Cell-fate transition and determination analysis of mouse male germ cells throughout development

Mammalian male germ cell development is a stepwise cell-fate transition process; however, the full-term developmental profile of male germ cells remains undefined. Here, by interrogating the high-precision transcriptome atlas of 11,598 cells covering 28 critical time-points, we demonstrate that cell-fate transition from mitotic to post-mitotic primordial germ cells is accompanied by transcriptome-scale reconfiguration and a transitional cell state. Notch signaling pathway is essential for initiating mitotic arrest and the maintenance of male germ cells’ identities. Ablation of HELQ induces developmental arrest and abnormal transcriptome reprogramming of male germ cells, indicating the importance of cell cycle regulation for proper cell-fate transition. Finally, systematic human-mouse comparison reveals potential regulators whose deficiency contributed to human male infertility via mitotic arrest regulation. Collectively, our study provides an accurate and comprehensive transcriptome atlas of the male germline cycle and allows for an in-depth understanding of the cell-fate transition and determination underlying male germ cell development.

showed a clear developmental trajectory. However, there were several small clusters of cells from E17.5, E18.5 and P0 locating away from the main trajectory. Do they represent different cell states? The authors could consider exploring more about this observation. In this regard, I would suggest the authors perform single cell RNA velocity analysis to see if it can add additional insights. Fig. 6a showed that Hhex was upregulated at both T-proSPG and Undiff.ed SPG stages, suggesting it may have a role in the transition. The authors used the culture derived from PND5.5 testis to examine the function of Hhex, which did not test this aspect directly. The authors should consider using culture derived from a corresponding earlier stage or at least discuss this limitation. In Fig. 5e and Supplementary Fig. 6f, the developmental delay in Helq null PGCs during E15.5 and E18.5 is obvious. However, it seems that in E12.5 and E13.5, the WT PGCs showed a delay compared with Helq null PGCs.  Reviewer #3: Remarks to the Author: In this study, Zhao et al. performed single-cell RNA-seq analysis of testicular cells at 28 timepoints throughout mouse germline development. Their data provide an integrated resource for understanding mouse germ cell development. Their analyses validate previous studies, as well as uncover some new gene markers and signaling pathways potentially involving in mouse germline development. They claim to have identified a new transitional germ cell stage between the mitotically active PGC and arrested pro-spermatogonia (ProSPG) stages. They provide evidence that the Notch signaling pathway promotes the transition of mitotic PGCs to mitotically arrested ProSPG. They also provide evidence that HELQ, a DNA damage-associated cell cycle regulator, drives the transition of mitotic PGCs to mitotically arrested ProSPG. Finally, they combine their mouse scRNA-seq datasets with analogous previously published human datasets to identify conserved gene regulatory networks.
Overall, this study provides a good integrated resource for the field. However, as explained below, novelty appears to be weak. In addition, there are several other concerns that need to be addressed. Major concerns: 1) While the authors examined some time points not previously examined by scRNA-seq, most of their scRNA-seq analyses are focused on known cell populations previously identified by published single cell RNA-seq analyses. While they claimed some "new" subsets, those are not convincing or were previously defined by others (see below).
2) The new identified "transitional PGC" subset is not convincing. Based on Fig. 3c, it's hard to believe this transitional PGC is a real cell subpopulation, as it does not form a clearly defined "cell cluster." Following this logic, it would appear that cells can be divided into as many as stages as the authors like. To make this claim, the authors need to provide more evidence that this is a new identified cell subpopulation, as well as provide some evidence for their functional roles. If not, related statements need to be corrected.
3) The novelty of the identification of the role of Notch in fetal germ cells is limited. First, Notch signaling has previously been reported to be active in developing mouse testes, both in germ cells and Sertoli cells (PMID: 18801836). In addition, roles of the Notch pathway in PGCs have previously been reported in chicken, C. elegans, Xenopus, sea urchin, and Drosophila (PMID: 29951200, PMID: 32008902, PMID: 20151992, PMID: 23533178, PMID: 20660750). None of these studies are cited in this study. Second, finding expression of NOTCH signaling components in a cell does not demonstrate that the NOTCH pathway is activate in this cell (PMID: 23907117). Indeed, the detection of Notch1 and Hes1 expression in mouse PGCs here does not mean the Notch pathway is activated in PGCs. Third, the in vivo effects observed by the authors in response to the Notch inhibitor, DAPT, do not necessarily reflect a defect in Notch signaling in germ cells. Indeed, it was previously shown that activating the Notch pathway in Sertoli cells influences fetal germ cell quiescence (PMID: 23907117, PMID: 23391689). In addition, several studies have shown that the Notch pathway is active exclusively in Sertoli cells in the perinatal testis, and that activating Notch is essential for fetal germ cell quiescence (PMID: 23907117, PMID: 23391689). 4) The study of the role of Helq in fetal germ cells is a novel work in this study, and thus this reviewer encourages the authors to study this further. Currently, the authors only show the cellular changes after Helq ko. While the authors tested potential mechanisms (as shown in Fig.  5l), these are mainly following previous publications (PMID: 24005329 and PMID: 24005041). Some further work can be done. For example, the authors could go deeper into how Helq influences fetal germ cell development. Currently, the authors stated that Helq depletion causes delayed development of fetal PGCs. However, based on the cell composition ( Fig. 5f), another possibility is that Helq depletion impacts PGC growth or maintenance. This is supported by the fact that there are less mitotic PGCs at E13.5 and E14.5 in Helq ko mice compared to WT mice. In addition, their finding that apoptosis is upregulated in Helq-/-PGCs also supports this hypothesis. The authors could go more deeply into other phenotypic defects. In addition, the authors could identify candidate HELQ-regulated genes from their scRNA-seq data and test their roles. 5) The scRNA-seq analysis of Helq-/-germ cells has only one biological replicate; at least one more replicate should be provided for rigor. This is important, as the authors find opposite effects at two stages of development (Figs. 5e and S6f), which could be due to sampling bias. Even if their finding is correct, their interpretation that inhibited development at one stage causes an accumulation of germ cells at later time point, is not necessarily correct. Another possibility is that loss of Helq inhibits germ cell maturation at one stage and promotes germ cell events later. These two possibilities should be distinguished or at least acknowledged. 6) More data analyses should be done to identify conserved gene regulatory networks between humans and mice. The authors integrated their own previously published datasets with that of Wang et al., but there are several resources in the field, e.g., PMID: 30726734, PMID: 31928944, and PMID: 33453151. While these datasets are from different platforms, the authors should at least compare their enriched genes/signaling pathways to validate their own findings. 7) Fig. 5c. Is the % at E13.5 and E14.5 really significantly changed? With only 4 samples, it is hard to believe that there is a significant difference. Please also check the statistical test in Fig. 5h. Minor concerns: 8) The authors often incorrectly refer to ProSPG as PGCs. PGCs convert into M ProSPG at ~E11.5-E13. 5 (PMID: 23843236). Thus, what they call "mitotic PGCs" during this period should contain both PGCs and M-ProSPG. What they refer to as "Arrest PGCs" are typically called T1-ProSPG (PMID: 23843236). The authors also refer to other ProSG stages that they call "Q-and T-ProSPG," without defining them. If they want to use this system, they should not only define them, but refer to a publication where this system is used. It is suggested to use the M-, T1-, and T2-ProSPG nomenclature instead. A final note: further confusion is rendered by the fact that the authors refer to gonocytes in the Introduction and never explain that gonocytes are the same as ProSPG. 9) Pg 9, line 11. This "post-arrest PGC" has been identified by Law et al. 2019. The authors should perform a bioinformatic comparison to test their similarities. 10) Potential doublets contamination? Pg 7, line 20. It is surprising that the authors found that round spermatids (RS) express pluripotency and SPG markers, given that RS are post-meiotic and certainly not pluripotent. One explanation is this is an artifact, and that these cells are actually doublets. The authors should investigate this possibility. 11) The authors refer to "transcriptional" regulation throughout the manuscript when they presumably instead mean "transcriptome." Changes in mRNA levels as detected by RNAseq or scRNA-seq could be due to either alterations or transcription rate or RNA half-life, not necessarily the former. Thus, the authors are incorrect when they state that changes detected by these methods are transcriptional in the Abstract ("transcriptional reconfiguration" and "transcriptional reprogramming" should be revised), on pgs 5, top of pg 7, etc. Search and replace should be conducted throughout the entire MS. 12) Protein acronyms should be all caps and not italicized; e.g., HELQ, not Helq. 13) Pg 29, line 21. The ref 80 has no mSSC culture method.

