Adult stem cells and niche cells segregate gradually from common precursors that build the adult Drosophila ovary during pupal development

  1. Amy Reilein
  2. Helen V Kogan
  3. Rachel Misner
  4. Karen Sophia Park
  5. Daniel Kalderon

  • Curated by eLife

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    Evaluation Summary:

    This manuscript describes a very extensive set of experiments charting the origin and fate of various cell populations in the Drosophila ovary that is a powerful system to explore interactions between adult stem cells and their niches. The authors put forward a new view of how different cell types acquire their fates during development. This is a more nuanced view than extant models, involving common progenitors from which different cell fates (stem cell, progeny and niche cells) arise gradually and relying on spatiotemporal cues.

    (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

Production of proliferative follicle cells (FCs) and quiescent escort cells (ECs) by follicle stem cells (FSCs) in adult Drosophila ovaries is regulated by niche signals from anterior (cap cells, ECs) and posterior (polar FCs) sources. Here we show that ECs, FSCs, and FCs develop from common pupal precursors, with different fates acquired by progressive separation of cells along the AP axis and a graded decline in anterior cell proliferation. ECs, FSCs, and most FCs derive from intermingled cell (IC) precursors interspersed with germline cells. Precursors also accumulate posterior to ICs before engulfing a naked germline cyst projected out of the germarium to form the first egg chamber and posterior polar FC signaling center. Thus, stem and niche cells develop in appropriate numbers and spatial organization through regulated proliferative expansion together with progressive establishment of spatial signaling cues that guide adult cell behavior, rather than through rigid early specification events.

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  1. Version published to 10.7554/elife.69749 on eLife
  2. eLife

    Author Response:

    Reviewer #1:

    Stem cells in the Drosophila ovary provide a great model to understand cell behavior and regulation due to its genetic tractability, organized morphological pattern, and ease to perform live imaging. Compared to the adult ovary which have been studied quite extensively, the pupae ovary is a much less explored stage. Here the authors extensively studied the cell lineage in the pupae ovary, which helps understanding the development of early cell fates and the formation of the first set of egg chambers. They first described the different stages of pupal ovary development, followed by several different lineage tracing experiments that conclude a subset of Intermingled Cells (ICs) as Escort Cell (EC)/ Follicle Stem Cell (FSC) common precursors. Then they described Extra-Germarial Crown Cells (EGCs) and basal stalk cells and showed by live imaging that they contribute to the first budding cyst.

    Strength:

    Several lineage tracing experiments and statistical calculations are performed to conclude that a subset of ICs function as EC/FSC common precursors. The methods are written in great detail to help understand the calculation.

    The finding that EGCs and basal stalk cells contribute to the first budding cyst is new and intriguing. This initial developmental process, different from what happens in the adult ovary, might provide insight into how germline and somatic cells are coordinated.

    Weakness:

    The authors showed in their 2017 NCB paper that FSCs contribute to ECs in adult ovary. Here they showed that there is a common precursor of EC/FSC. Are these two cell types the same? It has been shown in single cell analysis during third instar that the ICs and FSCPs present Con and bond as their specific markers (Slaidina et al. 2020). Both give rise to EC/FSC/FC in lineage tracing experiments. Therefore, the novelty of this finding is weakened.

    ECs and FSCs in adults have important different properties. ECs do not divide; FSCs do divide. ECs interact with developing germline cysts to support their progressive differentiation. FSCs are not known to have an analogous role. FSCs all have long processes that span the germarium. Most ECs have shorter processes, especially in anterior regions. Quite likely there are also many similarities between FSC and ECs, for example in gene expression profiles, since FSCs can readily become ECs.

    In adults, new ECs are continually produced from FSCs. There is evidence that the production of marked ECs from marked adult FSCs saturates over only a few days, suggesting that adult- produced ECs turnover by dying or returning to FSC status [1]. The same studies suggest that ECs produced during development do not turnover at a comparable rate. So, while there may be some conversion of adult-born ECs to FSCs it is unlikely that a significant proportion of ECs present at eclosion later become FSCs. So, overall, the relationship between adult FSCs and ECs appears to be a typical one of stem cell and maintained derivative cell.

