Single-cell transcriptomic atlas of enteroendocrine cells along the murine gastrointestinal tract

Christopher A. Smith; Elisabeth A. A. O’Flaherty; Nunzio Guccio; Austin Punnoose; Tamana Darwish; Jo E. Lewis; Rachel E. Foreman; Joyce Li; Richard G. Kay; Alice E. Adriaenssens; Frank Reimann; Fiona M. Gribble

doi:10.1371/journal.pone.0308942

Peer Review History

Original SubmissionJanuary 19, 2024
22 Feb 2024 Decision Letter - Mohammad Sadegh Taghizadeh, Editor PONE-D-24-02587Single-cell transcriptomic atlas of enteroendocrine cells along the murine gastrointestinal tractPLOS ONE Dear Dr. Gribble, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Apr 07 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Enteroendocrine cells (EECs) are rare hormone-producing cells in the intestinal epithelium. Distinct subtypes of EECs produce unique combinations of hormones, which regulate metabolism, bowel movement and appetite. Due to their scarcity, defining EEC heterogeneity at the transcriptome level has been challenging. In this study, the authors utilize a genetically encoded EEC reporter to generate a scRNAseq atlas comprising ~20K EECs from the proximal to distal mouse gastrointestinal tract. They describe regional variations in EEC subtype abundance, and showcase several GPCRs with a biased expression in specific EEC subtypes. As functional validation, they demonstrate that a specific GPCR agonist induces EEC activation in vitro. Overall, this study provides a comprehensive resource for the EEC field. Due to the existence of multiple single cell RNA atlases in the mouse gut that cover many EECs, this advance is however rather incremental. Please find below my point-by-point suggestions. - In figure 3: It is unclear to what extent reclustering based on GPCR expression allowed the authors to discover differences in GPCR distribution across EEC subtypes. It seems likely that differential expression on the initial clustering would have revealed the same. - Figure 4: The authors should include measurements of other hormones in response to Mc4r agonists, to stress the Cck-specific signaling. - The authors succinctly write in lines 418/419 that the identification of Mc4r in I cells is interesting, as previous work showed its role in GLP-1 release. Reference 24 does indeed show this/L cell expression, but also evidenced I cell expression using a CCK-reporter mouse. The authors should therefore mention Mc4r was previously found to be enriched in I cells. - It appears that in figure 4B the legend erroneously indicates that the dot size represents the percentage of cells expressing a certain hormone, instead of a GPCR. - Is there any bias in isolating certain EEC subtypes using the NeuroD1 tracer? All EEC subsets seem to be present, but a statement/reference on this would be helpful. Stomach endocrine cell distribution appears different than reported (should be more biased for EC/ECL). In addition: could the authors comment on the distribution of ECL cells across GI tract regions. It is present in equal numbers across the GI tract, while exclusive presence in the stomach is expected. This is also stated in line 294 (the authors similarly state this is absolute for G cells, which again is found in other regions). - I could not find any statement on the average number of reads/genes detected per cell. This would be important, including a comparison to previous murine gut sequencing studies. As much as the number of cells sequenced, the depth per cell is key to identify lowly expressed genes such as GPCRs. Reviewer #2: This manuscript from an eminent laboratory on enteroendocrine cell (EEC) biology compares mouse stomach, proximal and distal small intestine, caecum, and colonic EEC transcriptomes at single-cell resolution. Knowing that EEC physiology is tied to G protein-coupled receptor (GPCR) signaling, the authors highlight differences and overlaps in GPCR mRNA expression across EECs and GI regions. They show that a range of nutrients trigger Ca++ flux in ileal L cells differentiated in vitro. Several scRNA-seq profiles of EECs have been reported, including some by this group, but this is the first to consider the full length of the mouse GI tract together (colonic EEC data are taken from a previous study that is cited). The work is performed to the high standards of the senior authors, who are recognized experts in the field. The quality of the data seems high and the story is presented clearly. Although certain conclusions, such as that most EECs express more than one bioactive peptide, have been reported previously, these aggregate data from >20,000 cells will make a useful contribution to the literature. Demonstration of cholecystokinin (CCK) release in response to multiple stimulants (Fig. 3B) adds one physiologic dimension to the study. My only question pertains to the second physiologic dimension: Ca++ flux that various