Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4

Katherine Q. Chen; Aaron Anderson; Hiroko Kawakami; Jennifer Kim; Janaya Barrett; Yasuhiko Kawakami

doi:10.1371/journal.pone.0267273

Peer Review History

Original SubmissionJanuary 4, 2022
18 Feb 2022 Decision Letter - Atsushi Asakura, Editor PONE-D-22-00272Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4PLOS ONE Dear Dr. Kawakami, 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 04 2022 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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[Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. 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 ****** 4. 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 ****** 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: Chen et al. inserted Sall4 into the Rosa26 locus and overexpressed Sall4 in mice via mesodermal Cre-dependent recombination. Although Sall4a and Sall4b were overexpressed in the caudal part of the embryo and the entire forelimb bud, minimal abnormalities were detected in early embryogenesis and digit regeneration. Although this manuscript describes negative results, the included information and the generated mouse strain are useful for the research community. Major comments 1. The authors should describe the number of homologous recombinant ES clones and the number of independent mouse strains that were generated and analyzed. If this study involved only one ES clone and one mouse strain, those should be mentioned as limitations of the study; such information is necessary for objective interpretation by readers. 2. Figure 2: A description should be provided concerning the number of biologically independent experiments performed with reproducible results. This point applies to all figures in the manuscript. 3. Figure 2I: Because intensities differ among the loading controls (GAPDH bands), it is difficult to judge the quantitative increase in Sall4 protein expression. The ratio of Sall4 band intensity to GAPDH band intensity should be quantified, and the percentage increase should be provided. In addition, the results should be described in greater detail. For example, Sall4a overexpression is greater than Sall4b overexpression, while Sall4b expression is increased from baseline in the control. 4. Figure 3I, J: the definitions of the dots and double dots should be clarified; they also appear to be mislocated in the graphs. Because there are no significant differences between Cre-WT/Rosa-Tg and Cre-Tg/Rosa-Tg, the effect of Sall4 overexpression is negligible. Instead, the presence of Sall4 cassette itself may mildly affect limb development. Such interpretation should be provided in the Results section, not the Discussion section. 5. The Discussion section largely consists of repetitive descriptions of the results; it also contains many descriptions of loss-of-function studies that are already explained in the Introduction section. The authors should discuss the overexpression studies in greater detail, instead of simply mentioning that Sall4 is a “permissive” factor. Their data indicate that Sall4 overexpression alone is insufficient to evoke significant effects in their setting. They should discuss possible reasons based on the known molecular mechanisms of Sall4. They can compare their results with the previous reports with or without phenotypes; the findings may suggest technical differences or dependency on cellular context. The established mouse strain can also be used to address other biological questions. Such discussion will be informative for readers. Reviewer #2: This manuscript reports the finding that over expression of both Sall4a and Sall4b do not affect the embryonic development and digit regeneration in the mouse model. The finding shows that, contrary to expectation, Sall4 over expression does not impart regenerative capacity to the limb, despite of its requirement for limb regeneration in amphibian animal models. I reckon the work merits publication. Your title emphasizes neonatal digit regeneration, but I am still very curious about whether you have performed P2 amputation experiment in adult stage. I understand that one would expect the non-regeneration of adult P2 phalanges when the neonatal P2 fail to regenerate with Sall4 over expression. Another question is about Sall4a and Sall4b. You over-expressed both in your mouse models. Is there any difference between Sall4a versus Sall4b, in your expectation, to stimulate digit regeneration? The western results seem to suggest that expression of Sall4a is higher than Sall4b in the digit tip (with the same anti-Sall4). It was reported that Sall4b, but not Sall4a, "binds preferentially to highly expressed loci in ES cells" (doi: 10.1128/MCB.00419-10). You may want to discuss this. Some suggestions: 1. Line 83, "The regenerative ability is level specific and restricted to the terminal 84 phalangeal bone", I suggest delete "bone". 2.Line 310, legend, "neonatal stage", I suggest indicating the time of digit amputation (P0). "neonatal stage" does not necessarily equal to day 0. For example, “The neonatal period is the first 4 weeks of a child's life". ****** 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. 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.0267273.r001
Revision 1