Point-by-point response to the Reviewers' questions:
Below we have listed the point-by-point response to each of the questions, with the answers highlighted in blue.

Reviewer #1 (Remarks to the Author):
In mammals, the developmental process of germ cells is complex and delicate, involving Here I have some comments for this study and the carefully revisions are required for improving the quality of this paper.

Answer:
We thank the reviewer for these positive comments.
Major comments: 1.The title of the study is "Cell-fate transition and determination analysis of mouse male germ cells throughout the whole developmental program". However, the article is mainly written on SPG transition and from mitotic arrest PGCs to post-arrest PGC during PGC development.
Should the conversion from spermatogonia (SPG) to spermatocytes (SPC) and the conversion process from SPC to round spermatids (RS) be briefly described and analyzed? Answer: We appreciated the reviewer's suggestions and agree that more analyses should be included in this whole developmental program. To this aim, we have analyzed the global differentially expressed genes (DEGs) and gene ontology (GO) terms during spermatogenesis.
As expected, "Cell cycle", "Gamete generation", "Synaptonemal complex assembly", "Cilium organization", and "Sperm axoneme assembly" were significantly enriched in different stages Overall, these results characterized a dynamic developmental and cellular sequence of events during the conversion processes from SPG to SPC, and further to RS. And the main findings of this part were essentially consistent with previous studies 1,2 . 2.There were two articles on transcriptome sequencing of spermatogenesis of human, mouse, and non-human primates (1,2). Compared with them, how innovative is your article? Can the post-arrest PGCs, Q-ProSPG, and T-ProSPG mentioned in your article be reproduced based on the integration of the data from your research and them? (1). Hermann  Answer: We thank the reviewer for the comments. Our study makes essential progress in the field of germ cell development. Firstly, although precious studies have illuminated specific stages of germ cell development 1-10 , none of them monitored a full-term program of germ cell development in a single study. It is known that due to the batch effects which caused by different sequencing technologies, different library preparation protocols, and different data interpretation strategies, the integrated analyses of data from different platforms or labs was still technically challenging. It is therefore of profound significance to establish high-quality full-term transcriptome atlas of mouse male germ cell development at single-cell resolution. It would be convenient to know the dynamic expression patterns of any candidate genes in an accurate and consistent way. In addition, our dataset should be a high-quality comprehensive reference for comparison to inform the accurate cell type-specific transcriptional changes when any gene of interest is manipulated in male mouse germline.
Secondly, compared with 10×Genomics method, our single-cell single-tube amplification library construction method provides much higher gene detection rate (about 9,413 genes in each cell), and higher sensitivity in capturing RNA molecules (reviewed recently by directly comparing seven methods for single-cell and/or single-nucleus profiling) 11 , which can dig out more lowly-expressed functional genes such as transcription factors. Our method also showed much lower cross-contaminations between different individual cells due to its single-tube amplification library construction strategy.
Lastly, how the cell-fate transition and determination of male germ cells is regulated remain largely unexplored, especially during prenatal male germ cell development. Based on the unbiased single-cell transcriptome reference established in the present study, we uncovered that Notch signaling pathway and HELQ played critical roles in the proper cell-fate transition from mitotic to mitotic arrest PGCs to ensure male fertility. We believe that our data would offer novel insights into this field and provide new clues for further study on male germ cell development.