agonists trigger in Venus-labeled L cells isolated from differentiated ileal organoids (Fig. 4C-E). The range of values shown on the graph (Fig. 4D) raising questions about what is real signal and what is background. The implication of the dashed line on the graph is that any R/R0 value >1.0 is signal and therefore legitimate to represent in the Venn diagram (Fig. 4E) as a “reactive” cell. However, the example trace in Fig. 4C suggests that many of the values clustered near 1.0 may represent background noise. Only some of those cells may merit inclusion within the Venn representation. The authors should represent the data with clear and conservative definitions of real signals or establish that even small deviations over 1.0 are real. Data from a larger number of cells may also add clarity and rigor. Reviewer #3: The study entitled “Single-cell transcriptomic atlas of enteroendocrine cells along the murine gastrointestinal tract” by Smith et al. explores enteroendocrine cells (EECs) diversity and expression along the gastrointestinal tract by single-cell RNA-sequencing of GFP-labeled sorted EECs from different tissues and locations along the tract (from the stomach to rectum). The authors identified ten main EEC clusters within the different tissues with similar transcriptional profiles, showing multiple expressions of a few gut hormones in specific subsets. The authors investigated GPCRs of the different EEC types and showed that GPCR expression correlates with the gene expression of the cells. The authors propose to utilize this approach to test other specific factors within the cells, such as transcription factors, to investigate the diverse EEC population and properties. My only comment is the disadvantage of the EEC isolation method that relies on a GFP reporter under the specific TF associated with most EECs, NeuroD1. Using this method allows the enrichment of EECs from EpCAM+ cells, which are about 2-4% of total EpCAM, but could miss EEC subsets that are NeuroD1 negative. It would be interesting to compare the data obtained from this study to an unbiased analysis of EECs extracted from the total epithelial cells of one tissue. In conclusion, the authors performed elegant experiments to characterize the EEC subsets of different gastrointestinal compartments and test for hormones and GPCRs associated with them. This manuscript will surely provide valuable data and guidelines for further research in EEC, food intake, and metabolic disorders in general. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. 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Revision 1
4 Jun 2024 Author Response Reviewer #1: Enteroendocrine cells (EECs) are rare hormone-producing cells in the intestinal epithelium. Distinct subtypes of EECs produce unique combinations of hormones, which regulate metabolism, bowel movement and appetite. Due to their scarcity, defining EEC heterogeneity at the transcriptome level has been challenging. In this study, the authors utilize a genetically encoded EEC reporter to generate a scRNAseq atlas comprising ~20K EECs from the proximal to distal mouse gastrointestinal tract. They describe regional variations in EEC subtype abundance, and showcase several GPCRs with a biased expression in specific EEC subtypes. As functional validation, they demonstrate that a specific GPCR agonist induces EEC activation in vitro. Overall, this study provides a comprehensive resource for the EEC field. Due to the existence of multiple single cell RNA atlases in the mouse gut that cover many EECs, this advance is however rather incremental. Please find below my point-by-point suggestions. - In figure 3: It is unclear to what extent reclustering based on GPCR expression allowed the authors to discover differences in GPCR distribution across EEC subtypes. It seems likely that differential expression on the initial clustering would have revealed the same. Response: We agree with the reviewer that there are different ways in which the GPCR distribution can be analysed across different EEC populations. In S3 Fig we took a traditional approach and analysed individual GPCR expression across different EEC populations defined by their expression of a hormone of interest. As expected, this revealed differential GPCR across EEC populations. The aim of the experiment in figure 3 was to test the reverse hypothesis, that the EEC populations differ by how they signal. We believe this is an interesting alternative approach to compare cell populations, and the finding of differential Mc4r expression in CCK-cells was one of the key outcomes. - Figure 4: The authors should include measurements of other hormones in response to Mc4r agonists, to stress the Cck-specific signaling. Response: We thank the reviewer for making this suggestion and have performed additional experiments to test the hypothesis that Mc4r agonism would not affect