Bellymount-pulsed tracking: a novel approach for real-time in vivo imaging of Drosophila abdominal tissues

  1. Shruthi Balachandra
  2. Amanda A Amodeo

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Abstract

Quantitative live imaging is a valuable tool that offers insights into cellular dynamics. However, many fundamental biological processes are incompatible with current live-imaging modalities. Drosophila oogenesis is a well-studied system that has provided molecular insights into a range of cellular and developmental processes. The length of the oogenesis, coupled with the requirement for inputs from multiple tissues, has made long-term culture challenging. Here, we have developed Bellymount-pulsed tracking (Bellymount-PT), which allows continuous, noninvasive live imaging of Drosophila oogenesis inside the female abdomen for up to 16 h. Bellymount-PT improves upon the existing Bellymount technique by adding pulsed anesthesia with periods of feeding that support the long-term survival of flies during imaging. Using Bellymount-PT, we measure key events of oogenesis, including egg chamber growth, yolk uptake, and transfer of specific proteins to the oocyte during nurse cell dumping with high spatiotemporal precision within the abdomen of a live female.

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  1. Version published to 10.1093/g3journal/jkae271
  2. Review Commons

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    Reply to the reviewers

    The authors do not wish to provide a response at this time.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Balachandra and Amodeo presents Bellymount-Pulsed Tracking as a technique for continuous long-term imaging of Drosophila oogenesis. This approach modifies the existing Bellymount technique by exposing restrained female flies to pulses of CO2 anesthesia in combination with image acquisition. Flies that survived the restraint were kept alive for many hours by addition of a liquid diet in the restraint apparatus. This allowed for imaging and tracking of egg chamber development over longer time periods than capable with ex vivo culturing methods. However, the authors did report a 40% mortality rate and decreased fecundity compared to unrestrained flies. Using this method the authors were able to image and measure the growth rate of developing egg chambers in living flies, and capture events like vitellogenesis which relies on the interactions of multiple organ systems.

    This technique is a notable contribution to the fly community, as it could be useful for studying processes that require interactions between multiple tissues and organs, as well as for long-term imaging of other internal structures in the adult fly. The significance is somewhat reduced due to the relatively high mortality rate and the decreased fecundity and egg chamber growth rate reported. However, the authors should be commended for their diligence in documenting the limitations of the procedure, as this now provides a strong jumping off point to improve the technique if it becomes widely adopted by the fly community. Overall, the experiments appear to have been carefully performed and the manuscript is clearly written. However, there are several issues that should be addressed prior to publication.

    Major concerns

    1. The movies of egg chamber development are challenging to interpret. They could be improved by the addition of timestamps and other annotations. Having multiple example movies of the same process would also be valuable. It could be helpful to potential users of this technique to show the process the authors used for identifying the same egg chamber between such long time points.
    2. Figure 4 - Given that the Bellymount PT technique slows oogenesis and reduces egg chamber growth in vitellogenic stages (Figure 3E), it is possible that Bellymount PT slows yolk protein uptake. It would be important to establish a baseline for how much to expect yolk protein levels to change across stages to compare to measurements obtained with Bellymount PT. It would be a relatively simple experiment to show the change in yolk protein uptake across stages in fixed samples. This could also be performed for His2Av dynamics during nurse cell dumping.
    3. Movie 11 - The authors propose that Bellymount-PT can be used to visualize the process of border cell migration. However, there is no obvious movement of the cluster relative to the nurse cell nuclei over the course of the 3 hour long movie. The authors should either show a better movie of border cell migration, or remove this claim from the manuscript.
    4. Movie 13 - The authors claim that they see egg chamber rotation continue in stage 9 and 10 egg chambers. This movie is not convincing. There is also very strong evidence in the literature that egg chamber rotation ends at stage 8. Chen et al., Cell Reports, 2017 showed using a method that tracks follicle cell migration in vivo that rotational migration ends during stage 8. The only movement of follicle cells after stage 8 is due to the epithelial reorganization that occurs during the posterior movement of the follicle cells as the stretch cells flatten. Additionally, after stage 8 follicle cells lose their circumferentially oriented actin protrusions that drive rotation. This claim should be removed from the manuscript.

