss of Bmal1 leads to defects that interfere with normal expression patterns of lineage specification markers, which are detectable during in vitro differentiation. However, we cannot discard the possibility that during in vivo differentiation (i.e. by teratoma assays) there are also aberrant expression of specific markers of one or more germ layers even though Bmal1 KO teratomas seem grossly normal.
Additional Figure 4. 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 (n=3). Bars represent mean ± s.e.m. Significantly differences calculated using two-tailed unpaired Student's t-test analysis. ns: not significant. We apologize for not having included the statistical analysis in Figure 5D in the previous version of the manuscript. Following the reviewer´s suggestion we have now performed additional gastruloid formation assays and included statistical analysis (Additional Figure 6A) ( Figure 5E). Consistent with our previous data, we found that absence of BMAL1 significantly impact in gastruloid formation efficiency compared to wild-type (7.14% vs 41.07%, respectively). Additionally, to further characterize the effect of Bmal1 depletion in elongation during in vitro gastrulation, we have now analysed the size of Bmal1 WT or KO ESC-derived aggregates during gastruloid formation assay (Additional Figure 6B) ( Figure 5D). Our data supports that absence of BMAL1 impairs the elongation process in vitro, giving rise to cellular aggregates displaying reduced size at 120 hours. Likewise, we observed altered transcriptional dynamics of several members of the Hoxd gene cluster (Additional Figure 6C) ( Figure 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 by the seminal work of Beccari and collaborators [1]. Thus, these results are in line with the

Reviewer #2 (Comments to the Authors (Required)):
The studies by Ameneiro and colleagues investigate the function of circadian gene BMAL1 in regulating embryonic stem cells (ESC) differentiation and metabolism. data. However, their conclusion is some instances is premature or not supported by the data.
We thank the reviewer for these positive comments on our manuscript.
My comments are as follow: (2.1) Figure 6: please clarify how this figure supports BMAL1 control of transcriptional regulation of ESC differentiation. As the authors have noted loss of BMAL1 results in modulations of cellular status (pluripotency vs differentiation). The transcriptomic changes may be just a reflection of cellular differentiation and not the cause. The data does not support: "Thus, these results show that BMAL1 is involved in transcriptional regulation of developmental processes in ESCs" as stated by the authors.
We appreciate the reviewer´s comment and we apologize for the overstatement that "these results show that BMAL1 is involved in transcriptional regulation of developmental processes in ESCs". It is true that while BMAL1 has been widely reported to function as a transcriptional regulator in other cellular contexts (Reviewed in [2]), in this manuscript we have not directly  (Figures EV1A and 2D) or protein levels ( Figures   1B, 1D, EV1B Following the reviewer´s suggestion, we have now improved the section regarding Figure 3 (in the previous version) by providing more detailed information about the in silico analysis using our previously published genome-wide RNAi screening, which was designed to identify factors important in early stages of differentiation [6]. This is another interesting remark from the reviewer. Following his/her suggestion, we provide in the current manuscript version a more detailed analysis of the expression levels of metabolic-related genes affected by Bmal1 depletion in ESCs (Additional Figure 10A) ( Figure 7A).
On the other hand, although how cellular metabolism at the molecular level directly influences cell fate decisions is an emerging area of study, accumulating evidence have shown active roles of metabolism influencing gene expression that is key for self-renewal and differentiation of pluripotent cells, including mouse and human ESCs [7][8][9][10][11]. In particular, it is well documented that increased OXPHOS and ROS levels accompany the differentiation process [8,10,11]. Indeed, the balance between glycolysis and OXPHOS is critical for modulating the differentiation potential of pluripotent cells [8,10,11]. Importantly, as the reviewer mentioned, This is an interesting remark from the reviewer. As we mentioned in the text of the previous version of the manuscript: "Indeed, 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 to WT EBs (Fig 4E).These results indicate that Bmal1 is required for ESC differentiation to properly establish germ layer specific transcriptional programs." As layers, given that we observed a differentiation assay-dependent effect of BMAL1 on several ectoderm (i.e. Sox1) and mesoderm (i.e. Cxcl12, Mixl1 and T) markers. Altogether, from our data we can only conclude that BMAL1 loss influences the differentiation potential of ESCs in vitro, and future studies will be required to further characterize in detail the contribution of BMAL1 to the transcriptional regulation taking place during germ layers specification. Minor: (2.5) The authors should quantify the bands in WB ( Figure 1B, 2B etc).
Following the reviewer's suggestion, we have now quantified all WBs in the previous manuscript version: Figure 1B (Additional Figure 11) ( Figure 1B) and Figure 2B (Additional Figure 2) ( Figure 2B).
(2.6) Figure 2D, it is important to show protein expression.
While the protein expression of both NANOG and POU5F1 was already shown in Figure 2B (previous version), following the reviewer´s suggestion we have now included the western blot analysis of ZFP42 protein abundance in wild type and Bmal1 KO ESCs (Additional Figure 2) ( Figure 2B). (2.8) How do gene expressions in Figure 5B relate to that in 5E?
In Figure 5B (previous version) we depicted the expression level of Bmal1, the pluripotency marker Nanog and the differentiation markers Pax3, Gata6 and Mixl1 after 120h of gastruloid formation using wild type ESCs (Relative expression is shown as relative to 0h). On the other hand, in Figure 5E   Thank you for submitting your manuscript entitled "BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells" to Life Science Alliance. One of the original reviewers evaluated your revised manuscript. As you will see, the reviewer still raises significant concerns that preclude publication here at this stage.
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Thank you for this interesting contribution to Life Science Alliance. We are looking forward to receiving your revised manuscript. We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** This resubmitted version has addressed some of major concerns from reviewers and the authors have performed a number of additional assays according to suggestions or criticisms by the reviewers, which provides better support for the authors' claims that BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells. However, there are still some concerns which are related to improvement of the quality and presentation of the data to make the conclusions more convincing.
Major concerns: 1. Although the author argued about the quality of western blotting, the bands of ZFP42 in Fig 2B  ran into a line, which makes it difficult to quantify the intensity. The blotting of actin also seems uneven leading to inaccurate statistics. In addition, the blotting in Fig 1B looks like over-adjusted contrast. And at least three times blotting should be performed to obtain statistics. As main figures they seems a little sloppy and better quality of blotting should be provided. 2. To confirm the regulatory effect of BMAL1 on Nanog promoter activity, the quantifications of in Fig 3C should be provided. To further confirm that BMAL1 directly binds to Nanog promoter, ChIP-qPCR should also be performed.  Figure 7A show that the repeatability of some genes in the two WT and BMAL1 KO groups is not high. Based on this, it is not reliable to conclude that BMAL1 regulates the mitochondrial complex I-V and the tricarboxylic acid cycle (TCA or Krebs cycle). It is better to present with selected high repeatability genes in mitochondrial complex I-V and TCA and confirm some of them using RT-qPCR.

