Foxm1 regulates neural progenitor fate during spinal cord regeneration

Abstract Xenopus tadpoles have the ability to regenerate their tails upon amputation. Although some of the molecular and cellular mechanisms that globally regulate tail regeneration have been characterised, tissue‐specific response to injury remains poorly understood. Using a combination of bulk and single‐cell RNA sequencing on isolated spinal cords before and after amputation, we identify a number of genes specifically expressed in the spinal cord during regeneration. We show that Foxm1, a transcription factor known to promote proliferation, is essential for spinal cord regeneration. Surprisingly, Foxm1 does not control the cell cycle length of neural progenitors but regulates their fate after division. In foxm1 −/− tadpoles, we observe a reduction in the number of neurons in the regenerating spinal cord, suggesting that neuronal differentiation is necessary for the regenerative process. Altogether, our data uncover a spinal cord‐specific response to injury and reveal a new role for neuronal differentiation during regeneration.

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Review #1
1. How much time do you estimate the authors will need to complete the suggested revisions:

Estimated time to Complete Revisions (Required) (Decision Recommendation)
Between 1 and 3 months 2. Evidence, reproducibility and clarity:

Evidence, reproducibility and clarity (Required)
This manuscript describes the role of foxm1 in the regeneration of the Xenopus tadpole tail. Foxm1 is identified as upregulated in a well described and analysed single cell RNA seq of regenerating tails. In crispants (CRISPR/cas9 injected tadpoles in which much of foxm1 is mutated), the tail is regenerated, but there is a lower number of neurons. The data is overall well analysed and presented. **Major comments:** Although the authors performed some experiments to reveal the signalling pathway upstream of foxm1, including the inhibition of the sonic hedgehog pathway. Looking at the proliferation patterns in figure 4, it looks like the ventral, floor plate like domain is disturbed in foxm1 mutants. Is it possible that foxm1 is UPstream of shh signalling, rather than downstream? Showing the expression of hh pathway genes (e.g. patched, olig2) in the wt and mutant spinal cords might further inform about the position of foxm1 in the signalling cascade controlling regenerative neurogenesis. Higher magnifications of the tissue sections in figure 4 need to be shown. The in situ signal and distribution of cells is not well visible. Do the experiments on page 7 show that there is no difference in cell cycle length, or may there (also) be changes in the number of cells entering the cycle? A number of results are only borderline significant (e.g. p=0.04). It is not clear how group sizes were determined and what the power of the experiments is. **Minor comments:** dendogram should be dendrogram throughout Abstract: "reduction in THE NUMBER OF neurons" top of page 7: sequencing "revealed" (not leading to)

Significance (Required)
The main finding of this manuscript is that foxm1 has a regeneration-specific role and rather than control progenitor proliferation, it is important for neurogenesis/neuronal maturation. This is an interesting finding, of wider relevance for researchers in regenerative neurogenesis and stem cell differentiation. It appears that with relatively few more experiments, the position of foxm1 in the pro-neurogenesis cascade could be more clearly determined, in particular, the effect of its mutation on the hh pathway. Own expertise: regeneration, neurogenesis REFEREES CROSS COMMENTING Reading over our reviews, it looks like we all have the same concerns (in so many words) 1. that the cell cycle conclusions are not fully supported by the data and 2. that the disorganisation of the spinal ependymal zone/proliferating cells is insufficiently characterised. Both can be addressed experimentally and we have made suggestions how. What else would be important to strengthen the manuscript?

Review #2
1. How much time do you estimate the authors will need to complete the suggested revisions:

Estimated time to Complete Revisions (Required) (Decision Recommendation)
Between 3 and 6 months

