The single-cell chromatin accessibility landscape in mouse perinatal testis development
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Curated by eLife
Evaluation Summary:
Liao et al. aim to improve our understanding of the genetic networks that underlie mouse male gonadogenesis and germ cell maturation during the fetal to neonatal transition. This goal was pursued by performing scATACseq on multiple timepoints, followed by definition of cell types and identification of potential transcription factors that could control the progress of differentiation. This is an exciting paper that may have far-reaching implications in the field of reproductive biology and male infertility, but additional validation is needed to confirm the newly identified cell populations.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Spermatogenesis depends on an orchestrated series of developing events in germ cells and full maturation of the somatic microenvironment. To date, the majority of efforts to study cellular heterogeneity in testis has been focused on single-cell gene expression rather than the chromatin landscape shaping gene expression. To advance our understanding of the regulatory programs underlying testicular cell types, we analyzed single-cell chromatin accessibility profiles in more than 25,000 cells from mouse developing testis. We showed that single-cell sequencing assay for transposase-accessible chromatin (scATAC-Seq) allowed us to deconvolve distinct cell populations and identify cis -regulatory elements (CREs) underlying cell-type specification. We identified sets of transcription factors associated with cell type-specific accessibility, revealing novel regulators of cell fate specification and maintenance. Pseudotime reconstruction revealed detailed regulatory dynamics coordinating the sequential developmental progressions of germ cells and somatic cells. This high-resolution dataset also unveiled previously unreported subpopulations within both the Sertoli and Leydig cell groups. Further, we defined candidate target cell types and genes of several genome-wide association study (GWAS) signals, including those associated with testosterone levels and coronary artery disease. Collectively, our data provide a blueprint of the ‘regulon’ of the mouse male germline and supporting somatic cells.
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Author Response
Reviewer #1 (Public Review):
The authors have performed scATACseq on multiple timepoints during mouse male gonadogenesis and germ cell maturation during the fetal to neonatal transition (E18.5 and postnatal days 1,2,5). Clustering of thousands of cells revealed striking cellular diversity and led to the identification of cell populations that were not known before. This work may have far reaching implications, but additional validation is needed.
We would like to start by expressing our appreciation to the reviewer’s valuable comments and feedback on our manuscript. We would also like to express our sincere apologies for the delay in submitting our revised manuscript. The COVID-19 pandemic has had a significant impact on academic research and publication, and we encountered several challenges during this time. Both …
Author Response
Reviewer #1 (Public Review):
The authors have performed scATACseq on multiple timepoints during mouse male gonadogenesis and germ cell maturation during the fetal to neonatal transition (E18.5 and postnatal days 1,2,5). Clustering of thousands of cells revealed striking cellular diversity and led to the identification of cell populations that were not known before. This work may have far reaching implications, but additional validation is needed.
We would like to start by expressing our appreciation to the reviewer’s valuable comments and feedback on our manuscript. We would also like to express our sincere apologies for the delay in submitting our revised manuscript. The COVID-19 pandemic has had a significant impact on academic research and publication, and we encountered several challenges during this time. Both co-first authors of this manuscript were promoted to new roles, which required additional time and effort to transition into these new positions. Furthermore, we experienced significant delays in obtaining the necessary research materials due to longer shipment times for antibodies and other reagents during the pandemic, which further contributed to the delay. We understand that our delay may have caused inconvenience but we want to assure you that we have carefully addressed all of the reviewer comments and we deeply appreciate your understanding and patience during these challenging times.
The identification of novel transitional spermatogonia population in Figure 4D is intriguing. Independent validation by flow cytometry or in testis cross section to better allow the colocalization of nr5a1 and Oct4 and other germ cell markers would be important. Additional validation is needed to ensure that populations 1 and 2 in figure 4d are not to doublets. Providing violin plots for both soma and germ cell markers will be helpful. Is SF1 the only gene expressed in this unique germ cell population or are many other somatic markers expressed in the population. Do these cells express well recognized SPG markers like Oct4+ , PLZF, GFRA?
We have performed immunostaining of NR5A1 in testicular sections and showed that NR5A1+ germ cells (TRA98+ cells) exist in P5.5 testis (Figure 4D). We appreciate the reviewer's comment and understand the concern regarding potential doublets in figure 4d. We examined the expression of various markers in both scATAC-seq (gene score) and scRNA-seq (mRNA) datasets and provided violin plots. Sertoli cell markers and germ cell markers showed variable levels in unknown 1 and 2 populations while the Leydig cell marker did not (Supplementary figure S6D).
As additional evidence supporting our finding that a subset of somatic markers are expressed in the unique germ cell population we identified, we reference a study where cells in the spermatogonial signature 3 cluster showed high levels of mRNAs characteristic of Sertoli cells, including Nr5a1, Sox9, and Wt1 (PMID: 25568304). This indicates that cells with germ cell identity can express somatic cell genes, which is consistent with our findings. Additionally, another study reported the expression of the somatic cell marker WT1 in some germ cells through immunostaining (Figure 3B, PMID: 34815802). We have included this information in the revised manuscript to further support our conclusion (line 301). In addition, as we have isolated nuclei rather than whole cells, it is less likely that germ cells and sertoli cells are sticking together during single cell capture. We hope that the additional evidence and analysis provided will help to ease the reviewer's concerns and further support the conclusions drawn from our data.
The IF validation in 5F is not as convincing that these cells are potentially Sertoli stem cells. IF in cross-sections will be easier to interpret- especially when co-stained with several germ, somatic, or novel markers of that population. purification of these cells and further characterization is needed. A hallmark of fetal Sertoli cells is to mediate the migration of endothelial cells to the seminiferous tubules during testicular cord formation. Is it possible to purify these cells to determine whether they have functional Sertoli cells properties in vitro using human umbilical vein endothelial cells (HUVECs). Do these cells have immune privilege properties - can they suppress proliferation of Jurkat E6 cells.