    Our work here shows that the attribution (quoted by the reviewer) by Slaidina et al., (20-20) of “FSCP” status to cells specifically expressing bond is incorrect on two counts. First, there is no such thing as an “FSCP”, dedicated to produce just FSCs and FCs at the start of pupation. Almost all FSC-producing precursors at that stage also produce ECs. Second, all FSC precursors are within the IC population at the start of pupation and remain within the developing germarium throughout pupation. They are not posterior to ICs and the germarium, like the population of bond-expressing cells noted by Slaidina et al., in late third instar larvae. Our results refute the earlier conclusions that inappropriately assumed (i) bond-GAL4 labeled only cells posterior to ICs in lineage experiments (it does not) and (ii) ovarioles with marked ECs and FSC/FCs derive from two or more distinct precursors rather than a common precursor (which we deduced by looking at lineages derived from single cells).

    Our deduction of a common precursor of ECs and FSCs is new and opposite to the conclusions of Slaidina et al., who proposed separate precursors for each at the start of pupation.

    While the finding that EGCs and basal stalks contribute to the first budding egg chamber is intriguing, the definition of EGCs and basal stalks are quite vague. Do EGCs have a distinct feature that is worth noting as separate cell types, or are they simply early FCs locating posterior of the first germline cyst? Do basal stalks express mature stalk markers, or are they simply accumulating FCs that are not fully differentiated yet?

    We have clarified EGC and basal stalk definitions in the text. Prior to budding of the first egg chamber, there are some partially intercalated Fas3-positive cells posterior to the germarium previously termed the “basal stalk”. We noticed that the most anterior of those cells expressed Traffic-Jam, which was expected to be important for all ovariole cell types (ECs, FSCs and FCs), and therefore worthy of highlighting with a different name. Further analysis showed that not only cells in the EGC but also many in the basal stalk subsequently became FCs on the first egg chamber (acquiring Traffic-jam expression at some point along the way). Thus, EGC and basal stalk designations from 0-48h APF do not define different outcomes but the separate names are still useful descriptors of cell populations in specific locations and with slightly different expression profiles during the first half of pupation.

    We did not examine whether, or exactly when, pupal basal stalk cells express markers seen in the stalks between egg chambers. Most cells in the pupal basal stalk population contribute to the FC epithelium and, with the exception of polar cells, lose Fas3 expression by adulthood. The basal stalk cells that remain posterior to the first egg chamber form the basal stalk of a newly- eclosed adult and retain Fas3 expression. The stalks between egg chambers, like the FC epithelium, express Fas3 at early stages but lose Fas3 expression at later stages.

    Is the process of EGCs and basal stalk contributing to the first budding egg chamber similar to posterior FCs contributing to the budding egg chamber? Is it because the budding of the first germline cyst takes longer than normal, there are more FCs accumulating at the posterior, thus making this region look like EGCs and basal stalks?

    The two processes appear to be substantially different. In the adult, FCs are already associated with a germline cyst when it leaves the germarium. In pupae, the first germline cyst leaves the germarium without associated somatic cells and enters an accumulation of somatic cells, moving into and through the EGC and most of the basal stalk. Neither process is understood well enough to explain the underlying reasons for this difference. However, we believe that one important difference may be the absence of a posterior signaling center, provided by polar FCs of the previously budded egg chamber in adults, prior to budding of the first egg chamber in pupae. That could affect several germline and somatic cell properties relevant to adhesion among cells, accumulation of cells and cell movements.

    The presentation is too lengthy. A more concisely written paper would help the audience to get the key points that the authors hope to convey.

    We have trimmed and summarized substantially, spurred by specific comments of both reviewers.