30 Mar 2022 Author Response Responses to the reviewers Thank you for the critical and helpful comments regarding our manuscript (PONE-D-22-00272) entitled “Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4.” We have examined the comments carefully and revised our manuscript. We believe that the we have adequately addressed the Reviewers’ concerns and improved the revised manuscript. Below, we provide point-by-point responses. RESPONSE TO REVIEWER 1 Reviewer #1: Chen et al. inserted Sall4 into the Rosa26 locus and overexpressed Sall4 in mice via mesodermal Cre-dependent recombination. Although Sall4a and Sall4b were overexpressed in the caudal part of the embryo and the entire forelimb bud, minimal abnormalities were detected in early embryogenesis and digit regeneration. Although this manuscript describes negative results, the included information and the generated mouse strain are useful for the research community. We thank the reviewer for considering “the included information and the generated mouse strain are useful for the research community”. Below, we provide response to each comment. Major comments Comment 1. The authors should describe the number of homologous recombinant ES clones and the number of independent mouse strains that were generated and analyzed. If this study involved only one ES clone and one mouse strain, those should be mentioned as limitations of the study; such information is necessary for objective interpretation by readers. Response: Thank you for pointing out important information that was missing in the original manuscript. We picked up 64 ESC clones after G418 selection, and six of them were properly targeted by genomics PCR and Southern blotting. Among them, we used one clone for injection into the blastocyst after confirming normal karyotype. We have included this information as below (modified/new information is underlined) Line 185-196: Among the 64 clones isolated after G418 selection, six properly targeted embryonic stem cell colonies were confirmed by genomic Southern blotting and genomic PCR (Fig 1C, D). After confirming normal karyotype, chimeric mice were generated by injecting one ES cell clone into blastocysts, which were bred with C57BL/6 mice. Germline transmission was confirmed by genomic PCR of offspring. This study was performed using this one strain. Comment 2. Figure 2: A description should be provided concerning the number of biologically independent experiments performed with reproducible results. This point applies to all figures in the manuscript. Response: Thank you for pointing this out. The number of biologically independent experiments/samples, which were not described are as below. Fig. 2A-C (n=3), 2D-F (n=3), 2G, H (n=3) Fig. 4A (n=4), 4B (n=3), 4C (n=3), 4D (n=3), 4E (n=3) We have included this information in the main text. Comment 3. Figure 2I: Because intensities differ among the loading controls (GAPDH bands), it is difficult to judge the quantitative increase in Sall4 protein expression. The ratio of Sall4 band intensity to GAPDH band intensity should be quantified, and the percentage increase should be provided. In addition, the results should be described in greater detail. For example, Sall4a overexpression is greater than Sall4b overexpression, while Sall4b expression is increased from baseline in the control. Response: We have measured the Western blot band intensity by FIJI. The result shows that the ratio of SALL4A/GAPDH signal intensity was increased 1.7-fold in TCre+/Tg; R26-Sall4+/tg embryos, compared to TCre+/+; R26-Sall4+/tg embryos. We have included this information in the text as below. Regarding SALL4B, it does not show detectable signals in the control sample. So, following the reviewer’s comment, we have included the following description. Line 239 – 243: Specifically, the data show that SALL4A overexpression is greater than SALL4B overexpression, while SALL4B expression is increased from baseline in the control. The ratio of SALL4A/GAPDH signal intensity was increased 1.7-fold in TCre+/Tg; R26-Sall4+/tg embryos, compared with TCre+/+; R26-Sall4+/tg embryos. Taken together, these results show that Sall4 is overexpressed by TCre-dependent recombination. Comment 4. Figure 3I, J: the definitions of the dots and double dots should be clarified; they also appear to be mislocated in the graphs. Because there are no significant differences between Cre-WT/Rosa-Tg and Cre-Tg/Rosa-Tg, the effect of Sall4 overexpression is negligible. Instead, the presence of Sall4 cassette itself may mildly affect limb development. Such interpretation should be provided in the Results section, not the Discussion section. Response: Thank you for pointing this out. The dots were introduced to indicate statistical significance. However, we indicated the p values in the panel, so we removed the dots from Fig 3I and J.. Regarding the interpretation of bone lengths, as suggested by the reviewer, we have included the following description. Line 281 – 283: Given that the bone length between TCre+/+; R26-Sall4+/Tg and TCre+/Tg; R26-Sall4+/Tg did not show significant differences, the effect of Sall4 overexpression is negligible and the presence of the Sall4 cassette may mildly affect limb development. Comment 5. The Discussion section largely consists of repetitive descriptions of the results; it also contains many descriptions of loss-of-function studies that are already