    Minor comments

    1. Line 104 - The authors mention that CO2 affects fertility in flies. They should also reference Sustar et al., Genetics, 2023 and Zimmerman and Berg, PLoS One, 2024 for wider ranging effects of CO2 on oogenesis.
    2. Line 244 - Although it is true that the original paper describing egg chamber rotation reported that it starts at 5, subsequent studies from multiple labs have confirmed that it begins much earlier. First shown by Cetera et al., Nature Communications, 2014 but later confirmed by Bilder, Dahmann, and Mirouse labs. Chen et al., Cell Reports, 2016 has even published a movie of an egg chamber initiating rotation as it buds from the germarium.
    3. Figures of egg chambers are generally oriented anterior on the left and posterior on the right. Reorienting all the figures would be challenging, so the recommendation is to be clear in the figure legends the orientation of the images. This is important given they are shown in different orientations in Figure 1 than throughout the rest of the paper, and also will be helpful for readers who may not be familiar with the structure of the ovary/egg chambers.
    4. Figure 1B and Methods line 334 - Should "Rely" be "Relay"?
    5. Figure 1E - Oocyte nuclei are missing from the diagrams of stage 7, 13 and 14 egg chambers. Also, "G" looks like a figure panel label, could just say Germarium
    6. Figure 3F-H - "Stagee" should be "Stage"
    7. Figure 4B - Why is the fluorescence for egg chamber #6 so much higher than the others? It makes the slopes of the other samples hard to see.
    8. Figure 4D,E,G - For clarity, the labeled boxes should be the same color as the lines on the associated graphs. In line 790 "Note the steady increase of H2Av in all three regions as it exits the nurse cell nuclei" - this is not actually shown without the nurse cell nuclei average intensity being on the graph as well.
    9. Line 787 - "Note the flow of H2Av" - "flow" is not actually shown in these static images. Consider a more precise description.

    Referee Cross-commenting

    The other reviewers make several excellent points. We personally feel that it is beyond the scope of this initial report to ask the authors to show that they can see all aspects of oogenesis with this technique. If the method becomes widely adopted by the oogenesis community, individual researchers can optimize it to suit the exact process they want to study. If the authors want to claim they can see a particular process, it needs to be well documented and convincing. For example, we agree that the movies that claim to show egg chamber rotation (both during established stages and later) and border cell migration need to be improved or the claims need to be removed. However, we feel that the authors have documented enough other interesting processes to make the study worthy of publication. Likewise, asking the authors to determine the minimal time window that can be used for imaging could take months of open-ended work and is something that could be better tackled by subsequent users depending on the requirements of the biological process they want to study. It seems better to get the work out into the public sooner rather than later so that improvements can be crowd sourced.

    Finally, although Flp-out clones were used for cell tracking in the original Belly mount paper, this technique will be less effective during the first half of oogenesis when the egg chamber is rotating, as the clone is likely to rotate into and out of sight between imaging time points.

    Significance

    This technique is a notable contribution to the fly community, as it could be useful for studying processes that require interactions between multiple tissues and organs, as well as for long-term imaging of other internal structures in the adult fly. The significance is somewhat reduced due to the relatively high mortality rate and the decreased fecundity and egg chamber growth rate reported. However, the authors should be commended for their diligence in documenting the limitations of the procedure, as this now provides a strong jumping off point to improve the technique if it becomes widely adopted by the fly community. Overall, the experiments appear to have been carefully performed and the manuscript is clearly written. However, there are several issues that should be addressed prior to publication.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors describe an improvement of the Bellymount imaging method for internal tissues of the fly's abdomen. They are able to increase the total duration of the imaging by introducing pulsed anesthesia. This allows the immobilized flies to take up food in between the imaging; this increases survival rate and allows for longer total imaging times. The authors illustrate the technique by tracking the development of egg chambers.

