Single-cell RNA sequencing identifies regulators of differentiation and nutritional cues in Drosophila female germ cells

  1. Zhipeng Sun
  2. Todd G. Nystul
  3. Guohua Zhong

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Abstract

Drosophila ovarian germline stem cells (GSCs) are powerful model for stem cell research. However, due to the scarcity of GSCs in ovarian tissue, it is difficult to obtain the transcriptional profile of GSCs and identify novel GSC markers. In this study, we took advantage of single cell RNA sequencing (scRNA-seq) to profile the germline cells and somatic cells in wild type Drosophila ovary. We then performed an in vivo RNAi screen and network analysis to identify genes that are involved in the early stages of germ cell differentiation. We identified 33 genes with limited expression during early germ cell development and identified 19 genes that potentially regulate germ cell differentiation. Among these, an uncharacterized gene, which we named eggplant ( eggpl ), is highly expressed in GSC and downregulated in early daughter cells. Upon RNAi knockdown of eggpl , we observed an increase in germ cell proliferation, an accumulation of cysts in the early mitotic (2- and 4-cell) stages and an increase in overall ovary size compared to control when flies were maintained on a standard diet. In flies fed a rich yeast diet, the expression of eggpl was significantly lower and the effects of eggpl RNAi were suppressed, suggesting that downregulation of eggpl may link nutritional status to germ cell proliferation and differentiation. We also found that the matrix metalloproteases, Mmp1 and Mmp2 as well as the tissue inhibitor of metalloproteases ( Timp ) are additional regulators of eggpl . Collectively, this study provides new insight into the signaling networks that regulate early germ cell development and identifies eggpl as a key player in this process.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors have utilised single-cell RNA seq to profile cells in the Drosophila female germline cells and somatic cells within the ovary. Using a candidate approach, the authors assessed whether some of the candidate genes identified in this study play a role in germ cell differentiation. Amongst these, they found that an uncharacterized gene called eggplant is required for differentiation. On rich diets, Eggplant expression is reduced. Furthermore, Mmps and Timp are required to regulate Eggplant expression level.

    Major comments:

    Overall, the conclusions are supported by experimental evidence. The sc-RNA seq provides an important resource for the community, and some of the reagents generated such as the antibody against Eggplant or the tagged lines would provide valuable resource for future studies. The main criticism I have for this manuscript is that it lies half way between a resource paper and a mechanistic paper. If it was just a resource paper, then the sc data should suffice. Instead, the authors studied the effects of Eggplant and MMP/TIMP on GSC differentiation, however, not enough mechanism was gained through these studies.

    1. It is not clear what kind of protein Eggplant is, and its mode of action is unclear, despite that it was shown by the authors that Eggplant is required for GSC differentiation. The authors further showed that animlas fed a high yeast diet promotes GSC proliferation and increased egg production by inhibiting the expression of eggpl. The authors suggested that this may lie downstream of insulin signaling, however no genetic experiments were conducted. In order to support this line of conclusions, essential experiments should include: is there a genetic interaction between Eggplant and insulin signaling? Is this happening autonomously within the GSCs or at the GSC niche? In the discussion the authors mentioned that since egg production can occur upon Eggpl knockdown even on normal diet, thus protein is not involved in this process. So, what is the mechanism?
    2. Similarly, the authors mentioned that MMPs and TIMP are involved in GSC differentiation, and they discussed that it could be mediated via tissue stiffness. However, no experimental data was presented to support this. It would be good to offer some mechanistic insights. TIMP and Eggplant genetic interaction should be done.

    Minor comments:

    Line 376 and Line 437 are repetitive in explaining why the gene is called eggplant. I did not find the pictures in Figure7 F,G and Figure 8 B informative. Some quantifications should also proof the same point.

    Referees cross-commenting

    I also agree with the comments from both reviewers 1 and 2.

    Significance

    • The significance of this study lies in presentation of a thorough characterization of gene expression at the single cell level in the GSC and in identification of novel regulators of GSC differentiation.
    • My field of expertise is Drosophila stem cells and nutrition/metabolism.
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    Referee #2

    Evidence, reproducibility and clarity

    Brief summary

    The authors conducted single cell RNA sequencing on adult fly ovaries to survey the transcriptomic profile of individual ovarian cells. The datasets resulted in classification of 24 discrete cellular populations, including different types of stem cells, progenitors and differentiated cells. Genes that differentially expressed during germline differentiation were selected and then examined with RNA interference to determine their specific roles in regulating germline differentiation. Meanwhile, using the single cell transcriptomic profiles acquired from specific types of germ cells, gene regulatory networks were further characterized. Among the genes examined, the gene eggplant (eggpl) was found to act as a novel regulator mediating germline differentiation with its protein expression predominantly detected in germline stem cells (GSCs), CBs and early cysts. Interestingly, since the protein expression of Eggplant was decreased upon rich dietary intake, eggpl may function as a novel molecular link coupling the nutritional status and the process of germline differentiation during fly oogenesis.

