An improved organ explant culture method reveals stem cell lineage dynamics in the adult Drosophila intestine
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Evaluation Summary:
Marchetti and colleagues present a promising, ex vivo culture method for the Drosophila adult midgut and other abdominal organs. There are numerous advantages of the authors' method that will attract broad interest and enable real-time analysis of new and important scientific questions. There are concerns about the authors' interpretations of asymmetric/symmetric fate outcomes and terminal differentiation, more information about midgut viability is needed, and comparison of ex vivo vs in vivo regeneration would be useful.
(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. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
In recent years, live-imaging techniques have been developed for the adult midgut of Drosophila melanogaster that allow temporal characterization of key processes involved in stem cell and tissue homeostasis. However, these organ culture techniques have been limited to imaging sessions of < 16 hours, an interval too short to track dynamic processes such as damage responses and regeneration, which can unfold over several days. Therefore, we developed an organ explant culture protocol capable of sustaining midguts ex vivo for up to 3 days. This was made possible by the formulation of a culture medium specifically designed for adult Drosophila tissues with an increased Na + /K + ratio and trehalose concentration, and by placing midguts at an air-liquid interface for enhanced oxygenation. We show that midgut progenitor cells can respond to gut epithelial damage ex vivo, proliferating and differentiating to replace lost cells, but are quiescent in healthy intestines. Using ex vivo gene induction to promote stem cell proliferation using Ras G12V or string and Cyclin E overexpression, we demonstrate that progenitor cell lineages can be traced through multiple cell divisions using live imaging. We show that the same culture set-up is useful for imaging adult renal tubules and ovaries for up to 3 days and hearts for up to 10 days. By enabling both long-term imaging and real-time ex vivo gene manipulation, our simple culture protocol provides a powerful tool for studies of epithelial biology and cell lineage behavior.
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Author Response
Reviewer #1 (Public Review):
Marchetti and colleagues present an ex vivo culture method that enables live imaging studies of Drosophila adult midguts for periods up to 3 days. Important technical innovations include defining an optimized tissue culture media, placing midguts at an air-liquid interface for better oxygenation of the tissue, performing inducible gene expression while imaging, and performing multiplexed imaging of up to 12 midguts in a single culture. Using this ex vivo method, the authors show that midgut progenitor cells proliferate and differentiate in response to epithelial damage ex vivo. The authors exogenously activate the cell cycle, which enables them to trace and analyze the division behaviors of multiple generations of stem cell progeny, including the distances between sibling cell nuclei over …
Author Response
Reviewer #1 (Public Review):
Marchetti and colleagues present an ex vivo culture method that enables live imaging studies of Drosophila adult midguts for periods up to 3 days. Important technical innovations include defining an optimized tissue culture media, placing midguts at an air-liquid interface for better oxygenation of the tissue, performing inducible gene expression while imaging, and performing multiplexed imaging of up to 12 midguts in a single culture. Using this ex vivo method, the authors show that midgut progenitor cells proliferate and differentiate in response to epithelial damage ex vivo. The authors exogenously activate the cell cycle, which enables them to trace and analyze the division behaviors of multiple generations of stem cell progeny, including the distances between sibling cell nuclei over time. Finally, proof-of-principle imaging of adult renal tubules and egg chambers is provided.
Altogether, this ex vivo method offers important new advantages that will attract broad interest in the Drosophila community. My main concerns are (1) midgut viability when imaging is performed with a confocal microscope and (2) uncertainty about the authors' classification of asymmetric and symmetric fate outcomes.
Major comments:
- The ability to perform continuous imaging over periods as long as 72 hours is a significant achievement. As the authors point out, this time scale is several fold longer than the 16 hour viability window of in vivo imaging (Martin 2018). The multiday timescale is important because it could, in principle, enable live imaging of many fundamental, but slow, cellular behaviors such as enterocyte differentiation.