    Major Points

    • The Bellymount PT method results in decreased fecundity, which might affect the processes (oogenesis) the authors looked at. Indeed, the authors conclude that "oogenesis is not completely stalled under the Bellymount-PT protocol" (line 140). The authors do provide some data indicating that egg chambers develop (Fig. 2G,H; Fig. 3F,H), in particular a stage 10 egg chamber proceeding to a stage where dorsal appendages seem to form. However, for early stage egg chambers this is less convincing. The egg chambers show an increase in (cross-sectional) area, however, what is the evidence that they also mature? For example, during egg chamber maturation, the ratio of oocyte/nurse cell volume changes, follicle cells re-arrange, etc. The authors should test whether any of these characteristics can be observed in egg chambers imaged using Bellymount PT. This may include the imaging of egg chambers in which both nuclei and plasma membranes are visualized.
    • A potential advantage of the Bellymount PT method is the ability to follow the dynamics of processes. A current drawback, however, is the rather low temporal resolution as the fly needs to wake up between single images. The authors should provide an estimate for the minimal possible cycle time and should test whether flies imaged at 10 minutes interval show lower survival/fecundity than flies imaged at 2 hours interval.
    • The authors claim that they can track on a cellular level (based on nuclei), but it is unclear how accurate the tracking is. Especially cell tracking over very long times might be challenging here, as the time delay between two time points is big. The authors should test the accuracy of their tracking, potentially by creating Flip-out clones and using them as a control.
    • The authors show that they can visualize cell membranes (Moesin-GFP, Fig. 2C). Tracking cells over time based on their membranes would greatly widen the applicability of the method as it would enable to analyze the complex cellular dynamics during egg chamber maturation. The authors should test whether cells can be tracked over time (e.g. using Moesin-GFP) using their technique.
    • Movie 11. The authors claim that they can capture border cell migration. However, it is unclear whether the border cells actually migrate towards posterior. The authors should track and quantitatively analyze the migration path of the border cells in their movies.
    • Movie 12. The authors claim that they can observe egg chamber rotation. However, it is unclear whether the egg chambers actually rotate. The authors should track cells and quantify the angular velocity of movement.

    Minor Points

    • Please move the labels of the scale bars to the legends.
    • The figures (especially 2 and 3) would benefit from a clearer structuring. Moving part of them to supplementary figures would also help.
    • "stage" typo in figure 3

    Significance

    The authors describe here an improvement of an existing technique. The advantage of the improved technique is the longer imaging time, which potentially allows users to track cells/organelles/proteins over time. However, tracking requires the user to connect single time points with each other, which is somewhat unclear at this time. Moreover, the potential applicability (and significance) of the technique would be widened if visualization and tracking of cell membranes/organelles/vesicles would be possible. With these further optimizations, the technique would add a useful tool to the Drosophila community.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The Drosophila ovary is an established model system for many aspects of development and cell biology. In vitro culture of live ovaries has provided valuable insight, yet these methods do not accurately mimic oogenesis in vivo for some stages. Here the authors develop a new method that allows for sustained imaging of ovaries in intact flies, maintaining normal physiology.

    The method provides a valuable addition to the field. Processes such as growth, cell migration, egg chamber rotation, yolk uptake and nurse cell dumping can be observed in the intact fly. Time lapse and 3D reconstruction provide valuable tools. While the detail/resolution of the images is not as good as ex vivo or fixed samples, the ability to maintain normal development and homeostasis provides a novel advantage. The figures and movies are well-presented and sufficient detail is provided in the methods.

    Major comments

    1. Why do the authors think that growth is slowed? The imaging process or the trapping/anesthesia of the fly? For example, if the frequency of imaging was varied, it could reveal whether it was the actual imaging that affected development. Did the length of time the fly had been in the trap make a difference? The sentence on lines 190-191 is not clear.
    2. In Movie 6, the nurse cell nuclear shape does not look normal - more ovoid than round. Perhaps some settings are off in the 3D reconstruction.
    3. Movie 11 - why do the border cells seem stalled?
    4. There is no discussion of the earliest stages of oogenesis. Is it possible to see egg chambers forming from the germarium?