    Reviewer #2:

    Reilein, Kogan and co-workers chart the origin and fate of the different cell types in the Drosophila ovary throughout pupal stages using a combination of mosaic analysis, live imaging and immunohistochemistry. Their results challenge some of the assumed lineage and niche relationships between adult progenitors and support cells. The authors identify progenitors that can give rise to more cell types than previously thought (e.g. precursors that yield follicle stem cells and their adult niche and product cells) and revise some cell interactions/lineage relationships (for example, by providing evidence for separate precursors of follicle cells in the first-formed egg chamber, or by observing that germline progression can be supported by developing escort cells precursors rather than differentiated escort cells). Collectively, their data suggest a gradual and flexible adoption of these cell types according to the position of specific precursors during development.

    Although mosaic/clonal analyses do not always provide clear-cut answers, the authors are fully aware of possible caveats, and have done everything in their genetic power to ensure that their interpretations are as sound as they can possibly be. This includes, for example, extensive quantifications and statistical analyses/predictions based on clone frequency, the use of multicolour marking strategies to ensure that lineages are derived from single cells, and consideration of the effects of temperature shifts on developmental times. The resulting data are invariably comprehensive, have been documented and quantified extensively, and are often accompanied by stunning images.

    In a way, the comprehensive nature of this manuscript is also its Achilles heel: it is VERY long and dense. Any readers unfamiliar with the Drosophila ovary may not take the time to digest the data. This would be a pity as the manuscript's main messages are timely. They also resonate with observations in other systems such as the mouse gut field which, collectively, are beginning to challenge concepts such as hardwired "stemness" and "genetic programs", and to rediscover the importance of positional/mechanical cues in specifying cell fate.

    The power of description is somewhat underestimated in our post-genetic revolution era, and there is a lot to be learned by carefully observing and documenting "what does happen" - as opposed to exclusively relying on genetic loss/gain-of-function experiments. This manuscript is a good illustration of this. That said, the authors' observations make a number of predictions which could be genetically tested (e.g. through temporal ablation experiments to confirm flexibility/temporal requirements, or experiments targeting specific pathways to confirm their contribution as spatial organisers). These experiments would make their revised models much more compelling.

    We appreciate these comments, especially the value placed on studying how cells behave, the difficulty of ascertaining that through lineage studies, and the search for putting findings in a general context accessible to a broad group of scientists. We have tried to edit the manuscript further to achieve the last aim. However, the key virtues of thoroughness and the surprising difficulty of deducing “what does happen” from a combination of fixed and live imaging together with multiple lineage studies require careful and thorough explanations with accessible documentation. The result of our revisions is, I believe, analogous to keeping the qualities and achievements that earned Achilles’ reputation, while making the inevitable Achilles heel less obvious, though still present.

    Experiments testing the effects of altering specific signaling pathways are already underway. Some results certainly support the concept that cell locations and outcomes remain flexible, subject to external influences, through pupal development. However, such experiments, results and interpretations demand careful scrutiny and cannot practically be included in this already lengthy manuscript.

  3. eLife

    Evaluation Summary:

    This manuscript describes a very extensive set of experiments charting the origin and fate of various cell populations in the Drosophila ovary that is a powerful system to explore interactions between adult stem cells and their niches. The authors put forward a new view of how different cell types acquire their fates during development. This is a more nuanced view than extant models, involving common progenitors from which different cell fates (stem cell, progeny and niche cells) arise gradually and relying on spatiotemporal cues.

    (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.)

  4. eLife

    Reviewer #1 (Public Review):

    Stem cells in the Drosophila ovary provide a great model to understand cell behavior and regulation due to its genetic tractability, organized morphological pattern, and ease to perform live imaging. Compared to the adult ovary which have been studied quite extensively, the pupae ovary is a much less explored stage. Here the authors extensively studied the cell lineage in the pupae ovary, which helps understanding the development of early cell fates and the formation of the first set of egg chambers. They first described the different stages of pupal ovary development, followed by several different lineage tracing experiments that conclude a subset of Intermingled Cells (ICs) as Escort Cell (EC)/ Follicle Stem Cell (FSC) common precursors. Then they described Extra-Germarial Crown Cells (EGCs) and basal stalk cells and showed by live imaging that they contribute to the first budding cyst.