Evidence, reproducibility and clarity:
Evidence, reproducibility and clarity (Required) **Summary:** In the manuscript entitled "Foxm1 regulates neuronal progenitor fate during spinal cord regeneration "Pelzer and colleague described molecular events occurring during spinal cord regeneration in Xenopus tadpole. Using a transcriptomic approach, they show that the transcription factor Foxm1 is specifically and temporally upregulated in the spinal cord during regeneration. Foxm1 knockdown using CRISPR/cas9 technology reduces spinal regeneration indicating a requirement of this transcription factor in this process. Using single cell transcriptomic analyses, they show that Foxm1 is expressed only during early regeneration in a specific cluster of neural progenitor cells; these cells display upregulation of several genes related to the cell cycle and half of them are in S phase suggesting that they have specific proliferative features. Eventually, they show that Foxm1 controls the proliferation/differentiation balance of neural progenitor cells. **Major comments:** *Are the key conclusions convincing?* Yes, the key conclusions summarized above are convincing. The conclusions that deserve to be reinforced are those relating to the cell cycle length (see below). *Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?* The statement based on the pulse-chase experiments ( Figure 2F) that Foxm1 does not affect the overall length of the cell cycle is too strong. The same result could be obtained with a change in cell cycle length combined with self-renewing rather than differentiating divisions. A measure of the total cell cycle length would remove this ambiguity. Knowing that the total cell cycle length is not modified would also allow to conclude that at 3dpa the increase of the cells in S phase reflects a longer S phase. The same result would be obtained going from 45,7% at 3dpa to 12% at 5 dpa without changing the S phase length if the total cell cycle length increases 4 times between 3dpa and 5dpa. Literature data reporting cell cycle length at 3dpa and 5dpa would be sufficient to verify the credibility of the latter hypothesis. The authors claim that Foxm1 affects neurogenesis independently of its ability to control the proliferation rate contrary to what has been observed during Xenopus primary neurogenesis (Ueno et al., 2008). In their paper, Ueno and colleagues (2008) knock down Foxm1 in primary neurogenesis using RNAi and show a strong reduction of the number of mitotic cells using PH3 labelling and the total number of cells stained with Hoechst. Foxm1 controlling the G2/M transition it is an obvious readout. It would be useful to determine whether downregulating Foxm1 affects mitosis in regenerative spinal cord using PH3 labelling for example. If it is not the case, it should be verified that CRISPR/Cas9 that leads to 52% reduction of Foxm1 expression mimics the effect of RNAi at early stage inducing a reduction of PH3 cells in primary neurogenesis. *Would additional experiments be essential to support the claims of the paper?* Measurement of the total cell cycle length using cumulative EdU for example or BrdU/EdU dual labelling in Sox3+ cells will allow to show whether or not Foxm1 impacts on cell cycle length. Quantifying PH3 cells in primary neurogenesis as performed by Ueno and colleagues and in regeneration following CRISPR/Cas9 knockdown will allow to conclude whether the mechanisms are different. Note that PH3 detect cells in early mitotic phases that is more difficult with DAPI staining. *Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.* We are not familiar enough with experiments in Xenopus to give a clear answer. Measurement of cell cycle features can be time consuming. What is important here and is not mentioned is the total cell cycle length of regenerative neural progenitor cells. If it has been measured in other studies it should be mentioned at 3dpa and 5dpa because it will also help for interpretation of the data. Cumulative EdU experiments in a context of short cell cycle length (around 10 hours) is feasible. *Are the data and the methods presented in such a way that they can be reproduced?* In general yes. I would appreciate to have a precision regarding EdU treatment. It is written: "For EdU labelling, NF50 tadpoles at 3dpa were injected with 3 times 4.2nL of 10mM EdU (Life Technologies) in DMSO for 3hr". Is it 3 times an hour for a total of 3 hours? *Are the experiments adequately replicated and statistical analysis adequate?* yes **Minor comments:** -Specific experimental issues that are easily addressable. Can the authors comment on the detection of S phase markers in neurons? Figure 3F (right side bar; neur.) The main defect observed following Foxm1 downregulation is the disorganization of the structure (figure 4B) including in the anterior spinal cord area. The authors should comment on that. Is there ectopic mitosis in that context? *-Are prior studies referenced appropriately?* yes *-Are the text and figures clear and accurate?* Abstract: I do not understand the end: "In foxm1-/-tadpoles, we observed a reduction of neurons in the regenerating spinal cord, suggesting that neuronal differentiation is necessary for the regenerative process. Altogether, our data show that different tissues respond differently to injury and reveal a new role for neuronal differentiation during regeneration." What tissues are they referring to in the last sentence that were analyzed here? Paragraph Foxm1 regulates the fate ... "We next analysed Sox3 expression by immunofluorescence using anti-Sox3 ... As expected, in the non-regenerating spinal cord Sox3 is expressed in the cells lining the ventricle. We also observed Sox3+ extensions into the mantle zone of the spinal cord ( Figure 4B, white arrowheads)." Why do the authors state that it is in the mantle zone? It would be useful to have a marker for differentiating neurons to visualize the mantle zone. "In contrast, the proportion of self-renewal divisions increases significantly in Foxm1-/-tadpoles". What are the different mode of divisions in regenerative spinal cord? Self-renewal, asymmetric neurogenic and terminal neurogenic like in a developing spinal cord? Can the authors exclude also asymmetric neurogenic divisions if they exist? Some pictures should be improved: PCNA labelling Fig S3-E should be betterquality. The authors claim that they identified S versus G1/G2 phases using PCNA labelling that of course is possible but difficult to visualize on the pictures. A better resolution of the pictures would be more convincing. *-Do you have suggestions that would help the authors improve the presentation of their data and conclusions?* If the data in the literature permit, more information on what is known about the characteristics of the cell cycle during spinal cord regeneration in amphibians can be provided.