    Minor comments

    1. It would be helpful to mention if the egg chambers stay in similar locations or move around - is it challenging to locate the same egg chamber after 2 hours?
    2. Are any egg chambers degenerating? This could indicate stress in the fly.
    3. In Figure 4D, release of HisAV into the cytoplasm is described. Similar release of nuclear proteins was described by Cooley et al. 1992 so this paper could be cited.
    4. At 321 minutes in Figure 4D, a large nucleus is apparent in the oocyte. Is this an oocyte nucleus or evidence for nurse cell translocation to the oocyte as described in Ali-Murthy et al. 2021?

    Significance

    The technique provides a significant advance to the field, extending the time period currently possible to image ovaries through the Belly Mount method. It will immediately benefit researchers working on the ovary but could be extended to many other tissues in the fly abdomen such as the gut and tumor models.

  6. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time.

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Balachandra and Amodeo presents Bellymount-Pulsed Tracking as a technique for continuous long-term imaging of Drosophila oogenesis. This approach modifies the existing Bellymount technique by exposing restrained female flies to pulses of CO2 anesthesia in combination with image acquisition. Flies that survived the restraint were kept alive for many hours by addition of a liquid diet in the restraint apparatus. This allowed for imaging and tracking of egg chamber development over longer time periods than capable with ex vivo culturing methods. However, the authors did report a 40% mortality rate and decreased fecundity compared to unrestrained flies. Using this method the authors were able to image and measure the growth rate of developing egg chambers in living flies, and capture events like vitellogenesis which relies on the interactions of multiple organ systems.

    This technique is a notable contribution to the fly community, as it could be useful for studying processes that require interactions between multiple tissues and organs, as well as for long-term imaging of other internal structures in the adult fly. The significance is somewhat reduced due to the relatively high mortality rate and the decreased fecundity and egg chamber growth rate reported. However, the authors should be commended for their diligence in documenting the limitations of the procedure, as this now provides a strong jumping off point to improve the technique if it becomes widely adopted by the fly community. Overall, the experiments appear to have been carefully performed and the manuscript is clearly written. However, there are several issues that should be addressed prior to publication.

    Major concerns

    1. The movies of egg chamber development are challenging to interpret. They could be improved by the addition of timestamps and other annotations. Having multiple example movies of the same process would also be valuable. It could be helpful to potential users of this technique to show the process the authors used for identifying the same egg chamber between such long time points.
    2. Figure 4 - Given that the Bellymount PT technique slows oogenesis and reduces egg chamber growth in vitellogenic stages (Figure 3E), it is possible that Bellymount PT slows yolk protein uptake. It would be important to establish a baseline for how much to expect yolk protein levels to change across stages to compare to measurements obtained with Bellymount PT. It would be a relatively simple experiment to show the change in yolk protein uptake across stages in fixed samples. This could also be performed for His2Av dynamics during nurse cell dumping.
    3. Movie 11 - The authors propose that Bellymount-PT can be used to visualize the process of border cell migration. However, there is no obvious movement of the cluster relative to the nurse cell nuclei over the course of the 3 hour long movie. The authors should either show a better movie of border cell migration, or remove this claim from the manuscript.
    4. Movie 13 - The authors claim that they see egg chamber rotation continue in stage 9 and 10 egg chambers. This movie is not convincing. There is also very strong evidence in the literature that egg chamber rotation ends at stage 8. Chen et al., Cell Reports, 2017 showed using a method that tracks follicle cell migration in vivo that rotational migration ends during stage 8. The only movement of follicle cells after stage 8 is due to the epithelial reorganization that occurs during the posterior movement of the follicle cells as the stretch cells flatten. Additionally, after stage 8 follicle cells lose their circumferentially oriented actin protrusions that drive rotation. This claim should be removed from the manuscript.