If I understand correctly, the multi-day experiments in the study were captured using imaging conditions that were particularly gentle: (1) widefield epifluorescence, (2) an interval of 1 hour between time points, and (3) at most two fluorescent channels. By comparison, the 16 hour viability window defined in Martin 2018 was based on confocal imaging at 7-15 minute intervals in 3-4 channels. Can the authors provide information on midgut viability when their culture method is combined with confocal microscopy at minutes-long time intervals? This information is important to help users assess the types of questions that ex vivo culture can address given the widespread use of confocal imaging and the necessity of subcellular spatial or better temporal resolution for certain types of questions.
Viability during prolonged imaging is greatly dependent on the conditions used. If acquisition parameters are carefully set, intestines can be imaged using a confocal microscope with 15 minute intervals for a 48h interval. A movie has been added to demonstrate this (Video 23), and it has been discussed on lines 951-953. We also tested our live-imaging protocol on a Zeiss Lattice Lightsheet 7 microscope and observed no visible signs of phototoxicity after a 48 hours session with two channels (nlsGFP and His2Av.mRFP) 60µm z-stacks captured at 15 minute intervals. In regards to phototoxicity, it is hard to compare the different microscopes we tested quantitatively. As phototoxicity depends on the amount of light shone on a sample, we would need to carefully quantify the amounts of light delivered to the samples to determine the optimal frame rates and durations of different imaging methods, and have haven’t done this. Of course, since widefield, confocal, and lightsheet microscopes image samples in very different ways, comparing imaging quality is also a challenge. Therefore, all we can say with confindence is that, in our experience, if imaging parameters are set to minimize light exposure, all three different microscope types are viable for long term live-imaging. We have added some comments about microscopes to the Results section, lines 139-145, which we think will be helpful to users.
- The real-time tracing of multiple stem cell lineages through up to three generations is an impressive first for the midgut. The lineage trees are fascinating to examine. However I am unsure that division fate outcomes can be classified as symmetric or asymmetric using the data that are shown.
2a) Some divisions (9 of 25) were classified as asymmetric because exactly one sibling cell divided at a time point >6 hours before the end of the movie (lines 271-277). In my view, these fate outcomes are ambiguous because it cannot be excluded that the other sibling is a stem cell that would have divided after the movie ended. Although 6/7 sibling pairs that the authors observed exhibited temporally correlated divisions, failure to observe temporally correlated divisions is not a basis for concluding that sibling fates are asymmetric.
2b) Other divisions (8 of 25) were classified as asymmetric based on both the criteria in 2a and the observation that the non-dividing sibling showed increased nuclear size and decreased GFP intensity (lines 277-279). I agree with these criteria, but to my eye, the images in Fig 6 and Video 16 do not clearly show these changes. The nuclear size of the non-dividing sibling in panel G is not significantly different from the (presumably 4N) nuclei of the symmetrically dividing siblings in panel E. The GFP signal of the non-dividing sibling diminishes at the end of the movie, but without the His2Av::mRFP channel, I cannot tell whether the cell has lost GFP or, alternatively, has disappeared from view.
2c) The midguts lack markers to distinguish enteroblasts, enteroendocrine precursor cells, and stem cells. Without these, several types of fate outcomes are indiscernible: Asymmetric divisions that produce a stem cell and an enteroendocrine precursor (which remains diploid and can divide again), symmetric divisions (of enteroendocrine precursors) that produce two enteroendocrine cells (c.f. Chen 2018), and symmetric divisions (of stem cells) that produce two enteroblasts (c.f. de Navascues 2010, Guisoni 2017). Additionally, Kolhmaier 2015 has shown that stg/cycE manipulation results in divisions of SuH+ enteroblasts; these enteroblast divisions cannot be distinguished from stem cell divisions in the movies.
Can the authors either provide additional data that resolves the ambiguity of these fate classifications, or, alternatively, revise the text to describe these data in terms of division timing, displacement, and the other cell behaviors that are observed? In the latter case, speculation about fate outcomes could be added to the discussion.
As we were unable to collect additional data that could distinguish cell fates in our live lineage analyses, we have revised the text to describe division events based on the daughter cells’ actual behavior rather than their presumed cell identities and fates. We now refer to sister cells in lineages as either “co-dividing” and “non-co-dividing”, and define these terms (lines 277-283 and 301-307).