    Specific comments

    Major points:

    1. The authors chose a specific group of differentially upregulated genes (in GSCs) and performed germline specific RNA interference (RNAi) to determine their function in regulating germline differentiation. The RNAi results were then compiled and presented in Figure 3B-C. However, details shall be provided for the readers/reviewers to understand the phenotypic analyses. Specifically, as stated in text that "we found that RNAi knockdown of 19 upregulated genes induced disruption of GSCs/CB homeostasis. Of these, 12 genes were classified as "changes to the number of CSC/CB", 6 genes as "empty germarium" and 4 genes exhibited "differentiation defects"... ", there was neither specific descriptions nor representative examples (with proper labelling) explaining the bases of such classification. It was particularly difficult to appreciate the assorted phenotypes presented in Figure 3C without proper descriptions. Not to mention that the immunostaining needs to be optimized in some of the representative pictures. Meanwhile, it was not clearly stated in the text about how 19 candidate genes were then classified into (12+6+4=22) genes. Along the same line, it will be helpful to include a supplementary table of summarizing the genes tested and their corresponding phenotypes.
    2. The authors also performed germline specific RNAi to knockdown the expression of 21 out of 39 most highly expressing genes in GSCs to determine their function in regulating oogenesis. Some of the RNAi effects were presented in Figure S2B. Similarly, the effects of RNAi experiments will be better appreciated if some more descriptions on specific RNAi phenotypes are added. Also, inclusion of a supplementary table summarizing the RNAi lines utilized in this experiment is required.
    3. The authors performed in situ hybridization and immunostaining to discover that the eggpl transcripts were enriched in GSCs and CBs (Bam-GFP- germ cells), while the Eggpl::GFP expression (in the knock-in line, KI) was detected throughout region 1 within germarium. The authors then concluded that protein level of Eggpl was "actually highest in cells that have no detectable eggpl transcripts". However, as shown in Figure 5F, the highest level of Eggpl::GFP appeared in the cell that contains a spectrosome, suggesting that is either a GSC or a CB obtaining highest Eggpl::GFP (KI) expression. Furthermore, as shown in Figure 4E, similar level of Eggpl protein expression indicated by immunostaining was found in GSCs and CBs, indicating a nice coupling between the mRNA and protein level of eggpl found in GSCs/CBs. Therefore, the authors need to simultaneously perform in situ hybridization and immunostaining of eggpl to visualize the expression of eggpl transcripts and protein within the same germarium and to directly determine whether the statement that "protein level of Eggpl was actually highest in cells that have no detectable eggpl transcripts" holds true.
    4. The genetic experiments done by the authors show that disruption of eggpl led to an increase in the average number of Brdu+ cysts, the size of the ovary and ultimately the number of eggs produced. Interestingly, such phenotypes were reminiscent to the effects caused by rich dietary intakes. The authors interpret these results by proposing that eggpl negatively regulates GSC proliferation in regulating oogenesis. However, as shown in Figure 5J, the number of GSCs was not significantly altered upon eggpl RNAi but was increased when over-expressing Eggpl::GFP, indicating that eggpl promotes the proliferation of GSCs. To be able to determine the function of eggpl in regulating GSC proliferation during oogenesis and to support the stamen that "We also found that cell cycle in germ cell cysts was accelerated" (in introduction), the authors need to perform clonal analyses to monitor the progression rate of eggpl mutant cells (of both germline clones and follicle cell clones) compared to control cells. An accelerated rate of germline development shall be predicted by increased GSC proliferation. Moreover, to rule out the possibility that changes in the dynamics of germline apoptosis can affect the number of Brdu+ cysts observed upon eggpl loss, the authors should also perform experiments assaying the number of apoptotic cells in the germarium when manipulating the expression level of eggpl.
    5. The authors examined the expression level of Eggpl::GFP (KI) in different feeding conditions and found a reverse correlation between Eggpl::GFP (KI) protein level and the quality of dietary condition. Moreover, the enlarged ovary seen upon eggpl loss was recapitulated when over-expressing Timp and Mmp1/2. Interestingly, the authors also found that overexpression of Timp and Mmp1/2 led to lower protein expression of Eggpl::GFP (KI). The authors interpret these results by proposing that eggpl mediates the GSC differentiation via MMP-dependent Timp regulation pathway. However, the authors should perform experiments to directly examine if eggpl and Timp (and Mmp1/2) interact genetically to regulate GSC proliferation/egg production while monitoring the dietary status.