    Minor comments

    1. Line 104 - The authors mention that CO2 affects fertility in flies. They should also reference Sustar et al., Genetics, 2023 and Zimmerman and Berg, PLoS One, 2024 for wider ranging effects of CO2 on oogenesis.
    2. Line 244 - Although it is true that the original paper describing egg chamber rotation reported that it starts at 5, subsequent studies from multiple labs have confirmed that it begins much earlier. First shown by Cetera et al., Nature Communications, 2014 but later confirmed by Bilder, Dahmann, and Mirouse labs. Chen et al., Cell Reports, 2016 has even published a movie of an egg chamber initiating rotation as it buds from the germarium.
    3. Figures of egg chambers are generally oriented anterior on the left and posterior on the right. Reorienting all the figures would be challenging, so the recommendation is to be clear in the figure legends the orientation of the images. This is important given they are shown in different orientations in Figure 1 than throughout the rest of the paper, and also will be helpful for readers who may not be familiar with the structure of the ovary/egg chambers.
    4. Figure 1B and Methods line 334 - Should "Rely" be "Relay"?
    5. Figure 1E - Oocyte nuclei are missing from the diagrams of stage 7, 13 and 14 egg chambers. Also, "G" looks like a figure panel label, could just say Germarium
    6. Figure 3F-H - "Stagee" should be "Stage"
    7. Figure 4B - Why is the fluorescence for egg chamber #6 so much higher than the others? It makes the slopes of the other samples hard to see.
    8. Figure 4D,E,G - For clarity, the labeled boxes should be the same color as the lines on the associated graphs. In line 790 "Note the steady increase of H2Av in all three regions as it exits the nurse cell nuclei" - this is not actually shown without the nurse cell nuclei average intensity being on the graph as well.
    9. Line 787 - "Note the flow of H2Av" - "flow" is not actually shown in these static images. Consider a more precise description.

    Referee Cross-commenting

    The other reviewers make several excellent points. We personally feel that it is beyond the scope of this initial report to ask the authors to show that they can see all aspects of oogenesis with this technique. If the method becomes widely adopted by the oogenesis community, individual researchers can optimize it to suit the exact process they want to study. If the authors want to claim they can see a particular process, it needs to be well documented and convincing. For example, we agree that the movies that claim to show egg chamber rotation (both during established stages and later) and border cell migration need to be improved or the claims need to be removed. However, we feel that the authors have documented enough other interesting processes to make the study worthy of publication. Likewise, asking the authors to determine the minimal time window that can be used for imaging could take months of open-ended work and is something that could be better tackled by subsequent users depending on the requirements of the biological process they want to study. It seems better to get the work out into the public sooner rather than later so that improvements can be crowd sourced.

    Finally, although Flp-out clones were used for cell tracking in the original Belly mount paper, this technique will be less effective during the first half of oogenesis when the egg chamber is rotating, as the clone is likely to rotate into and out of sight between imaging time points.

    Significance

    This technique is a notable contribution to the fly community, as it could be useful for studying processes that require interactions between multiple tissues and organs, as well as for long-term imaging of other internal structures in the adult fly. The significance is somewhat reduced due to the relatively high mortality rate and the decreased fecundity and egg chamber growth rate reported. However, the authors should be commended for their diligence in documenting the limitations of the procedure, as this now provides a strong jumping off point to improve the technique if it becomes widely adopted by the fly community. Overall, the experiments appear to have been carefully performed and the manuscript is clearly written. However, there are several issues that should be addressed prior to publication.

  8. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The authors describe an improvement of the Bellymount imaging method for internal tissues of the fly's abdomen. They are able to increase the total duration of the imaging by introducing pulsed anesthesia. This allows the immobilized flies to take up food in between the imaging; this increases survival rate and allows for longer total imaging times. The authors illustrate the technique by tracking the development of egg chambers.