Reviewer #2 (Public Review):
This work provides a new protocol for extended culture of Drosophila midguts ex vivo and live-imaging for up to three days. This paper reveals a significant improvement of explanted intestines survival compared to previous protocols by optimizing the dissection procedure; by modifying the culture medium so that it approximates the adult hemolymph and by fine-tuning the live-imaging setup. In addition, this new protocol allows temperature-sensitive gene expression or knock-down ex vivo. By successfully performing intestinal stem cell lineage tracing experiments and cell tracking over time, authors demonstrate the potential and the robustness of this system in understanding key intestinal processes such as stem cell proliferation and cell differentiation over time. Interestingly, preliminary results demonstrate the possible use of this protocol for extended culture of other organs and its implication in other areas of research.
The relevance of the new protocol proposed by the authors in the improvement of the extended culture and live-imaging of intestines is well supported by the data. However, additional key control experiments would be needed to increase the confidence in using this protocol for the study and understanding of key intestinal processes.
- Authors tested the viability of explanted intestines over time by assessing different aspects such as the cell death or by testing the ability of cells to proliferate and differentiate. Adding control experiments to assess the state of the trachea and the visceral muscles, two major components of intestinal processes, would be needed.
We have now added considerations about trachea (lines 96 and 112) and visceral muscle (lines 984-996) to the main text.
- Authors tested GFP expression at permissive temperature in explanted intestines using intestinal stem cells or enterocytes GAL4 driver and detected no differences compared to in vivo condition.
Did the authors quantify the number of intestinal stem cells at different time points in explanted intestines? Did they see a difference compared to in vivo conditions?
Same questions for other cell types?
This analysis could be a good control to further validate the use of this system for the study and understanding of key intestinal processes.
Since in undamaged intestines we did not observe cell death, the number of cells of any type remains constant in our cultured intestines. Therefore, cell composition is the same before (i.e. in vivo) and after (i.e. ex vivo) dissection. When using the esgTS>UAS-GFP line, we did not observe any difference in GFP+ cell numbers between intestines shifted to the permissive temperature in vivo or ex vivo.
- Authors tested the ability of explanted intestines to regenerate following intestinal damage induced by SDS feeding. SDS feeding results in stem cell proliferation and progenitors differentiation in explanted intestines. Adding control experiments comparing stem cell proliferation and cell differentiation upon control feeding or upon SDS treatment in explanted intestines versus in in vivo conditions would reinforce the use of this system.
A new figure (Figure 3 – figure supplement 1) has been added to provide a SDS treatment in vivo comparison to our observation ex vivo. Results are discussed in the main text at lines 210-220.
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Evaluation Summary:
Marchetti and colleagues present a promising, ex vivo culture method for the Drosophila adult midgut and other abdominal organs. There are numerous advantages of the authors' method that will attract broad interest and enable real-time analysis of new and important scientific questions. There are concerns about the authors' interpretations of asymmetric/symmetric fate outcomes and terminal differentiation, more information about midgut viability is needed, and comparison of ex vivo vs in vivo regeneration would be useful.
(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. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Marchetti and colleagues present an ex vivo culture method that enables live imaging studies of Drosophila adult midguts for periods up to 3 days. Important technical innovations include defining an optimized tissue culture media, placing midguts at an air-liquid interface for better oxygenation of the tissue, performing inducible gene expression while imaging, and performing multiplexed imaging of up to 12 midguts in a single culture. Using this ex vivo method, the authors show that midgut progenitor cells proliferate and differentiate in response to epithelial damage ex vivo. The authors exogenously activate the cell cycle, which enables them to trace and analyze the division behaviors of multiple generations of stem cell progeny, including the distances between sibling cell nuclei over time. Finally, …
Reviewer #1 (Public Review):
Marchetti and colleagues present an ex vivo culture method that enables live imaging studies of Drosophila adult midguts for periods up to 3 days. Important technical innovations include defining an optimized tissue culture media, placing midguts at an air-liquid interface for better oxygenation of the tissue, performing inducible gene expression while imaging, and performing multiplexed imaging of up to 12 midguts in a single culture. Using this ex vivo method, the authors show that midgut progenitor cells proliferate and differentiate in response to epithelial damage ex vivo. The authors exogenously activate the cell cycle, which enables them to trace and analyze the division behaviors of multiple generations of stem cell progeny, including the distances between sibling cell nuclei over time. Finally, proof-of-principle imaging of adult renal tubules and egg chambers is provided.