Significance (Required)
*-Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.* RNAseq data is clearly a resource for the scientific community. Moreover, this work extends our knowledge of Foxm1 function in primary neurogenesis to tadpole spinal cord regeneration. *-Place the work in the context of the existing literature (provide references, where appropriate)* The research of the molecular cues responsible for controlling the choice between proliferation and differentiation, the role of the cell cycle in this process in the developing or regenerating nervous system is clearly of interest. -State what audience might be interested in and influenced by the reported findings. Neurobiologists, cell biologists particularly those working in the role of the cell cycle in cell fate, and people interest in the process of regeneration including those working on stem cells. *-Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.* Neurodevelopment, cell cycle, neurogenesis, spinal cord, vertebrate Lower expertise: regeneration, Xenopus, RNAseq single cell analyses

Review #3
1. How much time do you estimate the authors will need to complete the suggested revisions:

Estimated time to Complete Revisions (Required) (Decision Recommendation)
Less than 1 month

Evidence, reproducibility and clarity (Required)
The main aim of this study is to investigate the role of Foxm1 during spinal cord regeneration in Xenopus tropicalis. The authors first identified Foxm1 as the top upregulated gene in the regenerating spinal cord tissue by bulk RNA-seq analysis. Interestingly, they found that knocking out Foxm1 resulted in regeneration defect of the tail, while the proportion of spinal cord progenitor cells in S phase in the regenerating spinal cord was not affected. The authors then compared the proportion of cells in different cell cycle stages between uninjured and regenerating spinal cord and found that there was a significant increase in the proportion of S phase in the 3-day post-amputated spinal cord, which correlated with the expression timing of Foxm1. Lastly, they showed that the progenitor pool is increased, and the neuron pool decreased, in the regenerating spinal cord in the absence of Foxm1. This observation lead to the hypothesis that Foxm1 might play roles in controlling the fate switch from progenitors to neuron, a key feature of regenerative species. Overall, it is laudable that the authors took on this challenging topic with state-of-the-art scRNA-seq analysis and took time to generate Foxm1 knock-out frog line with CRISPR/Cas9 technology. However, there are several points the authors should address in the study to make a convincing story. The authors also have to be more cautious about interpretation of the data (see below). It is intriguing that frogs can survive without Foxm1, as it has been reported that Foxm1 knock-out mice is embryonic lethal. Therefore, the authors should confirm that foxm1 transcript and protein are indeed absent in foxm1-null tadpoles with ISH and IHC on the cross-section of tail. It was not shown in the manuscript. This is important because the deletion is made in the beginning of the coding sequence but there is an ATG present just after the deletion, so initiation from this downstream methionine may be occurring. In addition, whether the same regeneration phenotype exists in foxm1-null tadpoles as in F0 is also not mentioned or shown. The documentation of the regeneration phenotype should be in more detail in addition to length. This is important because, if the foxm1-null tadpoles also have the regeneration phenotype, comparing the progenitor cells with cross section in Figure4B would be tricky. For example, the morphology of cells near the amputation plane and those near the terminal vesicle is very different even in wildtype. Without knowing the corresponding regions of the regenerating spinal cords between wildtype and foxm1-null tadpoles, it is difficult to make a meaningful comparison. The authors state at the end of second section "...Foxm1 does not affect the overall length of cell cycle" based on the one-time EdU pulse labeling (Figure2D-F). The data only shows the percentage of cells in S-phase is the same but does not tell us about the length of cell cycle. In order to make this claim, the author would have to do multiple EdU pulse labeling and deduce the cell cycle length from the saturation