Following the reviewer’s suggestions, we conducted further immunostaining of MBD3 and AMH in Sertoli cells (Figure 5F). The observed staining results not only confirm the properties of MBD3+ cells (MBD3-high/AMH-high) but also highlight the heterogeneity of Sertoli cells, as evidenced by the presence of various expression patterns such as MBD3-low/AMH-high (cluster SC3 in Figure 5A) and MBD3-low/AMH-low (cluster SC2/4/5/6 in Figure 5A). This further emphasizes the complexity and diversity within the Sertoli cell population.
However, we understand that it is premature to definitively conclude that MBD3-high cells are Sertoli stem cells without functional studies. We appreciate the suggestion of using additional functional assays such as in vitro co-culture with HUVECs and immune privilege assays to further characterize the potential Sertoli stem cell population. These are valuable experiments to consider for future research in order to gain a deeper understanding of the properties and functions of these cells. To more accurately reflect the scope of our study and avoid potential misinterpretation, we have revised the language to reflect that we have identified subpopulations of Sertoli cells with unique characteristics, rather than using the term "stem cell". We hope that our revised data adequately addresses the reviewer’s concerns.
Reviewer #2 (Public Review):
Liao et at performed single cell ATAC sequencing to reveal chromatin status in various cell types in the perinatal mouse testes. The chromatin status was then used to define cell types and identify potential transcription factors that control the progress of differentiation. This work could provide new insights into how various cell types acquire their fate in early testis development and establish a genomic framework that can be used to correlate with human data for infertility. The strength lies on the novelty of single cell analyses. The weaknesses include a lack of statistical power, the uncertainty on the correlation between chromatin status, gene expression, and transcription factor activity, and insufficient information and confirmation on some of the experiments and results.
We would like to start by expressing our appreciation to the reviewer’s valuable comments and feedback on our manuscript. We would also like to express our sincere apologies for the delay in submitting our revised manuscript. The COVID-19 pandemic has had a significant impact on academic research and publication, and we encountered several challenges during this time. Both co-first authors of this manuscript were promoted to new roles, which required additional time and effort to transition into these new positions. Furthermore, we experienced significant delays in obtaining the necessary research materials due to longer shipment times for antibodies and other reagents during the pandemic, which further contributed to the delay. We understand that our delay may have caused inconvenience but we want to assure you that we have carefully addressed all of the reviewer comments and we deeply appreciate your understanding and patience during these challenging times.
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Evaluation Summary:
Liao et al. aim to improve our understanding of the genetic networks that underlie mouse male gonadogenesis and germ cell maturation during the fetal to neonatal transition. This goal was pursued by performing scATACseq on multiple timepoints, followed by definition of cell types and identification of potential transcription factors that could control the progress of differentiation. This is an exciting paper that may have far-reaching implications in the field of reproductive biology and male infertility, but additional validation is needed to confirm the newly identified cell populations.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The authors have performed scATACseq on multiple timepoints during mouse male gonadogenesis and germ cell maturation during the fetal to neonatal transition (E18.5 and postnatal days 1,2,5). Clustering of thousands of cells revealed striking cellular diversity and led to the identification of cell populations that were not known before. This work may have far reaching implications, but additional validation is needed.
The identification of novel transitional spermatogonia population in Figure 4D is intriguing. Independent validation by flow cytometry or in testis cross section to better allow the colocalization of nr5a1 and Oct4 and other germ cell markers would be important.
Additional validation is needed to ensure that populations 1 and 2 in figure 4d are not to doublets. Providing violin plots for both …
Reviewer #1 (Public Review):
The authors have performed scATACseq on multiple timepoints during mouse male gonadogenesis and germ cell maturation during the fetal to neonatal transition (E18.5 and postnatal days 1,2,5). Clustering of thousands of cells revealed striking cellular diversity and led to the identification of cell populations that were not known before. This work may have far reaching implications, but additional validation is needed.
The identification of novel transitional spermatogonia population in Figure 4D is intriguing. Independent validation by flow cytometry or in testis cross section to better allow the colocalization of nr5a1 and Oct4 and other germ cell markers would be important.
Additional validation is needed to ensure that populations 1 and 2 in figure 4d are not to doublets. Providing violin plots for both soma and germ cell markers will be helpful. Is SF1 the only gene expressed in this unique germ cell population or are many other somatic markers expressed in the population. do these cells express well recognized SPG markers like Oct4+ , PLZF, GFRA?
The IF validation in 5F is not as convincing that these cells are potentially Sertoli stem cells. IF in cross-sections will be easier to interpret- especially when co-stained with several germ, somatic, or novel markers of that population. purification of these cells and further characterization is needed. A hallmark of fetal Sertoli cells is to mediate the migration of endothelial cells to the seminiferous tubules during testicular cord formation. Is it possible to purify these cells to determine whether they have functional Sertoli cells properties in vitro using human umbilical vein endothelial cells (HUVECs). Do these cells have immune privilege properties - can they suppress proliferation of Jurkat E6 cells.
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Reviewer #2 (Public Review):
Liao et at performed single cell ATAC sequencing to reveal chromatin status in various cell types in the perinatal mouse testes. The chromatin status was then used to define cell types and identify potential transcription factors that control the progress of differentiation. This work could provide new insights into how various cell types acquire their fate in early testis development and establish a genomic framework that can be used to correlate with human data for infertility. The strength lies on the novelty of single cell analyses. The weaknesses include a lack of statistical power, the uncertainty on the correlation between chromatin status, gene expression, and transcription factor activity, and insufficient information and confirmation on some of the experiments and results.
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