Altogether, this ex vivo method offers important new advantages that will attract broad interest in the Drosophila community. My main concerns are (1) midgut viability when imaging is performed with a confocal microscope and (2) uncertainty about the authors' classification of asymmetric and symmetric fate outcomes.
Major comments:
1. The ability to perform continuous imaging over periods as long as 72 hours is a significant achievement. As the authors point out, this time scale is several fold longer than the 16 hour viability window of in vivo imaging (Martin 2018) . The multiday timescale is important because it could, in principle, enable live imaging of many fundamental, but slow, cellular behaviors such as enterocyte differentiation.
If I understand correctly, the multi-day experiments in the study were captured using imaging conditions that were particularly gentle: (1) widefield epifluorescence, (2) an interval of 1 hour between time points, and (3) at most two fluorescent channels. By comparison, the 16 hour viability window defined in Martin 2018 was based on confocal imaging at 7-15 minute intervals in 3-4 channels. Can the authors provide information on midgut viability when their culture method is combined with confocal microscopy at minutes-long time intervals? This information is important to help users assess the types of questions that ex vivo culture can address given the widespread use of confocal imaging and the necessity of subcellular spatial or better temporal resolution for certain types of questions.
2. The real-time tracing of multiple stem cell lineages through up to three generations is an impressive first for the midgut. The lineage trees are fascinating to examine. However I am unsure that division fate outcomes can be classified as symmetric or asymmetric using the data that are shown.
2a. Some divisions (9 of 25) were classified as asymmetric because exactly one sibling cell divided at a time point >6 hours before the end of the movie (lines 271-277). In my view, these fate outcomes are ambiguous because it cannot be excluded that the other sibling is a stem cell that would have divided after the movie ended. Although 6/7 sibling pairs that the authors observed exhibited temporally correlated divisions, failure to observe temporally correlated divisions is not a basis for concluding that sibling fates are asymmetric.
2b. Other divisions (8 of 25) were classified as asymmetric based on both the criteria in 2a and the observation that the non-dividing sibling showed increased nuclear size and decreased GFP intensity (lines 277-279). I agree with these criteria, but to my eye, the images in Fig 6 and Video 16 do not clearly show these changes. The nuclear size of the non-dividing sibling in panel G is not significantly different from the (presumably 4N) nuclei of the symmetrically dividing siblings in panel E. The GFP signal of the non-dividing sibling diminishes at the end of the movie, but without the His2Av::mRFP channel, I cannot tell whether the cell has lost GFP or, alternatively, has disappeared from view.
2c. The midguts lack markers to distinguish enteroblasts, enteroendocrine precursor cells, and stem cells. Without these, several types of fate outcomes are indiscernible: Asymmetric divisions that produce a stem cell and an enteroendocrine precursor (which remains diploid and can divide again), symmetric divisions (of enteroendocrine precursors) that produce two enteroendocrine cells (c.f. Chen 2018), and symmetric divisions (of stem cells) that produce two enteroblasts (c.f. de Navascues 2010, Guisoni 2017). Additionally, Kolhmaier 2015 has shown that stg/cycE manipulation results in divisions of SuH+ enteroblasts; these enteroblast divisions cannot be distinguished from stem cell divisions in the movies.
Can the authors either provide additional data that resolves the ambiguity of these fate classifications, or, alternatively, revise the text to describe these data in terms of division timing, displacement, and the other cell behaviors that are observed? In the latter case, speculation about fate outcomes could be added to the discussion.
Marchetti and colleagues present an ex vivo culture method for the Drosophila adult midgut. Numerous, important advantages of their method will attract broad interest in the Drosophila midgut community. In addition, proof-of-principle imaging of adult renal tubule, heart, and egg chambers is also provided. My main concerns are (1) midgut viability when imaging is performed with a confocal microscope and (2) uncertainty about the authors' classification of asymmetric and symmetric fate outcomes in cultured guts.