curve, as describe in Nowakowski et al. 1989. This is important as the main conclusion of this manuscript is based on this observation. The fact that the ratio of Edu positive spinal cord progenitors remains the same (Figure2E) and that the shortening of regenerating spinal cord ( Figure 2B) does not come together and require more explanation. It goes back to the previous point of incomplete documentation of the regeneration phenotype. In the third section about scRNA-seq analysis, the authors state that "...whilst some cell types cluster together, neural progenitors display a big shift in their transcriptome" based on the tSNE plot in Figure3D. However, it is also likely due to batch effect, which is common in scRNA-seq data. The authors could computationally correct the batch effect with the Seurat or Harmony package. However, to completely exclude the possibility of batch effect, the authors would have to do multiplexing of these two samples using MULTI-seq or CITE-seq technique before making a definitive conclusion. In general, caution has to be taken when naming a cluster by a single marker. It would be a better measure to show the unbiased clustering result of Figure 3D and to see if foxm1 is the representative marker of this cluster. It is also not advisable to define a cluster with cell cycle-related genes, as it represents a cell state rather than a cell type. On the same note, it is not surprising that foxm1+ cells are mostly in S and G2/M phase as shown in Figure3F because foxm1 is known to be highly expressed in S and G2/M phase. In figure4C and figure 4E, the authors showed progenitor pool increased and neuron pool decreased. There are other possible explanations in addition to the failure of neuron differentiation as the authors suggested. It can be that the progenitors are over proliferating, or the neurons are dying. Different possibilities should be tested in order to make a strong conclusion. It will be helpful for the readers by showing the representative image of mty1 staining and to show the absolute number of counted cells in addition to percentages. To sum up, the manuscript requires a major improvement to strengthen the finding and novelty. One possible direction is to delineate the molecular pathways of progenitor-to-neuron transition with their scRNA-seq data in combination with in vivo validation. For example, trajectory analysis could give us insights about how the transition happens, and which cells express foxm1 and when. The authors listed the potential downstream factors in figureS2C, but unfortunately did not pursue this direction. The authors should also take advantage of their foxm1-null frog line to compare the scRNA-seq data between wildtype and foxm1-null tadpoles during tail regeneration. There are some minor comments following the order of Figures

Significance (Required)
This is one of a very small number of manuscripts that successfully male CRISPR-induced mutants to study regeneration in Xenopos. The authors even report F1 homozygous animals. This would be of great interest to the community. Unfortunately the work verifying the null mutation is lacking and more experiments with the F l homozygotes are needed. A better molecular definition of the phenotype would be needed to attain the highest potential of excitement for this work. Expertise: regeneration, genome engineering REFEREES CROSS COMMENTING A strange part of the manuscript is that the growth defect is only shown using f0 transgenics. In my view, they need to clarify and quantitate the major phenotypes including the spinal cord growth defects using the f1 homozygotes that exist.

Reviewer #1
1-S/he was wondering whether Foxm1 might be upstream of Shh signalling rather than downstream. Shh is known to be important for tail regeneration much earlier than the onset of foxm1 expression (Taniguchi et al. 2014; Hamilton et al. 2021). Furthermore, impairing Shh signalling during tail regeneration affects proliferation in the spinal cord, a phenotype not consistent with the one we observed for Foxm1 (Taniguchi et al. 2014). We agree that the role of Shh in neuronal differentiation during regeneration is an interesting question but we felt it was outside the scope of this manuscript. figure 4 need to be shown. We apologise for the fact that the pdf compression led to a loss of resolution. This has been rectified throughout the manuscript.