secretion of GIP. As predicted, setmelanotide increased secretion of CCK but not of GIP from primary mouse upper small intestinal epithelial cultures. The additional CCK results have been added to the original data, increasing the n-number, and the GIP results are shown in a new subfigure (S3 Fig). As these were upper small intestinal cultures, GLP-1 and PYY levels were too low to quantify, so we restricted the new analysis to CCK and GIP. - The authors succinctly write in lines 418/419 that the identification of Mc4r in I cells is interesting, as previous work showed its role in GLP-1 release. Reference 24 does indeed show this/L cell expression, but also evidenced I cell expression using a CCK-reporter mouse. The authors should therefore mention Mc4r was previously found to be enriched in I cells. Response: We thank the reviewer for noticing that we had not referred to the CCK-cell data in this reference. This is now included, and the text reads: “The identification of Mc4r in I-cells was interesting as this receptor has previously been implicated in GLP-1 release from the distal gut, and has been identified but not functionally characterised in EECs purified from Cck-Cre mice (25, 26)” - It appears that in figure 4B the legend erroneously indicates that the dot size represents the percentage of cells expressing a certain hormone, instead of a GPCR. Response: Thank you, this is now corrected. - Is there any bias in isolating certain EEC subtypes using the NeuroD1 tracer? All EEC subsets seem to be present, but a statement/reference on this would be helpful. Stomach endocrine cell distribution appears different than reported (should be more biased for EC/ECL). In addition: could the authors comment on the distribution of ECL cells across GI tract regions. It is present in equal numbers across the GI tract, while exclusive presence in the stomach is expected. This is also stated in line 294 (the authors similarly state this is absolute for G cells, which again is found in other regions). Response: As also questioned by reviewer 2, we have now systematically examined whether the NeuroD1-Cre reporter labelled EECs in an unbiassed manner. As shown in the new S1 Fig (D, E), we combined our new scRNAseq data with published scRNAseq data from the epithelium of the upper SI (Haber et al 2017) and large intestine (Xu et al 2023). Our EEC populations showed good overlap with the (relatively small) EEC populations in these unbiassed datasets, and we did not identify any EEC populations in the unbiassed data that had not been detected using the NeuroD1 marker. The revised version contains additional methods, and the following text in the results: “We combined the data from our USI and LSI NeuroD1-labelled EECs with published unbiassed datasets (7, 18). EECs from the current study showed good overlap with EEC populations in the unbiassed datasets (S1 Fig), and no additional EEC populations were evident in the full epithelial data that had not been picked up using the NeuroD1-Cre approach.” We thank the reviewer for noticing that the Huang thresholding method (shown in S2 Fig as the method used to define positive cells in Fig 2), calculated that there are more ECL cells than expected in regions beyond the stomach. This is because the method looks at the range of expression of the marker gene across all the cells in the region, and calls those with the highest expression as “positive” even if the expression level is very low. Use of a thresholding method is necessary because transcripts for hormones that are highly expressed in a tissue are detectable in the majority of negative cells, likely because high abundance RNA released from a few burst cells can be picked up in all droplets during the 10X analysis. We prefer not to tweak the Huang thresholding programme manually to generate an expected outcome, so have instead added extra colour coded circles in figure 2 to show average expression of the marker gene in its positive cell population in each region. This shows up as a series of circles across the diagonal, with the colour varying by region e.g. Hdc is now seen to be more highly expressed in the Hdc-positive cells in the stomach than in Hdc-positive cells in the colon/rectum (where it is co-expressed at low levels in L-cells). We hope this provides some additional clarity, and have added the following text to explain the data: “Use of the Huang thresholding method identified similar numbers of EECs as the clustering method for most cell types, but over-estimated ECL cell numbers in regions beyond the stomach where low Hdc expression in other EEC types was called positive by the algorithm (Fig 2 A). “ We have also now presented the cell numbers generated from the clustering in fig1A, as an alternative estimate of cell cluster sizes in different regions of the gut (S1 Fig). This again reveals that the Huang thresholding method has