For the second question that "Can the post-arrest PGCs, Q-ProSPG, and T-ProSPG mentioned in your article be reproduced based on the integration of the data from your research and them?"
According to the suggestion of the reviewer, we have tried to validate the robustness of our data by integrated analysis with these two publicly available scRNA-seq data of germ cells 5,10 .
Firstly, by mapping the purified P6 (Postnatal day 6) and adult Id4-GFP positive SPG onto scRNA-seq based developmental trajectory constituted by the cells from post-arrest PGCs to diff.ed SPG, we found that the distribution of P6 and adult Id4-GFP positive SPG was generally merged with the corresponding cell clusters ranged from T-ProSPG to Diff.ing SPG, which confirmed our cell cluster assignment results (Rebuttal Secondly, we obtained similar results by integrating our data and a previous study 5 (Rebuttal Overall, we provide an accurate, high-quality and more importantly, robust single-cell transcriptome atlas of male germline cycle, which provided the opportunity to uncover how the cell-fate transition and determination were regulated during male germ cell development in an unbiased way. 3. The spermatocytes in C7 were very heterogeneous (Fig. 1c), especially the group on the right, which only have R1 cells and not R2-4 (Supp. Fig. 1b). Please briefly describe the reason for this heterogeneity in the discussion or result part.
Answer: Thank the reviewer for this question. We have checked our data carefully and re- Answer: Thank the reviewer for this question. We are sorry that the original manuscript might somehow lead to misunderstanding and confusion due to lacking of essential description of Bar value and more details for determining the cell cycle. We have revised them in the revised manuscript accordingly (see Fig. 1c of the revised Manuscript). Figure 3e   As we know, Helq encodes a DNA helicase, which might regulate many downstream genes directly or indirectly. Thus, the DEGs revealed here should result from Helq knockout itself or further downstream effects.

5.In
Concerning on the novel role of HELQ in the regulation of germ cell development, we also tried to dissect certain interesting downstream target. In the DEG list when Helq was knocked out, we found that Helq -/germ cells highly expressed Cdh1 (encoding E-cadherin) (Rebuttal Mean ± SEM, n = 4 per group, **** P < 0.0001, unpaired two-tailed t test. e, Relative proportions of of p-CHK1 + germ cells in the WT-and Helq -/mouse male germ cells at E14.5. Mean ± SEM, n = 4 per group, ** P < 0.01, unpaired two-tailed t test. f, Relative proportions of of p-CHK1 + germ cells in the WT-and Helq -/mouse male germ cells at E15.5. Mean ± SEM, n = 4 per group, * P < 0.05, unpaired two-tailed t test. g, (Top) Immunofluorescence of WEE1 co-stained with MKI67 and BLIMP1 (GFP) in WT-and Helq -/mouse male gonads at E14.5.

11.In Fig.7a, human PGC development was not continuous. Obviously, its sampling time was very close. whether the development of human PGC is really discontinuous, or it is because the analysis method is not suitable.
Answer: Thank the reviewer for this comment. PCA is a linear dimension reduction method that can preserve the global structures well, which has been widely used for the dimension reduction analysis. In fact, our previous study of human PGCs also presented a discontinuous developmental trajectory, which was reconstructed using both t-SNE and 3D PCA plots 14 (Rebuttal Fig. R12). The discontinuity was probably caused by the sparse sampling time of human PGCs, which lacked the time points from 11 to 18 weeks. This sampling strategy might lead to the failed collection of certain transitional states between mitotic PGCs and mitotic arrest PGCs; by contract, our study here was performed with much denser sampling points. Answer: Thank the reviewer for this suggestion. We have checked all the acronyms used in our paper and marked them in the position when they were first mentioned. Fig. 6m is a group with only lentivirus or nothing added.