    Minor points:

    1. Please clarify what you mean in this sentence in the introduction as the message is not clear: "Mei-P26 suppress transcripts that promote differentiation in CB by antagonizing miRNA pathway
    2. In Figure 3B, for the genes that were listed for more than once (CG42250, CG10295, CG6904 and CG15845), please note that those are different RNAi lines.
    3. In Figure S2A, for the genes that were listed for more than once, please note that those are different RNAi lines.
    4. If applicable, more than one RNAi line of each candidate gene should be examined to validate the specific effects caused by gene KD.
    5. As shown in Figure S2B, knocking down CG31666 and CG12743 led to impaired oogenesis. Please add their gene name (CG31666 (chinmo) and CG12743 (otu)) for acknowledging their well-documented role in regulating oogenesis.
    6. How was the RNAi generally performed. Please add into material and method.
    7. In Figure 7A, it is difficult to tell how many of the BrdU+ cysts was shown in Eggpl[1] mutant germarium and whether it is qualitatively/quantitively different from control.
    8. The authors compared their scRNA-Seq dataset with other 4 public available Drosophila ovary scRNA-Seq datasets and concluded that comparable features were found among these datasets by UMAP analysis. The authors should also comment on what are the potential differences among these adult ovary scRNA-Seq datasets in the discussion section. Providing such information will not only highlight the importance of this dataset and will also be beneficial to the fly community for future research.

    Referees cross-commenting

    I also agree with the comments from both reviewers 1 and 3.

    Significance

    In summary, the characterization of single-cell atlas of adult fly ovary from this study adds additional datasets for future investigation on cell-specific differentiation in vivo. Meanwhile, the identification of a novel molecular link between nutritional status and germline differentiation is of broad interest to readers in the field of dietary/metabolic control on stem cell function. However, there are specific concerns about how the results were presented and interpreted while some important controls are missing. I believe this article would be much improved if the points mentioned can be effectively addressed.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Sun et al. conduct single-cell RNAseq on germline stem cells in the Drosophila ovary to better understand the transcriptional profile of this small, but important, population of stem cells. This resulted in the identification of a subset of genes and gene networks proposed to be involved in early germ cell development and differentiation. A subsequent RNAi screen of these candidate genes indicated that an uncharacterized one (renamed eggplant) was expressed at the transcriptional level in GSCs and at the protein level in the germarium. RNAi-mediated knockdown or CRISPR/Cas9-mediated knockout of eggplant resulted in an accelerated cell cycle in germ cell cysts and flies with overall larger ovaries. These results were not observed, however, when flies were fed a rich yeast-based diet. The authors also draw a connection between the MMP-Timp pathway and the regulation of eggplant in ovarian germ cells. Collectively, the findings presented in this manuscript detail novel modulators of germ cell differentiation and proliferation that are regulated by nutrient availability. While the data presented are intriguing (especially in the first part of the manuscript), there are several major and minor issues that should be addressed prior to publication.

    Major issues:

    1. The bioinformatic analysis of the scRNA-seq data is elegant and well done. However, the data from the follow-up studies on eggplant are mostly observational, correlative, and not convincing. There is little to no mechanism described regarding the function of eggplant. This may be beyond the scope of this paper, but it should at least be addressed in more detail in the Discussion section.
    2. Along the lines of point 1, what is the amino acid sequence of eggplant? What is its predicted molecular weight? Does it contain any predicted domains that may hint at its function in the germarium? The answers to these questions should be included in the manuscript.
    3. There is no validation of the novel eggplant antibody that was generated. This needs to be demonstrated in order to be confident that the antibody is working as expected to interpret the immunofluorescence data.
    4. In Figure 8, the connection between MMPs, TIMPs, and eggplant is very weak. It is unclear how the regulation is occurring. Is it direct or indirect? And by what mechanism? Also need to show representative images to go along with the graphs. But as is, this is the weakest data in the manuscript and needs more clarification.

    Minor issues:

    1. It is unclear what it is being shown in Figure 5M.
    2. In Figure 7B, how was the egg-laying assay performed. This information was not included in the Materials and Methods section.
    3. There are some grammar issues in the Introduction section.

    Referees cross-commenting

    My comments align closely with those of Reviewer #3. I also agree that the specific points raised by Reviewer #2 should be addressed to strengthen the manuscript.

    Significance

    The significance of this work lies in the transcriptional profiling of the Drosophila ovarian germline stem cells. These cells are scarce and are notoriously difficult to isolate and study. This work makes headway in that regard, laying the foundation for future studies, and as mentioned, the bioinformatic analysis of the scRNA-seq data is the strength of the study.

  5. Version published to 10.1101/2022.08.15.504025v1 on bioRxiv