    Strength:

    Several lineage tracing experiments and statistical calculations are performed to conclude that a subset of ICs function as EC/FSC common precursors. The methods are written in great detail to help understand the calculation.

    The finding that EGCs and basal stalk cells contribute to the first budding cyst is new and intriguing. This initial developmental process, different from what happens in the adult ovary, might provide insight into how germline and somatic cells are coordinated.

    Weakness:

    The authors showed in their 2017 NCB paper that FSCs contribute to ECs in adult ovary. Here they showed that there is a common precursor of EC/FSC. Are these two cell types the same? It has been shown in single cell analysis during third instar that the ICs and FSCPs present Con and bond as their specific markers (Slaidina et al. 2020). Both give rise to EC/FSC/FC in lineage tracing experiments. Therefore, the novelty of this finding is weakened.

    While the finding that EGCs and basal stalks contribute to the first budding egg chamber is intriguing, the definition of EGCs and basal stalks are quite vague. Do EGCs have a distinct feature that is worth noting as separate cell types, or are they simply early FCs locating posterior of the first germline cyst? Do basal stalks express mature stalk markers, or are they simply accumulating FCs that are not fully differentiated yet? Is the process of EGCs and basal stalk contributing to the first budding egg chamber similar to posterior FCs contributing to the budding egg chamber? Is it because the budding of the first germline cyst takes longer than normal, there are more FCs accumulating at the posterior, thus making this region look like EGCs and basal stalks?

    The presentation is too lengthy. A more concisely written paper would help the audience to get the key points that the authors hope to convey.

  5. eLife

    Reviewer #2 (Public Review):

    Reilein, Kogan and co-workers chart the origin and fate of the different cell types in the Drosophila ovary throughout pupal stages using a combination of mosaic analysis, live imaging and immunohistochemistry. Their results challenge some of the assumed lineage and niche relationships between adult progenitors and support cells. The authors identify progenitors that can give rise to more cell types than previously thought (e.g. precursors that yield follicle stem cells and their adult niche and product cells) and revise some cell interactions/lineage relationships (for example, by providing evidence for separate precursors of follicle cells in the first-formed egg chamber, or by observing that germline progression can be supported by developing escort cells precursors rather than differentiated escort cells). Collectively, their data suggest a gradual and flexible adoption of these cell types according to the position of specific precursors during development.

    Although mosaic/clonal analyses do not always provide clear-cut answers, the authors are fully aware of possible caveats, and have done everything in their genetic power to ensure that their interpretations are as sound as they can possibly be. This includes, for example, extensive quantifications and statistical analyses/predictions based on clone frequency, the use of multicolour marking strategies to ensure that lineages are derived from single cells, and consideration of the effects of temperature shifts on developmental times. The resulting data are invariably comprehensive, have been documented and quantified extensively, and are often accompanied by stunning images.

    In a way, the comprehensive nature of this manuscript is also its Achilles heel: it is VERY long and dense. Any readers unfamiliar with the Drosophila ovary may not take the time to digest the data. This would be a pity as the manuscript's main messages are timely. They also resonate with observations in other systems such as the mouse gut field which, collectively, are beginning to challenge concepts such as hardwired "stemness" and "genetic programs", and to rediscover the importance of positional/mechanical cues in specifying cell fate.

    The power of description is somewhat underestimated in our post-genetic revolution era, and there is a lot to be learned by carefully observing and documenting "what does happen" - as opposed to exclusively relying on genetic loss/gain-of-function experiments. This manuscript is a good illustration of this. That said, the authors' observations make a number of predictions which could be genetically tested (e.g. through temporal ablation experiments to confirm flexibility/temporal requirements, or experiments targeting specific pathways to confirm their contribution as spatial organisers). These experiments would make their revised models much more compelling.

  6. Version published to 10.1101/2020.06.25.171447v2 on bioRxiv
  7. Version published to 10.1101/2020.06.25.171447v1 on bioRxiv