5.In page 15, Line 14-16, Please describe exactly whether the Control in
Answer: Thank the reviewer for this suggestion. The control in this test is a group with no shRNA added, the sh-NC is a group with lentivirus carrying shRNA against no target, and sh-Hhex-1 and sh-Hhex-2 are groups with lentivirus carrying shRNA against Hhex. We have added these descriptions in the Method part, and oligonucleotides used in this study were listed in the Supplementary Data 7 of the revised Manuscript.
6.The order of the author's figures is quite chaotic, which may cause inconvenience to read, so it is suggested to improve it.
Answer: We thank the reviewer for the suggestion. We have fine-tuned the order of Figures and text to make them more logical, which will make it easier to understand.

Reviewer #2 (Remarks to the Author):
The authors are the first to perform scRNA-seq capturing the germ cell development from embryo to adult in mice. In  PGCs. Lastly, they integrated the mouse scRNA-seq data with previous human scRNA-seq data to identify conserved regulators of the two species. Altogether, the high-resolution data serve as a very useful resource for future germ cell investigation.

Answer:
We thank the reviewer for these positive comments of our work.
2-1：Despite the well-known role of Gfra1 in SSC maintenance, Gfra1 expression has not been reported in round spermatids. Therefore, the authors could consider to provide more evidence at mRNA or protein level to validate the finding from scRNA-seq.
Answer: Thank the reviewer for this comment. We have validated the mRNA levels of these genes in round spermatids and spermatogonia as well as spermatocytes isolated from adult male Oct4-EGFP reporter mice. In brief, after testis dissociation and Hoechst staining, we sorted GFP-positive diploid, tetraploid (4N), and haploid (1N) cells, which were spermatogonia, spermatocytes and round spermatids, respectively. FACS-sorted populations were first confirmed by detecting the expression of well-known germ cell marker genes via q-PCR.
Finally, we detected Gfra1 expression in round spermatids, which was consistent with our scRNA-seq data (Rebuttal Fig. R14). Answer: We thank for this comment. As far as we know, fetal germ cell are with low mitotic activity; thus, the in vitro culture of fetal germ cell is still very difficult. Although a previous study 15 had reported that mouse male germ cells derived from E12.5 to E18.5 could be expanded in vitro, the offspring from the cultured cells showed growth abnormalities and were defective in genomic imprinting. As suggested by the reviewer, we have discussed this limitation in the Discussion part of the revised manuscript.

2-5：The authors could consider including UMAP plots showing gene expression and cell
annotation on their website.
Answer: Thank for this suggestion. Accordingly, we have updated all the gene expression UMAP featureplots on our website. We believe it should be more convenient for researchers to access our data at https://tanglab.shinyapps.io/Mouse_Male_Germ_Cells/. Minor:

Supp fig. 1b -some clusters distinct from the main trajectory only present in 1 replicate
Answer: Thank the reviewer for this question. We checked our data carefully and re-analyzed cells in C7 by re-clustering (Rebuttal Fig. R4a, already shown in our original manuscript). In addition, the developmental trajectory was reconstructed using Monocle (Rebuttal Fig. R4b).
Our results showed that germ cells in C7 were developed along the ordered developmental trajectory. And the cells from Repeat 1 exhibited similar molecular characteristics like other cells of the same cell cluster (Rebuttal Fig. R4c and 4d). Based on these results, the certain heterogeneity of C7 does not affect our conclusions and the subsequent analysis.

The appearance of figures of supp fig. 4 in text is not in order
Answer: Thank the reviewer for this suggestion. We have revised this in the revised manuscript.

Page 10 line 19 -"HES1"
Answer: We thank for this suggestion. We have checked all the names of mouse genes and mouse proteins in our manuscript and revised them according to the international standard.

All proteins should be in capital letter for mouse
Answer: We thank for this suggestion. We have checked all the names of mouse genes and mouse proteins in our manuscript and revised them according to the international standard.

Page 11 line 20 -Supplementary Data 2
Answer: Thank the reviewer and we have revised it in the revised manuscript. Fig. 5f Answer: Thank the reviewer and we have revised it in the revised manuscript.

Fig. 5c appears after
In Fig. 5e and Supplementary Fig. 6f, the developmental delay in Helq null PGCs during E15.5 and E18.5 is obvious. However, it seems that in E12.5 and E13.5, the WT PGCs showed a delay compared with Helq null PGCs.
Answer: We thank the reviewer for this comment. Our results demonstrated that Helq knockout resulted in the hyperactivation of p-ATR/p-CHK1 regulatory axis (Rebuttal Fig. R11), which might further cause the abnormal cell cycle switch of PGCs (Rebuttal Fig. R11). Therefore, the emergence of transitional PGCs at E12.5 when Helq was knocked out indicate that E12.  Overall, this study provides a good integrated resource for the field. However, as explained below, novelty appears to be weak. In addition, there are several other concerns that need to be addressed.