3-the reviewer asked if there is no difference in cell cycle length, or may there (also) be changes in the number of cells entering the cycle?
We have now shown by Dual Pulse Labelling that there is no change in the duration of the overall cell cycle length between wild type and mutant tadpoles (Fig. 3G/H). We have also shown that the percentage of cycling Sox3+ve cells (PCNA+) at 3dpa is the same independently of the genotype (Appendix FigS1). It is therefore very unlikely that our results could be explained by a change in the number of cells entering the cycle.

4-A number of results are only borderline significant (e.g. p=0.04). It is not clear how group sizes were determined and what the power of the experiments is.
The main result with a borderline significance is the regrowth of the tail when comparing wild type and knockdown tadpoles. This might be due to a subtle phenotype or the fact that these experiments were done on knockdown tadpoles rather than knockout. The new data on tail regeneration on knockout now has a p_value<0.0001. The other experiment with a small significance, is the RT-qPCR in Figure 4A (new Figure EV5A), also done comparing the knockdown and wild type. However, in this case we feel that the protein work (immunofluorescence on Figure 5A-F) supports the qPCR data and is much more revealing in characterising the phenotype of the foxm1 mutant. In general, all experiments were done from three independent crosses with at least three different animals per cross.

5-minor comments
-dendogram should be dendrogram throughout: this has been change -Abstract: "reduction in THE NUMBER OF neurons" : corrected -top of page 7: sequencing "revealed" (not leading to): corrected

1-Measurement of the total cell cycle length using cumulative EdU for example or BrdU/EdU dual labelling in Sox3+ cells will allow to show whether or not Foxm1 impacts on cell cycle length.
We have done these experiments (see above) and they do not show differences in overall length between mutant and wild type tadpoles.

2-Quantifying PH3 cells in primary neurogenesis as performed by Ueno and colleagues and in
regeneration following CRISPR/Cas9 knockdown will allow to conclude whether the mechanisms are different.

20th May 2021 1st Authors' Response to Reviewers
Detailed response to the reviewers Pelzer et al.
We have performed these experiments by comparing the proliferation in the neural plate at NF13 in embryos injected with either a morpholino preventing the splicing of foxm1 RNA or our Cas9/gRNA against the foxm1 locus used to generate the knockout. In all conditions, we do not observe a difference in proliferation quantified using anti-pH3 staining. These data suggest that in Xenopus tropicalis, Foxm1 does not regulate the proliferation of neuronal progenitor cells.
3-The reviewer wanted precision regarding EdU treatment. This is now explicitly described in the methods section: we do one injection of 12.6nL (3 times 4.2nL), then chased for 2 days.