worked well for most cell types, but has broken down for ECL cells in regions where both cell abundance and Hdc expression are low. - I could not find any statement on the average number of reads/genes detected per cell. This would be important, including a comparison to previous murine gut sequencing studies. As much as the number of cells sequenced, the depth per cell is key to identify lowly expressed genes such as GPCRs. Response: Apologies for the omission of this important piece of information. The following text has now been added to the methods: “Of the filtered data, the median (min – max) number of reads per cell were 35,679 (2,140 – 389,084) in the stomach, 15,440 (718 – 261,381) in the USI, 6,462 (650 – 216,128) in the LSI, 5,664 (1,648 – 104,978) in the caecum, and 4,438 (1,402 – 40,537) in the LI dataset. The median (min – max) number of genes per cell were 3,276 (1,002 – 9,330) in the stomach, 3,663 (504 – 9,350) in the USI, 2,487 (501 – 9,367) in the LSI, 2,043 (1,001 – 8,308) in the caecum, and 1,943 (1,001 – 5,935) in the LI dataset.” Reviewer #2: This manuscript from an eminent laboratory on enteroendocrine cell (EEC) biology compares mouse stomach, proximal and distal small intestine, caecum, and colonic EEC transcriptomes at single-cell resolution. Knowing that EEC physiology is tied to G protein-coupled receptor (GPCR) signaling, the authors highlight differences and overlaps in GPCR mRNA expression across EECs and GI regions. They show that a range of nutrients trigger Ca++ flux in ileal L cells differentiated in vitro. Several scRNA-seq profiles of EECs have been reported, including some by this group, but this is the first to consider the full length of the mouse GI tract together (colonic EEC data are taken from a previous study that is cited). The work is performed to the high standards of the senior authors, who are recognized experts in the field. The quality of the data seems high and the story is presented clearly. Although certain conclusions, such as that most EECs express more than one bioactive peptide, have been reported previously, these aggregate data from >20,000 cells will make a useful contribution to the literature. Demonstration of cholecystokinin (CCK) release in response to multiple stimulants (Fig. 3B) adds one physiologic dimension to the study. My only question pertains to the second physiologic dimension: Ca++ flux that various agonists trigger in Venus-labeled L cells isolated from differentiated ileal organoids (Fig. 4C-E). The range of values shown on the graph (Fig. 4D) raising questions about what is real signal and what is background. The implication of the dashed line on the graph is that any R/R0 value >1.0 is signal and therefore legitimate to represent in the Venn diagram (Fig. 4E) as a “reactive” cell. However, the example trace in Fig. 4C suggests that many of the values clustered near 1.0 may represent background noise. Only some of those cells may merit inclusion within the Venn representation. The authors should represent the data with clear and conservative definitions of real signals or establish that even small deviations over 1.0 are real. Data from a larger number of cells may also add clarity and rigor. Response: We thank the reviewer for noticing that we had not included a description of how we defined a cell as a “responder”. The cut-off for defining responders was set at a relatively conservative increase by 10% in the fura-2 ratio during addition of the test compound. Based on extensive previous experience, this cut-off is more likely to generate false negatives than false positives. This has been added to the methods, which now reads: “Cells exhibiting increases in the fura2 fluorescence ratio of >10% during perfusion of the test agent were counted as responders – a definition that is more likely to generate false negatives than false positives.” Reviewer #3: The study entitled “Single-cell transcriptomic atlas of enteroendocrine cells along the murine gastrointestinal tract” by Smith et al. explores enteroendocrine cells (EECs) diversity and expression along the gastrointestinal tract by single-cell RNA-sequencing of GFP-labeled sorted EECs from different tissues and locations along the tract (from the stomach to rectum). The authors identified ten main EEC clusters within the different tissues with similar transcriptional profiles, showing multiple expressions of a few gut hormones in specific subsets. The authors investigated GPCRs of the different EEC types and showed that GPCR expression correlates with the gene expression of the cells. The authors propose to utilize this approach to test other specific factors within the cells, such as transcription factors, to investigate the diverse EEC population and properties. My only comment is the disadvantage of the EEC isolation method that relies on a GFP reporter under the specific TF associated