Answer:
We thank the reviewer for the positive comments and his/her appreciation that our work provides a good integrated resource for the field. To summarize, our work offers novel insights into research of cell-fate transition and determination during male germ cell development.
2) The new identified "transitional PGC" subset is not convincing. Based on Fig. 3c, it'  (2) On the other hand, to evaluate the heterogeneity of identified cell clusters, we performed ROGUE-guided analysis to calculate the purity score of cell types before and after re-clustering of the mitotic arrest PGCs as previously reported 20 . Our results showed that the purity score of mitotic arrest PGCs was increased from 0.26 to 0.50 when re-clustering was performed  . Based on these data, HELQ depletion could disrupt the mitotic to mitotic arrest transition of PGCs, wherein HELQ deficiency might hyperactivate the p-ATR/p-CHK1 regulatory axis, which then prolonged the cell cycle checkpoint and resulted in PGC arrest (Also see the answer to major question 10 raised by Reviewer #1).
In conclusion, based on all these data, we believe that transitional PGCs is a new cell population, which is critical for the cell-fate transition from mitotic to post-arrest PGCs. in the WT-versus Helq -/mouse male gonads at E14.5. Mean ± SEM, n = 4 per group, ns, not significant, * P < 0.05, unpaired two-tailed t test. To verify the germ cell identity, the sorted PGCs and somatic cells without treatment were used as the controls. Relative expression levels are shown with normalization to Gapdh. Error bars indicate mean ± SEM from at least two independent biological replicates. ** P < 0.01, unpaired two-tailed t test. g, Immunofluorescence of NOTCH1 co-stained with DDX4 in LY411575treatment and control mouse male gonads at E15.5. Scale bar, 10 μm. h, Immunofluorescence of HES1 co-stained with DDX4 in LY411575-treatment and control mouse male gonads at E15.5. Scale bar, 10 μm. i, Immunofluorescence of MKI67 co-stained with DDX4 in LY411575-treatment and control mouse male gonads at E15.5. Solid yellow arrowheads indicate MKI67 Positive subtypes in DDX4 + cells. Scale bar, 10 μm. j, The quantification of relative fluorescence intensity of NOTCH1 in LY411575-treatment and control mouse male gonads at E15.5. Mean ± SEM, n = 3 per group, **** P < 0.0001, unpaired two-tailed t test. k,

3) The novelty of the identification of the role of Notch in fetal germ cells is limited. First, Notch signaling has previously been reported to be active in developing mouse testes, both in germ cells and Sertoli cells (PMID: 18801836). In addition, roles of the Notch pathway in
The quantification of relative fluorescence intensity of HES1 in LY411575-treatment and control mouse male gonads at E15.5. Mean ± SEM, n = 3 per group, **** P < 0.0001, unpaired two-tailed t test. l, Proportion of MKI67 + cells in DDX4 + cells in LY411575-treatment and control mouse male gonads at E15.5. Mean ± SEM, n = 3 per group, ** P < 0.01, unpaired two-tailed t test.   Fig. 5f), another possibility is that Helq depletion impacts PGC growth or maintenance. This is supported by the fact that there are less mitotic PGCs at E13.5 and E14.5 in Helq ko mice compared to WT mice. In addition, their finding that apoptosis is upregulated in Helq-/-PGCs also supports this hypothesis. The authors could go more deeply into other phenotypic defects.

4) The study of the role of Helq in fetal germ cells is a novel work in this study, and thus this reviewer encourages the authors to study this further. Currently, the authors only show the cellular changes after Helq ko. While the authors tested potential mechanisms (as shown in
In addition, the authors could identify candidate HELQ-regulated genes from their scRNA-seq data and test their roles.

Answer:
We thank the reviewer for these comments and suggestions. Accordingly, we have performed more experiments to investigate the potential mechanisms of HELQ in the regulation of germ cell development. We found that there were several interesting phenotypes and mechanisms about HELQ ablation in germ cells by combined analysis of the original data and the newly obtained data. The conclusions were listed below: (1) Compared to the WT control, the higher ratio of MKI67-positive germ cells but lower ratio of total number of germ cells was detected in seminiferous cords of Helq -/mice (Rebuttal   which was critical for the differentiation of prenatal male germ cells 12,13 . Based on these data, it is most likely that HELQ also functions in the regulation of PGC differentiation. (Also see the answer to major question 9 raised by Reviewer #1). Answer: Thank the reviewer for this suggestion. Accordingly, we have integrated our dataset in this study with the human scRNA-seq datasets from different platforms, spanning infancy to adulthood 25 (Rebuttal Fig. R19a, b). Clustering analysis showed that human undiff.ed SPG-1 and undiff.ed SPG-2 were well-merged with mouse Q-ProSPG and T-ProSPG, respectively (Rebuttal Fig. R19a-c), suggesting that they represent a more 'naïve' state; while human undiff.ed SPG-3 were merged with mouse undiff.ed SPG. And human and mouse diff.ing SPG and diff.ed SPG could be also well-merged, respectively. Further analysis showed that the expression patterns of the highly conserved stage-by-stage matched regulators identified in our study were highly conserved between mouse and human (Rebuttal Fig. R19d). Overall, these analyses emphasized its potential conserved regulatory roles in mouse and human germ cell development.  Fig. 5c. Is the % at E13.5 and E14.5 really significantly changed? With only 4 samples, it is hard to believe that there is a significant difference. Please also check the statistical test in Fig.   5h.