4-minor comments:
-Can the authors comment on the detection of S phase markers in neurons? Figure 3F (right side bar; neur.): we have corrected an error: some bars labelled neurons were in fact differentiated neurons. To clarify this figure (as requested by reviewer 3 also), we have now indicated which bar corresponds to which cluster ( Figure 4B). There are still some cells in S/G2/M phases in the neurons cluster, likely due to the presence of some sox2+ cells in these cluster ( Figure 3D) -The main defect observed following Foxm1 downregulation is the disorganization of the structure (figure 4B) including in the anterior spinal cord area. The authors should comment on that. Is there ectopic mitosis in that context?: whilst we agree that this is the case for the regenerating spinal cord, we do not think that the anterior (non-regenerating) spinal cord architecture is different in foxm1-/-. We do not see differences in term of the number of Sox3+, Myt1+ and EdU+ cells. Furthermore the distribution of DAPI and Sox3 cells along the dorsoventral axis is similar in wild type and mutants in the non-regenerating spinal cord ( Figure EV5C-D). Figures 2D and 5E shows very similar architecture of the nonregenerating spinal cord in mutant and wild type tadpoles. We think that the apparent difference in Figure 4B (new Figure 5A) is due to the deformation of the tissue during the sectioning process. -Abstract: I do not understand the end: "In foxm1-/-(…) data show that different tissues respond differently to injury and reveal a new role for neuronal differentiation during regeneration." What tissues are they referring to in the last sentence that were analyzed here? The last sentence of the abstract refers to the fact we do not observe an upregulation of foxm1 expression and its transcriptional targets when the whole tail is analysed (Fig.1D), suggesting that we are reporting a spinal cord -specific response. We have changed the sentence to "Altogether, our data uncover a spinal cord -specific response to injury and reveal a new role for neuronal differentiation during regeneration", removing the reference to other tissues. -Paragraph Foxm1 regulates the fate " We also observed Sox3+ extensions into the mantle zone of the spinal cord ( Figure 4B, white arrowheads)." Why do the authors state that it is in the mantle zone? It would be useful to have a marker for differentiating neurons to visualize the mantle zone. This has been done using anti-acetylated Tubulin antibodies ( Figure 5B) -"In contrast, the proportion of self-renewal divisions increases significantly in Foxm1-/tadpoles". What are the different mode of divisions in regenerative spinal cord? Selfrenewal, asymmetric neurogenic and terminal neurogenic like in a developing spinal cord? Can the authors exclude also asymmetric neurogenic divisions if they exist? This is an interesting question but at this stage we cannot differentiate between PP, PN and NN divisions. Here, we consider any cells having incorporated EdU and still expressing Sox3 at the end of the chase period as a self-renewal division (i.e. remains a progenitor after division). -Some pictures should be improved: this has been done (see response to reviewer 1).
-If the data in the literature permit, more information on what is known about the characteristics of the cell cycle during spinal cord regeneration in amphibians can be provided: changes in cell cycle dynamics have been well described in Axolotl but nothing has been shown in the Xenopus regenerating spinal cord. We have referenced the Axolotl work and our data are in agreement with theirs. -Reviewer #3

1-the reviewer wanted us to confirm that our Crispr/Cas9 model is a true knockout.
We agree with the reviewer that we needed to characterise the foxm1 KO much better. We have now performed RT-qPCR to quantify precisely the expression of foxm1 during regeneration in wildtype and mutant tadpoles ( Figure EV2E). These data show more than 80%reduction of foxm1 in the knockout compared to wildtype. We have also generated F2 full knockout tadpoles and show that they display impaired regeneration (Figure 2A/B, see response to reviewer 1). We would love to do IHC but we have not found an antibody against Foxm1 that works in Xenopus yet.

2-The reviewer asked for a better documentation of the regeneration phenotype, in particular asking how we identified equivalent area of regeneration in the wild type versus the mutant tadpoles
We have added more details in the methods, in brief we mark the amputation plane during the embedding process to be able to identify it when sectioning. We then only consider the first 10 sections posterior to amputation plane (120µm), to avoid the neural ampulla which is quite different as the reviewer noted. Furthermore, there is not really a growth phenotype at 3dpa, allowing us to compare proliferation, differentiation etc in the mutant an. d wildtype situation

3-S/he asked us to quantify the length of the cell cycle.
See response to reviewer 1, we have performed these experiments using Dual Pulse Labelling as described in Thuret et al. 2015. (Figure2E) and that the shortening of regenerating spinal cord ( Figure 2B) does not come together and require more explanation. It goes back to the previous point of incomplete documentation of the regeneration phenotype. We agree with the reviewer that this present us with an apparent paradox. We suggest in the manuscript that this might have to do with a loss of directionality of division. It has been clearly shown in Axolotl that the main driver for growth of the tail in the fact that upon amputation, division align along the anteroposterior axis. We show here that the distribution of cells along the AP axis is different in wild type and mutants in the regenerate ( Fig  EV5 C-D), providing a possible explanation for this paradox.

5-The review asked us to correct our scRNAseq for batch effect.
We would like to thank the reviewer for pointing this out and we have tested multiple batch corrections algorithm ( Figure EV4A-B). This has led us to identify two clusters specific for 3dpa: a neuronal cluster characterised by the expression of leptin (and reported in Aztekin et al. 2019 and Kakebin et al. 2020) and a progenitor cluster expressing foxm1.