March 30, 2020
We would like to thank the reviewer for his/her positive comments on our work, and his/her feedback, which has helped further clarify and enhance our manuscript. Here we provide our responses to each specific comment and suggestion raised by the reviewer and the editor.

Reviewer #1 (Comments to the Authors (Required)):
This resubmitted version has addressed some of major concerns from reviewers and the authors have performed a number of additional assays according to suggestions or criticisms by the reviewers, which provides better support for the authors' claims that BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells. However, there are still some concerns which are related to improvement of the quality and presentation of the data to make the conclusions more convincing.
Major concerns: 1. Although the author argued about the quality of western blotting, the bands of ZFP42 in Fig   2B ran into a line, which makes it difficult to quantify the intensity. The blotting of actin also seems uneven leading to inaccurate statistics. In addition, the blotting in Fig 1B looks like overadjusted contrast. And at least three times blotting should be performed to obtain statistics.
As main figures they seems a little sloppy and better quality of blotting should be provided.
Following the reviewer´s suggestion we provide now a better ZFP42 blot in Fig S2E and have replaced ACTIN by βTUBULIN as a loading control in Fig 2B. On the other hand, we realized that during the conversion of the figures to .pdf format there was a decrease in the quality of some panels which could give a bad impression. We now provide better image quality for all figures including the Western Blots, and we also provide statistics from three independent experiments for Fig 1B. 2. To confirm the regulatory effect of BMAL1 on Nanog promoter activity, the quantifications of in Fig 3C should be provided. To further confirm that BMAL1 directly binds to Nanog promoter, ChIP-qPCR should also be performed.
We provide now the quantification of three independent experiments in Fig 3D, further supporting that BMAL1 loss significantly decreases the percentage of Nanog-GFP positive cells under mild retinoic acid-induced differentiation conditions. Thus, these results identify BMAL1 as a regulator of Nanog expression during the exit of pluripotency. However, as we mention in the current manuscript version, future studies are warranted to investigate whether BMAL1 regulates Nanog promoter activity in a direct or indirect manner.
3. The two photographs of BMAL1 WT and BMAL1 KO teratoma in Fig 4A differ significantly in magnification.
We apologize for this, and we have now fixed this issue. 4. The structure of the three germ layers in Fig 4B is unclear. Three germ layers should be labelled with specific markers of mesoderm, ectoderm and endoderm.
We apologize for the bad quality of the final Fig 4B after  5. Statistical analysis should be performed in Fig 5G. We provide now statistical analysis in Fig 5G. 6. The RNA-seq heatmap results in Figure 7A show that the repeatability of some genes in the two WT and BMAL1 KO groups is not high. Based on this, it is not reliable to conclude that BMAL1 regulates the mitochondrial complex I-V and the tricarboxylic acid cycle (TCA or Krebs cycle). It is better to present with selected high repeatability genes in mitochondrial complex I-V and TCA and confirm some of them using RT-qPCR.
In the current manuscript version we provide a new heatmap in Fig 7A and validation of some of them in Fig S4C following the reviewer´s suggestion.

Editor:
-Please make sure that the author order in our submission system matches the one depicted in the manuscript text We have made sure that the author order both in the submission system and the manuscript is the same.
-Please provide a "summary blurb" within our submission system We now provide a summary blurb in the submission system. We have fixed this accordingly.
- Figure EV1 (should be Figure S1) should have a callout for panel C (spelling mistake) This has been corrected in page 32.
-Please provide the manuscript text in docx format The manuscript text has been provided in docx format.
-Please add a scale bare to Figure 4B The scale bars in Fig 4B have  Thank you for submitting your revised manuscript entitled "BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells". We appreciate the introduced changes and would thus be happy to publish your paper in Life Science Alliance. Before sending you the official acceptance letter, please log in one more time to move all files to the next manuscript version and to fill in the electronic license to publish associated with that version.
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