7)
Answer: Thank the reviewer for this comment. We have carefully checked our raw data. Firstly, for the statistics of the percent of MKI67 + cells in DDX4 + fraction, we have counted 4 independent samples from different embryos, and there were significant differences between the WT and Helq -/during E13.5 and E16.5 (Rebuttal Fig. R20). Secondly, we have checked the original data and the proportion of S population was 47.84% as shown in the text. And we have modified the number in the Supplementary Fig. 8c of the revised Manuscript. We make sure that the proportion of G1 and G2/M were correct. Our raw data of G1, S, and G2/M phase in the WT and Helq -/-mESCs (powered by GraphPad Prism) were shown in Rebuttal Fig. R13. Indeed, the difference was significant between the WT and Helq -/group.
(1) First of all, according to a classical naming system 26 , germ cells before colonization in gonads are all called PGCs. After colonization, male germ cells are called ProSPG (prospermatogonia), but different stages of ProSPG have its own name. In detail, germ cells have just colonized in gonads, which are still rapidly multiplying, they are called M-ProSPG, but when male germ cells enter mitotic arrest, they are called T1-ProSPG (transitional 1 ProSPG). After birth, the male germ cells resume proliferation, at this time they are called T2-ProSPG, which finally develop into spermatogonia.
(2) In another typical naming system 27 , the germ cells from the specification PGCs to the proliferating germ cell after colonization in gonads (~E13.5) are collectively referred to as PGCs, and the germ cells thereafter until the spermatogonia after birth are collectively referred to as gonocytes (prospermatogonia).
(3) Given the fact there are many naming systems in this field, which always make it not easy to accurately understand the meaning of each word. Therefore, in previous studies of our group about human germ cell development 28,29 , human male germ cells were classified into migrating FGCs (fetal germ cells, 4W, before colonization in gonad), mitotic FGCs (~4W-35W, after colonization in gonad), and mitotic arrest FGCs (9W-25W, after colonization in gonad).
Thus, we follow the similar principle in the present study.
(4) In this study, we focused on the transition of germ cells after colonization in gonads, especially the transition of germ cells from the mitotic to mitotic arrest stage. In order to ensure the continuity for understating the story, we divided the PGCs before colonization in gonads into specification PGCs and migrating PGCs; male germ cells upon the entrance into the gonads, which still multiplied, were defined as mitotic PGCs; germ cells that exited from cell cycle after colonization were divided into mitotic arrest PGCs, post-arrest PGCs, Q-ProSPG, and T-ProSPG. Furthermore, during the transition from mitotic to mitotic arrest PGCs, we discovered a new subpopulation-the transitional PGCs.
Based on the classical nomenclature systems and the scRNA-seq data as well as experimental verification data in this study, we named the germ cells of mice in more detail to ensure the completeness and continuity of this study, which was mainly consistent with previous studies of our group 28,29 . At the same time, to make it easier for reviewers and readers to understand the development sequence of germ cells, we have compared our nomenclature with the classic nomenclatures aforementioned (Rebuttal Fig. R21). Answer: We thank for this suggestion. Accordingly, we have downloaded the data of Law et al. 30 and performed graph-based clustering to subdivide the E16.5 population. Consistent with this study 30 , three major cell clusters were found (Rebuttal Fig. R22a and b). Then, we extracted the mitotic arrest PGCs and post-arrest PGCs from our data to compare their similarities.
According to the expression of selective marker genes, E16.5 C1 (Law et al. 30 ) were more like the mitotic arrest PGCs in this study, which highly expressed the germline pluripotency and stem cell markers such as Nanog, Id4, Etv5, and Ret (Rebuttal Fig. R22c and d). In contrast, the differentiation-associated markers such as Dnmt3a, Dnmt3b, Sohlh1, and Sox3 were highly expressed in post-arrest PGCs of this study as well as E16.5 C2 and C3 (especially C3) (Rebuttal Fig. R22c and d). In conclusion, C2 and especially C3 of E16.5 in the previous study were more similar to the post-arrest PGCs of our study. 12) Protein acronyms should be all caps and not italicized; e.g., HELQ, not Helq.