6-
The reviewer is asking for the unbiased clustering of the scRNAseq dataset and is concerned that we may be using cell state rather than cell type for clustering the foxm1+ cells.
We are now providing the full dataset of differentially expressed genes for each cluster against the rest of the dataset (Supplementary Table 2). Whilst we agree with the reviewer that the foxm1+ cluster is enriched with cell cycle -related genes, GO analysis also show over-representation of terms associated with signalling, Development and Neurogenesis (Fig 3J).

5-S/he wants us to exclude other possibilities for our observation that there is an increased number of progenitors and a decreased number of neurons in our mutant compared to wildtype.
We have shown now that neuronal progenitors are not over proliferating and we show evidence of a very low level (and no difference depending on genotype) of apoptosis in the regenerating spinal Detailed response to the reviewers Pelzer et al. Figure S3). Altogether, these data support very strongly our hypothesis that it is the fate of dividing progenitors that changes in our mutant.

6-It will be helpful for the readers by showing the representative image of mty1 staining and to show the absolute number of counted cells in addition to percentages.
We are now showing images of Myt1 staining ( Figure 5E). We have the absolute numbers if the reviewer wants them, but given the fact that we have an equivalent number of cells (DAPI) in wildtype and mutants ( Figure EV5F), the difference only comes from the number of Myt1+ cells. The absolute number of Myt1+ cells show exactly the same result at the data normalised over DAPI.
9-the reviewer suggests to delineate the transition from progenitor-to-neurons and the transition to foxm1+ cells. This is now shown on Figure EV4C-E. S/he also asks for in vivo validation. This will take a very long time from the identification of genes on the foxm1 path, followed by generating their knockout/knockdown and characterisation of the phenotype. Whilst we agree with the reviewer that this is an interesting direction of research, that we feel it is outside the scope of this manuscript.

10-
The authors should also take advantage of their foxm1-null frog line to compare the scRNAseq data between wildtype and foxm1-null tadpoles during tail regeneration. We agree with the reviewer but this will be quite an undertaking (more than 60 amputations, genotyping in 2 days before spinal cord dissection) and a very expensive experiment. We think that at this point, it is outside the scope of this manuscript.

Minor comments
-In order to make a strong statement on the "spinal cord-only" expression of Foxm1, Foxm1 ISH or IHC on the cross-section of the regenerate is appreciated (Figure1E). This has been added on Figure 1E -The statistics is missing in Figure1F: the statistics are now shown -Figure3F and Figure 4D require more explanation or the figures have to be simplified: we have added explanation to Figure 3F (now Figure 4B) and explain better the method for Figure 4D (now Figure 5D). Thank you for the submission of your revised manuscript. We have now received the enclosed reports from the referees, and I am happy to say that all support the publication of your study now. Referee 2 only has a minor suggestion that I would like you to incorporate before we can proceed with the official acceptance of your manuscript.
A few other editorial changes will also be required: -A callout to Fig 3A and Fig EV3A is missing. The Appendix Figs S1+S3 panels are not called out. Please correct.
-The 2 Tables should be called Dataset EV1 and Dataset EV2, and the legends need to be added to the files. Please also correct the callouts in the manuscript text.
-Please add a table of content with page numbers to the Appendix file.
EMBO press papers are accompanied online by A) a short (1-2 sentences) summary of the findings and their significance, B) 2-3 bullet points highlighting key results and C) a synopsis image that is exactly 550 pixels wide and 200-600 pixels high (the height is variable). You can either show a model or key data in the synopsis image. Please note that text needs to be readable at the final size. Please send us this information along with the revised manuscript.
I would like to suggest a few changes to the abstract that needs to be written in present tense. Please let me know whether you agree with this: Xenopus tadpoles have the ability to regenerate their tails upon amputation. Although some of the molecular and cellular mechanisms that globally regulate tail regeneration have been characterised, tissue-specific response to injury remains poorly understood. Using a combination of bulk and single cell RNA sequencing on isolated spinal cords before and after amputation, we identify a number of genes specifically expressed in the spinal cord during regeneration. We show that Foxm1, a transcription factor known to promote proliferation, is essential for spinal cord regeneration. Surprisingly, Foxm1 does not control the cell cycle length of neuronal progenitors but regulates their fate after division. In foxm1-/-tadpoles, we observe a reduction in the number of neurons in the regenerating spinal cord, suggesting that neuronal differentiation is necessary for the regenerative process. Altogether, our data uncover a spinal cord -specific response to injury and reveal a new role for neuronal differentiation during regeneration.
I look forward to seeing a final version of your manuscript as soon as possible. Please use this link to submit your revision: https://embor.msubmit.net/cgi-bin/main.plex