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Reviewer #2 (Public Review):
This work provides a new protocol for extended culture of Drosophila midguts ex vivo and live-imaging for up to three days. This paper reveals a significant improvement of explanted intestines survival compared to previous protocols by optimizing the dissection procedure; by modifying the culture medium so that it approximates the adult hemolymph and by fine-tuning the live-imaging setup. In addition, this new protocol allows temperature-sensitive gene expression or knock-down ex vivo. By successfully performing intestinal stem cell lineage tracing experiments and cell tracking over time, authors demonstrate the potential and the robustness of this system in understanding key intestinal processes such as stem cell proliferation and cell differentiation over time. Interestingly, preliminary results …
Reviewer #2 (Public Review):
This work provides a new protocol for extended culture of Drosophila midguts ex vivo and live-imaging for up to three days. This paper reveals a significant improvement of explanted intestines survival compared to previous protocols by optimizing the dissection procedure; by modifying the culture medium so that it approximates the adult hemolymph and by fine-tuning the live-imaging setup. In addition, this new protocol allows temperature-sensitive gene expression or knock-down ex vivo. By successfully performing intestinal stem cell lineage tracing experiments and cell tracking over time, authors demonstrate the potential and the robustness of this system in understanding key intestinal processes such as stem cell proliferation and cell differentiation over time. Interestingly, preliminary results demonstrate the possible use of this protocol for extended culture of other organs and its implication in other areas of research.
The relevance of the new protocol proposed by the authors in the improvement of the extended culture and live-imaging of intestines is well supported by the data. However, additional key control experiments would be needed to increase the confidence in using this protocol for the study and understanding of key intestinal processes.
Authors tested the viability of explanted intestines over time by assessing different aspects such as the cell death or by testing the ability of cells to proliferate and differentiate. Adding control experiments to assess the state of the trachea and the visceral muscles, two major components of intestinal processes, would be needed.
Authors tested GFP expression at permissive temperature in explanted intestines using intestinal stem cells or enterocytes GAL4 driver and detected no differences compared to in vivo condition.
Did the authors quantify the number of intestinal stem cells at different time points in explanted intestines? Did they see a difference compared to in vivo conditions?
Same questions for other cell types?
This analysis could be a good control to further validate the use of this system for the study and understanding of key intestinal processes.Authors tested the ability of explanted intestines to regenerate following intestinal damage induced by SDS feeding. SDS feeding results in stem cell proliferation and progenitors differentiation in explanted intestines.
Adding control experiments comparing stem cell proliferation and cell differentiation upon control feeding or upon SDS treatment in explanted intestines versus in in vivo conditions would reinforce the use of this system.
-
Reviewer #3 (Public Review):
In this paper, Marchetti and colleagues present an improved protocol to sustain midguts for up to 3 days for live imaging. Using this system, they examined the homeostasis and regeneration in response to damage and genetic induction. In addition, they identified symmetric and asymmetric division through tracing with multiple cell divisions. At last, this improved protocol can be applied to ovary and renal tubules. Therefore, the overall idea is significant. But they examined symmetric and asymmetric division when RasG12V or stg and CycE is activated by esg-Gal4. Because esg-Gal4 is expressed in both ISCs and EBs and expression of stg and CycE in EBs can drive EBs to divide (Kohlmaier et al. Oncogene 2015), the analysis of different divisions (symmetric verse asymmetric) is not precise based on the cell …
Reviewer #3 (Public Review):
In this paper, Marchetti and colleagues present an improved protocol to sustain midguts for up to 3 days for live imaging. Using this system, they examined the homeostasis and regeneration in response to damage and genetic induction. In addition, they identified symmetric and asymmetric division through tracing with multiple cell divisions. At last, this improved protocol can be applied to ovary and renal tubules. Therefore, the overall idea is significant. But they examined symmetric and asymmetric division when RasG12V or stg and CycE is activated by esg-Gal4. Because esg-Gal4 is expressed in both ISCs and EBs and expression of stg and CycE in EBs can drive EBs to divide (Kohlmaier et al. Oncogene 2015), the analysis of different divisions (symmetric verse asymmetric) is not precise based on the cell division of their progenies.
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