with most EECs, NeuroD1. Using this method allows the enrichment of EECs from EpCAM+ cells, which are about 2-4% of total EpCAM, but could miss EEC subsets that are NeuroD1 negative. It would be interesting to compare the data obtained from this study to an unbiased analysis of EECs extracted from the total epithelial cells of one tissue. Response: We thank the reviewer for this comment, and have now integrated our data with the single cell data from upper small intestine (Haber 2017) and large intestine (Xu 2023). The merged clustering analysis is included in a new S1 Fig D,E. The analyses showed good overlap between the EECs from the different studies, but did not identify any EEC populations in the unbiassed datasets that were not picked up using NeuroD1-Cre as a reporter. We conclude that our analysis did successfully identify all the different EEC populations derived from the gut epithelium in the small intestine, and have added this to the methods and results. The new text in the results is: “We combined the data from our USI and LSI NeuroD1-labelled EECs with published unbiassed datasets (7, 18). EECs from the current study showed good overlap with EEC populations in the unbiassed datasets (S1 Fig), and no additional EEC populations were evident in the full epithelial data that had not been picked up using the NeuroD1-Cre approach.” In conclusion, the authors performed elegant experiments to characterize the EEC subsets of different gastrointestinal compartments and test for hormones and GPCRs associated with them. This manuscript will surely provide valuable data and guidelines for further research in EEC, food intake, and metabolic disorders in general. Response: Thank you for the positive comments Attachments Attachment Submitted filename: Responses to reviewers.docx https://doi.org/10.1371/journal.pone.0308942.r002
21 Jun 2024 Decision Letter - Mohammad Sadegh Taghizadeh, Editor PONE-D-24-02587R1Single-cell transcriptomic atlas of enteroendocrine cells along the murine gastrointestinal tractPLOS ONE Dear Dr. Gribble, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please submit your revised manuscript by Aug 05 2024 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Mohammad Sadegh Taghizadeh, Ph.D. Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed Reviewer #2: (No Response) ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors have addressed my questions and I recommend publication in Plos One. I am convinced this will be a very useful resource to the community. A remaining note on the cell clustering in Figure 2: The authors comment that the Huang thresholding calls cell types based on transcript levels are relative to cell in that particular GI tract region. It may be better to combine all regions for the tresholding and then separately present the data? To me it is still somewhat confusing to present these high HDC+ numbers in the distal gut. This may also resolve some other 'stretched' annotations (e.g., now it appears there are relatively equal number of Gcg+ cells in the proximal and distal gut, INSL5 in the proximal gut..). More GIP than GCG in the distal gut? This is perhaps more a point of preference of how to present this, and I appreciate the addition of average gene expression as a clarification. Reviewer #2: I was the most positive of the 3 referees, with significant concern only about the Fura2A ratios represented in Fig. 4C-E. The authors’ response is that a 10% increase in the ratio is “relatively conservative” and that “based on extensive previous experience,” that “cut-off is more likely to generate false negatives than false positives.” This response and insertion of a sentence to that effect in the Methods are less than satisfactory. Unless I don’t understand the tracing (Fig. 4C) or how values are graphed in Fig. 4D, the distribution of values within any group seems to vary by more than 10% and the responses to Glucose or Tryptophan look very different in Fig. 4C. I don’t challenge the authors’ conservatism, nor do I need to stand in the way of publication, but other readers may share my concern (confusion?) and deserve more clarity about the assay and its signal/noise parameters. I otherwise like this paper and believe it makes a useful contribution to the field. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0308942.r003
Revision 2