Reviewers' Comments:
Reviewer #1: Remarks to the Author: Comments for Author According to the revised manuscript and response files, the author has provided detailed answers to our proposed amendments, and added a lot of data analysis and experimental content, which is more conducive to understanding the development trajectory of male germ cells. Additionally, the author re-analyzed the dynamic development and cellular events during the transition from SPG to SPC and further to RS according to our suggestions. This part of the analysis allows readers to fully understand the transcriptome changes throughout the entire developmental process of mouse male germ cells. This work is of great significance for understanding the development process of male germ cells in detail. Compared with other work, this article focuses on the analysis of gene expression patterns in the transition process between different developmental stages, which is highly innovative. Moreover, according to the homology analysis of gene expression in human and mouse spermatogenesis, many conserved genes were obtained, and 435 genes related to male infertility and testicular germ cell tumors were screened out. The screening of these homologous genes helps In-depth understanding of the pathogenesis of human male infertility and testicular germ cell-related diseases. In order to verify the accuracy of the transcriptome data, a variety of molecular biology methods were used to verify the work, and the construction of a knockout mouse model made it clear that the HELQ protein is essential for the cell fate transition from mitosis to PGC arrest. Furthermore, cell experiments verified that Notch signaling pathway regulates the mitotic arrest of male PGCs. The verification of these new results can already support its biological functions. There are two minor comments on data analysis (the last part), I hope the author will explain a little bit in the discussion. In the revised manuscript methodological description, the method steps and details are described in more detail. Minor comments: For the second question that "Can the post-arrest PGCs, Q-ProSPG, and T ProSPG mentioned in your article be reproduced based on the integration of the data from your research and them?" Q: In Rebuttal Fig. R3, we can't see any post-arrest PGCs, Q-ProSPG, and T ProSPG, which you find in your article in paper (PMID:30146481), hope to explain it in the discussion. In Figure 3e, the authors found that there were thirteen modules of DEGs, while the range of the ordinate is quite small, from -0.4 to 0.4. Was the trend significant? Did the three categories really make sense? Q: We know you scaled TPM to log(TPM/10+ 1) of each gene among these cells and then scaled log(TPM/10+ 1) to z-score. You did too much calculation on expression levels, and the gene trend may not be the original expression trend. Meanwhile, there were spelling errors in Rebuttal Fig.  R5.
Reviewer #2: Remarks to the Author: The revised version is much improved and has addressed the questions I raised earlier. As such, the manuscript can be released for publication.
Reviewer #3: Remarks to the Author: In this revised manuscript, the authors addressed some of my concerns, however, there are still couple that are not addressed well. 1) The evidence supporting "transitional PGCs" as a newly identified cell subset is not convincing. a) As shown in Fig. 3a, the defined "transitional PGCs" clearly contain some cells similar with mitotic PGCs (~80%) and other cells with mitotic arrest PGCs (~20%). That said, this transitional PGCs cells are a mixture that take part of mitotic PGCs and part of mitotic arrest PGCs. This is well supported by FiG. 3C, where the left part of the defined subcluster is close to mitotic PGCs, while the right part is close to mitotic arrest PGCs. b) If the authors further sub-cluster this defined "transitional PGCs" based on cell cycle status, they will definitely increase their ROGUE score as well, thus this is not a clear evidence. c) With regard to the immunostaining result, it cannot support that WEE1+Ki67+ cells are transitional PGCs as well. This is because: i) whether Wee1 RNA level is specific for this subset+mitotic arrest PGCs is unclear; ii) whether WEE1 protein (or signal defined by the WEE1 Ab) has the same expression pattern with their RNA. If the authors do want to confirm this transitional PGC is a new cell subset, they should perform RNA-scope (identified marker genes) +IF (cell-cycle markers, such as EdU) to confirm that transitional PGCs share transcriptome with both mitotic PGCs and mitotic arrest PGCs. Otherwise, the authors should omit this statement. 2) While the authors provide some evidence that fetal germ cells have active Notch signaling, whether germ cell-Notch pathway plays the role in germ cell development is unclear, as the evidence is from the treatment with two global Notch inhibitors. This cannot exclude the possibility that Sertoli cell-Notch signaling pathway plays the role in fetal germ cell development, as reported previously (PMID: 23907117, PMID: 23391689), the authors should at least discuss about this in the MS.
3) With regard to the naming of germ cells, while the authors argued there are two naming system, this is not the reason they generated new ones here, which makes the field more confusing. Anyway, they should add the table (Rebuttal Fig. R21) to their MS, in case readers will understand the study better.

Point-by-point responses to the Reviewers' questions:
Below we have listed the point-by-point responses to each of the questions, with the answers highlighted in blue.

Comments for Author
According to the revised manuscript and response files, the author has provided Answer: Thanks for the question. According to the routine scRNA-seq analysis, we performed pre-processing (quality control, normalization, feature selection, scaling, and dimensionality reduction) and cell-and gene-level downstream analysis in our data 3 .
In the Seurat pipeline 4 , gene scaling is always generally applied. In Fig. 3e, gene counts were scaled to have zero mean and unit variance (z scores); therefore, all genes were weighted equally. To avoid the similar concern that might be raised by the readers, we calculated the statistical significance between two adjacent cell types in each trend. The magnitudes of significance agreed with smooth or sharp changes of the DEGs (Fold change≥1.5, p-value<0.01) in the trends (Rebuttal 2 Fig. R1). In addition, the normalized data (unscaled) was used to show the original expression trend, and we found that it was consistent with the scaled data (Rebuttal 2 Fig. R1). Based on these data, the scaled data could reflect the gene expression patterns of DEG modules.
In addition, the spelling errors in Supplementary Fig. 5d have been corrected in the revised Manuscript.

Reviewer #2 (Remarks to the Author):
The revised version is much improved and has addressed the questions I raised earlier.
As such, the manuscript can be released for publication.

Answer:
We thank the reviewer for the positive comments.