explained in the Introduction section. The authors should discuss the overexpression studies in greater detail, instead of simply mentioning that Sall4 is a “permissive” factor. Their data indicate that Sall4 overexpression alone is insufficient to evoke significant effects in their setting. They should discuss possible reasons based on the known molecular mechanisms of Sall4. They can compare their results with the previous reports with or without phenotypes; the findings may suggest technical differences or dependency on cellular context. The established mouse strain can also be used to address other biological questions. Such discussion will be informative for readers. Response: Thank you for this suggestion. We have extended our discussion as below. First, we have discussed that Sall4 may require highly regenerative cellular or tissue context to contribute to limb regeneration (line 418-421). Second, we have discussed the possibility that SALL4 requires molecular partner to evoke significant effects in the tissue we examined (line 423 -433). Third, we have discussed other biological questions that the Sall4 overexpression mouse strain may be useful (line 439 - 445). We hope that the reviewer acknowledges our new discussion. RESPONSE TO REVIEWER 2 Reviewer #2: This manuscript reports the finding that over expression of both Sall4a and Sall4b do not affect the embryonic development and digit regeneration in the mouse model. The finding shows that, contrary to expectation, Sall4 over expression does not impart regenerative capacity to the limb, despite of its requirement for limb regeneration in amphibian animal models. I reckon the work merits publication. We thank the reviewer for his/her words “I reckon the work merits publication”. Below, we provide response to each comment. Comment 1: Your title emphasizes neonatal digit regeneration, but I am still very curious about whether you have performed P2 amputation experiment in adult stage. I understand that one would expect the non-regeneration of adult P2 phalanges when the neonatal P2 fail to regenerate with Sall4 over expression. Response: Thank you for this comment. As we showed in the manuscript, we did not observe regeneration after P2 amputation at the P0 stage. We think it is highly unlikely that the digit regenerates after P2 amputation in the adult stage, and therefore, we did not perform P2 amputation at the adult stage. Comment 2: Another question is about Sall4a and Sall4b. You over-expressed both in your mouse models. Is there any difference between Sall4a versus Sall4b, in your expectation, to stimulate digit regeneration? The western results seem to suggest that expression of Sall4a is higher than Sall4b in the digit tip (with the same anti-Sall4). It was reported that Sall4b, but not Sall4a, "binds preferentially to highly expressed loci in ES cells" (doi: 10.1128/MCB.00419-10). You may want to discuss this. Response: As the reviewer indicated, a previous study by the Orkin lab provided evidence that SALL4A and SALL4B have overlapping but non-identical binding sites in the mouse ES cell. However, we do not know functional differences between SALL4A and SALL4B in the Brachyury lineage during embryonic development and neonatal digit regeneration. Therefore, we simultaneously overexpressed both Sall4a and Sall4b in our study. We have clarified this point in the Result as below (line: 177-180). A previous study in mouse ES cells provided evidence that SALL4A and SALL4B have overlapping but non-identical binding sites (37). Because functional differences between SALL4A and SALL4B during mouse development are unknown, we expressed both Sall4a and Sall4b. Some suggestions: Comment 3 (Suggestion 1). Line 83, "The regenerative ability is level specific and restricted to the terminal phalangeal bone", I suggest delete "bone". Response: Thank you for the suggestion. We have deleted “bone” from the sentence (new line 86). Comment 4 (Suggestion 2) .Line 310, legend, "neonatal stage", I suggest indicating the time of digit amputation (P0). "neonatal stage" does not necessarily equal to day 0. For example, “The neonatal period is the first 4 weeks of a child's life". Response: Thank you for the suggestion. We have indicated the amputation was performed at the P0 stage in line 344 in the revised manuscript. Attachments Attachment Submitted filename: Responses to Reviewers.docx https://doi.org/10.1371/journal.pone.0267273.r002
6 Apr 2022 Decision Letter - Atsushi Asakura, Editor Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4 PONE-D-22-00272R1 Dear Dr. Kawakami, 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 for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, 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, Atsushi Asakura, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0267273.r003
Formally Accepted
8 Apr 2022 Acceptance Letter - Atsushi Asakura, Editor PONE-D-22-00272R1 Normal embryonic development and neonatal digit regeneration in mice overexpressing a stem cell factor, Sall4 Dear Dr. Kawakami: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. 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 plosone@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. Atsushi Asakura Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0267273.r004

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