Kind regards, Esther
Esther Schnapp, PhD Senior Editor EMBO reports Referee #1: The authors have carefully revised the manuscript, adding a significant amount of new data and comprehensively dealing with/addressing all points of criticism.
Referee #2: The main objective of this manuscript is to investigate key factors that regulate the spinal cord regeneration in Xenopus tropicalis. The authors elegantly identified the spinal cord regenerationspecific upregulation of foxm1 by bulk RNA-sequencing and determined that ROS as an upstream of foxm1 pathway. scRNA-seq analysis revealed foxm1+ve population emerged in 3dpa tail regenerate and is associated with cell cycle-related genes. Interestingly, foxm1-KO does not affect the length of cell cycle. Phenotypic analysis on foxm1 knock-out/knock-down animals showed that foxm1 depletion in Xenopus tropicalis does not affect the proliferation of neuroprogenitors during embryogenesis and the foxm1-null tropicalis develop normally. However, after tail amputation foxm1 mutants showed impaired regeneration measured by length. The authors then attributed the phenotype to the change of progenitor orientation and the failure of progenitor-to-neuron transition.
I appreciate authors' effort to address most of my precious comments, including the characterization of the mutant lines, determining overall cell cycle length, scRNA-seq batch correction, improving image quality, and so on. Overall, I find the revised manuscript provides a convincing story.
A minor comment is that the authors use "dividing" and "proliferating" interchangeably in line 2, line 6 and line 9 of page 13. If I understand it correctly, the assumption was drawn from the conclusion that Tc was 50 hours (around 2 days), so a 2-day chase should encompass one cell cycle. However, this was not immediately apparent to me. The authors could improve the clarity in this paragraph.

Referee #3:
The new version of the Pelzer et al. manuscript includes the additional experiments that I suggested in the first review of the manuscript. These data reinforce the conclusions of the paper which is now appropriate for publication. We have incorporated all the changes that you and referee two asked for. In the case of referee two's comment, whilst I understood what they were asking (I think), I could not see where we used dividing / proliferating in the page / lines mentioned. However, I have modified the sentence on page 13 lane 10/11 as follow: "Because we estimated the cell cycle to be approximately 2 days (Fig 2H), we labelled cycling cells with EdU at 3 dpa and determined their fate after one cell cycle at 5 dpa using immunofluorescence." Finally, for consistency, we now refer NPCs as neural progenitor cells throughout the text as before we used both neuronal and neural. At the end of this email I include important information about how to proceed. Please ensure that you take the time to read the information and complete and return the necessary forms to allow us to publish your manuscript as quickly as possible.
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Is there an estimate of variation within each group of data?
For most experiments, at least three tadpoles from three different matings were analysed. For experiments where less than nine tadpoles were analysed, one to three tadpole(s) were used from three independent matings. no samples or animals were excluded from our analyses For regeneration experiments: the individuals were genotyped after the end of the experiment to avoid any bias in measuring re-growth. For quantification from immunofluorescence data, the images were randomised using a blind macro from FIJI and unblinded at the end of the quantification.
Manuscript Number: EMBOR-2020-50932V1 yes normal distribution was tested using a Shapiro-Wilk test in all experiments no no randomization was used genotypes were only revealed at the end of the experiments and images were randomised in FIJI For regeneration experiments: the individuals were genotyped after the end of the experiment to avoid any bias in measuring re-growth. For quantification from immunofluorescence data, the images were randomised using a blind macro from FIJI and unblinded at the end of the quantification.

Data
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