Reviewer #3 (Remarks to the Author):
In this revised manuscript, the authors addressed some of my concerns, however, there are still couple that are not addressed well.
1) The evidence supporting "transitional PGCs" as a newly identified cell subset is not convincing. a) As shown in Fig. 3a,  Answer: Thank the reviewer for these questions and suggestions.
Firstly, for the comment "The evidence supporting "transitional PGCs" as a newly identified cell subset is not convincing. a) As shown in Fig. 3a . 3C, where the left part of the defined subcluster is close to mitotic PGCs, while the right part is close to mitotic arrest PGCs.", we are sorry for this misunderstanding due to the lack of essential descriptions and more details for cell cluster definition results. And we have performed more thorough analyses and experiments to provide evidences that the transitional PGC were an independent cell population. The evidence includes: Automatically dimension reduction (Fig. 1c of the revised Manuscript) could firstly cluster the gonadal PGCs at E11.5 to ~E16.5 into two cell clusters. According to previous reports 5 and cell cycle status by immunostaining of MKI67 ( Fig. 2b and Supplementary Fig. 4a of the revised Manuscript), the first one in earlier stages was mitotic PGCs, while the second one in later stages was mitotic arrest PGCs. Cell cycle analysis showed that almost all male mitotic PGCs were indeed actively proliferating, which was similar to the specification and migrating PGCs; but a part of mitotic arrest PGCs expressed a high level of cell cycle-related genes (Fig. 2b of the revised Manuscript), indicating that a subpopulation or an independent cell population existed in mitotic arrest PGCs. Hence, unsupervised hierarchical clustering analysis was performed on mitotic arrest PGCs using cell cycle-related genes (Rebuttal 2 Fig. R2a).
Notably, two cell populations that expressed high-or low expression levels of cell cycle-related genes were identified; therefore, the cell population that highly expressed cell cycle-related genes was named transitional PGCs and the cell population that lowly expressed cell cycle-related genes was the ultimate mitotic arrest PGCs (Rebuttal 2 Fig.   R2a). In addition, as this panel (Rebuttal 2 Fig. R2a) R2e and f). We found that these cells could also be automatically sub-divided into three independent cell populations, which was comparable with the previous results by unsupervised hierarchical clustering analysis. In details, the mitotic PGCs, transitional PGCs and mitotic arrest PGCs corresponded to cluster C1, C2 and C3, respectively (Rebuttal 2 Fig. R2e and f). Coupled with these data and the newly acquired RNAscope and IF results (Rebuttal 2 Fig. R3, discussed below) Collectively, based on thorough bioinformatics analyses and more detailed description of our data, we confirmed that the transitional PGCs were indeed an independent cell population, which was further supported by the RNAscope and IF results (Rebuttal 2 Fig. R3, discussed below).  in the revised Manuscript to support the conclusion that the transitional PGCs were indeed an independent cell population which shared transcriptome signatures of both mitotic PGCs and mitotic arrest PGCs.
As we explained above, the transitional PGCs were an independent cell population according to scRNA-seq results, and Wee1 was identified to be a marker for transitional PGCs (Rebuttal 2 Fig. R3a), which has been validated by immunostaining using the WEE1 antibody in our previous revised Manuscript. According to the suggestion of this reviewer, RNAscope (identified marker genes) +IF (cell-cycle markers) were performed to prove that the transitional PGCs were a new cell population. We have conducted IF (immunofluorescence) & RNAscope (mRNA expression level could be relatively quantified according to criteria following RNAscope manual, see Method).
First, we confirmed that the expression patterns of WEE1 protein and Wee1 mRNA were generally consistent (Rebuttal 2 Fig. R3c). This result indicated that our previous immunostaining of WEE1 co-stained with MKI67 could be used to distinguish the Answer: Thank the reviewer for this suggestion. We have discussed the roles that Sertoli cell-Notch signaling pathway plays in fetal germ cell development 6,7 in the Results and Discussion sections of the revised manuscript.
3) With regard to the naming of germ cells, while the authors argued there are two naming system, this is not the reason they generated new ones here, which makes the field more confusing. Anyway, they should add the table (Rebuttal Fig. R21) to their MS, in case readers will understand the study better.
Answer: Thank the reviewer for this suggestion. We have added this table to the revised manuscript following the reviewer's suggestion.

Method Simultaneous RNAscope and immunofluorescence assay
To visualize the transcription of mRNA and distinguish subtypes of PGCs, the RNAscope probe targeting Wee1 was designed and synthesized by Advanced Cell Diagnostics company, and the assay of RNAscope and IF were performed by using RNAscope® Multiplex Fluorescent Reagent Kit v2. For each experiment, POLR2A, PPIB, UBC and HPRT were used as the positive controls; dapB was used as the negative control.
Briefly, after fixing in 10% NBF (Neutral buffer formalin) for 24 h, fresh mouse male gonadal sections (5-μm-thin) were prepared and then pretreated with hydrogen peroxide solution, target retrieval solution, and stained with primary antibodies overnight at 4°C. Sections were further treated with protease plus and finally hybridized with the RNA probe of target gene for 2 h at 40°C in hybrid furnace, followed with a series of signal amplifications. After RNAscope, sections were stained with secondary antibody 30 min at room temperature. And Nuclei were counterstained with Hoechst for 10 min at room temperature. Images were obtained with ZEISS LSM880 confocal microscope.
The signal dots were visually counted following the RNAscope manual (as outlined at https://acdbio.com/how-correctly-interpret-your-rnascope%C2%AE-images), and single dot equals to single mRNA. Robust cutoff for determining the "High expression level" and "Low expression level" of Wee1 mRNA was set up using the following criteria: (1) The number of dots in each cell is an integer, so the cutoff must also be an integer; (2) The mean dot in random PGCs is 5.35, which means PGCs with more than 5 (less than and nearest to 5.35) are Wee1 mRNA high expression, otherwise are low expression; (3) The 25% percentile of the dot numbers in WEE1 High (WEE1 protein high expression) PGCs is 5.25, representing the lower threshold of expected expression is 5 (less than and nearest to 5.25). Overall, PGCs with less than or equal to 5 dots are defined as Wee1 Low (Wee1 mRNA low expression) ones, and PGCs with more than 5 dots are defined as Wee1 High (Wee1 mRNA high expression) ones. In all cases, statistics was performed following the above criteria.