17 Jul 2024 Author Response Reviewer #1: A remaining note on the cell clustering in Figure 2: The authors comment that the Huang thresholding calls cell types based on transcript levels are relative to cell in that particular GI tract region. It may be better to combine all regions for the tresholding and then separately present the data? To me it is still somewhat confusing to present these high HDC+ numbers in the distal gut. This may also resolve some other 'stretched' annotations (e.g., now it appears there are relatively equal number of Gcg+ cells in the proximal and distal gut, INSL5 in the proximal gut..). More GIP than GCG in the distal gut? This is perhaps more a point of preference of how to present this, and I appreciate the addition of average gene expression as a clarification. Response: We thank the reviewer for this question, which triggered us to look yet again at the Huang thresholding method. We tried several ways of combining the data, and have now made 2 adjustments to the method: (1) we have combined data from all regions, as the reviewer suggested, with the exception of Ghrl and Sst in the stomach which were so highly expressed that they resulted in background levels in other stomach EECs that were higher than in Ghrl and Sst-expressing cells in the small intestine – this was a major reason we had originally treated each region independently; (2) we have used CPM instead of raw counts when combining the data. The new calculations still identify Hdc-expressing cells in the distal gut, but these are not a distinct cell type, rather there is some Hdc expression in other EECs, particularly enterochromaffin cells and L-cells. We have changed the description of this result to make it clear that this is not a distinct ECL population. The new text reads: “Low levels of Hdc expression were unexpectedly identified in some EECs in the distal intestine, predominantly colocalised in EC cells in the caecum and with L-cell hormones in the large intestine.” Reviewer #2: I was the most positive of the 3 referees, with significant concern only about the Fura2A ratios represented in Fig. 4C-E. The authors’ response is that a 10% increase in the ratio is “relatively conservative” and that “based on extensive previous experience,” that “cut-off is more likely to generate false negatives than false positives.” This response and insertion of a sentence to that effect in the Methods are less than satisfactory. Unless I don’t understand the tracing (Fig. 4C) or how values are graphed in Fig. 4D, the distribution of values within any group seems to vary by more than 10% and the responses to Glucose or Tryptophan look very different in Fig. 4C. I don’t challenge the authors’ conservatism, nor do I need to stand in the way of publication, but other readers may share my concern (confusion?) and deserve more clarity about the assay and its signal/noise parameters. I otherwise like this paper and believe it makes a useful contribution to the field. Response: There is no perfect way of distinguishing a responding cell from a non-responder, since some responses can be subtle and background noise in the traces varies from cell to cell. Attempting to address the reviewer’s concern, we have taken a different approach in this revision, taking 10 readings of the fura2 ratio from just before addition of the test reagent, and 10 centred around the maximum response during perfusion with the test compound, for each cell. We have then applied a t-test between these pairs of readings and called a cell as a responder if there was a significant increase (with p<0.01). I’m not sure this is particularly superior to the 10% method, although it does call some additional cells as responders. I don’t think the exact numbers in each set of the Venn diagram are critical, as the major question was whether the cells are capable of responding to more than one stimulus type. Overall the conclusion of this section is unchanged by the method of analysis: that EECs are multi-responsive to different types of nutrient signal. Attachments Attachment Submitted filename: Responses to reviewers 2.docx https://doi.org/10.1371/journal.pone.0308942.r004
2 Aug 2024 Decision Letter - Mohammad Sadegh Taghizadeh, Editor Single-cell transcriptomic atlas of enteroendocrine cells along the murine gastrointestinal tract PONE-D-24-02587R2 Dear Dr. Gribble, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Mohammad Sadegh Taghizadeh, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #2: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #2: (No Response) ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #2: (No Response) ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #2: (No Response) ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #2: (No Response) ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #2: (No Response) ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0308942.r005
Formally Accepted
14 Aug 2024 Acceptance Letter - Mohammad Sadegh Taghizadeh, Editor PONE-D-24-02587R2 PLOS ONE Dear Dr. Gribble, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, if your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. If we can help with anything else, please email us at customercare@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Mohammad Sadegh Taghizadeh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0308942.r006

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