    Major Points

    • The Bellymount PT method results in decreased fecundity, which might affect the processes (oogenesis) the authors looked at. Indeed, the authors conclude that "oogenesis is not completely stalled under the Bellymount-PT protocol" (line 140). The authors do provide some data indicating that egg chambers develop (Fig. 2G,H; Fig. 3F,H), in particular a stage 10 egg chamber proceeding to a stage where dorsal appendages seem to form. However, for early stage egg chambers this is less convincing. The egg chambers show an increase in (cross-sectional) area, however, what is the evidence that they also mature? For example, during egg chamber maturation, the ratio of oocyte/nurse cell volume changes, follicle cells re-arrange, etc. The authors should test whether any of these characteristics can be observed in egg chambers imaged using Bellymount PT. This may include the imaging of egg chambers in which both nuclei and plasma membranes are visualized.
    • A potential advantage of the Bellymount PT method is the ability to follow the dynamics of processes. A current drawback, however, is the rather low temporal resolution as the fly needs to wake up between single images. The authors should provide an estimate for the minimal possible cycle time and should test whether flies imaged at 10 minutes interval show lower survival/fecundity than flies imaged at 2 hours interval.
    • The authors claim that they can track on a cellular level (based on nuclei), but it is unclear how accurate the tracking is. Especially cell tracking over very long times might be challenging here, as the time delay between two time points is big. The authors should test the accuracy of their tracking, potentially by creating Flip-out clones and using them as a control.
    • The authors show that they can visualize cell membranes (Moesin-GFP, Fig. 2C). Tracking cells over time based on their membranes would greatly widen the applicability of the method as it would enable to analyze the complex cellular dynamics during egg chamber maturation. The authors should test whether cells can be tracked over time (e.g. using Moesin-GFP) using their technique.
    • Movie 11. The authors claim that they can capture border cell migration. However, it is unclear whether the border cells actually migrate towards posterior. The authors should track and quantitatively analyze the migration path of the border cells in their movies.
    • Movie 12. The authors claim that they can observe egg chamber rotation. However, it is unclear whether the egg chambers actually rotate. The authors should track cells and quantify the angular velocity of movement.

    Minor Points

    • Please move the labels of the scale bars to the legends.
    • The figures (especially 2 and 3) would benefit from a clearer structuring. Moving part of them to supplementary figures would also help.
    • "stage" typo in figure 3

    Significance

    The authors describe here an improvement of an existing technique. The advantage of the improved technique is the longer imaging time, which potentially allows users to track cells/organelles/proteins over time. However, tracking requires the user to connect single time points with each other, which is somewhat unclear at this time. Moreover, the potential applicability (and significance) of the technique would be widened if visualization and tracking of cell membranes/organelles/vesicles would be possible. With these further optimizations, the technique would add a useful tool to the Drosophila community.

  9. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The Drosophila ovary is an established model system for many aspects of development and cell biology. In vitro culture of live ovaries has provided valuable insight, yet these methods do not accurately mimic oogenesis in vivo for some stages. Here the authors develop a new method that allows for sustained imaging of ovaries in intact flies, maintaining normal physiology.

    The method provides a valuable addition to the field. Processes such as growth, cell migration, egg chamber rotation, yolk uptake and nurse cell dumping can be observed in the intact fly. Time lapse and 3D reconstruction provide valuable tools. While the detail/resolution of the images is not as good as ex vivo or fixed samples, the ability to maintain normal development and homeostasis provides a novel advantage. The figures and movies are well-presented and sufficient detail is provided in the methods.

    Major comments

    1. Why do the authors think that growth is slowed? The imaging process or the trapping/anesthesia of the fly? For example, if the frequency of imaging was varied, it could reveal whether it was the actual imaging that affected development. Did the length of time the fly had been in the trap make a difference? The sentence on lines 190-191 is not clear.
    2. In Movie 6, the nurse cell nuclear shape does not look normal - more ovoid than round. Perhaps some settings are off in the 3D reconstruction.
    3. Movie 11 - why do the border cells seem stalled?
    4. There is no discussion of the earliest stages of oogenesis. Is it possible to see egg chambers forming from the germarium?

    Minor comments

    1. It would be helpful to mention if the egg chambers stay in similar locations or move around - is it challenging to locate the same egg chamber after 2 hours?
    2. Are any egg chambers degenerating? This could indicate stress in the fly.
    3. In Figure 4D, release of HisAV into the cytoplasm is described. Similar release of nuclear proteins was described by Cooley et al. 1992 so this paper could be cited.
    4. At 321 minutes in Figure 4D, a large nucleus is apparent in the oocyte. Is this an oocyte nucleus or evidence for nurse cell translocation to the oocyte as described in Ali-Murthy et al. 2021?

    Significance

    The technique provides a significant advance to the field, extending the time period currently possible to image ovaries through the Belly Mount method. It will immediately benefit researchers working on the ovary but could be extended to many other tissues in the fly abdomen such as the gut and tumor models.

  10. Version published to 10.1101/2024.03.31.587498 on bioRxiv