Kinetics of blood cell differentiation during hematopoiesis revealed by quantitative long-term live imaging

Article activity feed

1. 

   Version published to 10.7554/elife.84085 on eLife

   Mar 31, 2023
2. 

   eLife

   Feb 15, 2023

   Author Response

   Reviewer #2 (Public Review):

   The authors sought to be able to examine what cellular mechanisms underlie increases in mature blood cell production upon immune challenge. To this end they devised a new in vitro organ culturing system for the lymph gland, the main hematopoietic organ of the fruit fly Drosophila melanogaster; the fly serves as an excellent model for studying fundamental questions in immunology, as it allows live imaging combined with genetic manipulation, and the molecular pathways and cellular functions of its innate immune system are highly conserved with vertebrates.

   The authors provide compelling evidence that the cultured lymph gland shows a similar time scale, dynamics, and capacity for cell division as was observed in vivo, and does not undergo undue oxidative stress in their optimized culture …

   Author Response

   Reviewer #2 (Public Review):

   The authors sought to be able to examine what cellular mechanisms underlie increases in mature blood cell production upon immune challenge. To this end they devised a new in vitro organ culturing system for the lymph gland, the main hematopoietic organ of the fruit fly Drosophila melanogaster; the fly serves as an excellent model for studying fundamental questions in immunology, as it allows live imaging combined with genetic manipulation, and the molecular pathways and cellular functions of its innate immune system are highly conserved with vertebrates.

   The authors provide compelling evidence that the cultured lymph gland shows a similar time scale, dynamics, and capacity for cell division as was observed in vivo, and does not undergo undue oxidative stress in their optimized culture conditions. This technique will prove extremely useful to the large community studying the fly lymph gland, and potentially vertebrate immunologists seeking to expand the models they utilize.

   In these cultured glands, the authors identify progenitors undergoing symmetric cell divisions and provide some evidence that is consistent with, but does not prove, that these two cells maintain their proliferative capacity. They detect equivalent levels in the two equally sized daughter cells of dome-Meso-GFP, a marker for JAK-STAT activity; however, this could be due to an equal inheritance of the protein from the mother, not an equivalent maintenance of a proliferative capacity.

   This is an interesting question. A close look at the our movie (Video 4) of the dome-Meso-GFP marker shows the following sequence of events: the marker is nuclear, the mother cell divides and the nuclear envelope breaks down, cell division is completed, the dome-Meso-GFP re-accumulates at the nucleus of the daughter cells. This sequence of events implies that JAK-STAT is still active in the daughter cells. But as the reviewer points out there is a possibility of inheritance of the signal from the mother. If one of the cells were to differentiate, we would expect two things to occur, a differentiation marker to turn on in one of the daughter cells, and likely a slow decrease in the signal level of dome-Meso-GFP in one of the cells over time. We failed to mention that we accounted for both of those possibilities in our experiments such as the one shown in Video 5. We did this by first, including the eater-dsRed in the genetic background (see Figure 2 figure legend) in which these experiments were undertaken, if differentiation took place dsRed level would go up, an occurrence which we did not observe. Second, long-term tracking of dome-Meso-GFP levels for extended periods of time after completion of cell division did not show divergence or significant decrease of protein levels in the two daughter cells (Figure 2 - figure supplement 2). In any case, to directly make readers aware of this important caveat raised by the reviewer concern we added to the Results section in line 225-230 an explanation mentioning the possibility of inheritance of the marker and why we did not think this was the case.

   The authors develop a technique to conduct tracking of progenitor cell size over time in the cultured lymph glands and identify a switch increase in growth after division, as well as two orientations of the divisions, with the main one occurring 90% of the time.

   They show that bacterial infection results in a significant decrease in the division of Blood progenitors and the elimination of the minor orientation of division, but no obvious change in the rate of division.

   By imaging two markers, Dome-GFP for the progenitor state and Eater dsRed for the differentiated one, they examine the trajectories by which differentiation occurs in the wild-type lymph gland. They describe two main categories of fate transitions. In one that they call linear, the blood cells express high levels of the differentiation marker along with the progenitor marker before turning off the progenitor marker. The dynamics of how these progenitor cells get to the state of expressing both the differentiation and progenitor marker at high levels is not described. In the other, which they call sigmoidal, cells express only high levels of the progenitor marker, and the differentiation marker increases after or as the progenitor marker decreases. The authors show that upon infection there is a large increase in the amount of the linear type of differentiation. But how this change in the type of differentiation upon infection explains the increased amount of differentiation is not clear.

   A potential explanation comes from an aspect of their data that the authors don't comment upon. In their live analysis of lymph glands at a distinct time point in the uninfected state (Fig 7M-N), 95% of the cells they analyze traversing the sigmoidal path are in the intermediate step. This would predict that the cells on this path spend a much longer time stuck in this intermediate state before traversing to the final differentiated one, or that only a small fraction of the cells that become sigmoidal intermediate cells progress onwards to full differentiation. But this does not match the trajectories observed in the real-time analysis for uninfected cultured lymph glands (Fig 7A'-D') marker. Perhaps their algorithm discarded traces from the live imaging in which the differentiation marker did not come up quickly and was thus not analyzed in the trajectories.

   If my interpretation of the single time point analysis is true, this would argue that the linear path is actually much faster/more fruitful than the sigmoidal one and this would explain why a higher level of total progenitor differentiation infection is the result of infection-inducing more differentiation by the linear path. Otherwise, I don't understand how their data explains that observation.

   We understand the reviewer concern here and would like to state categorically that we did not use an algorithm to “discard” traces. As the reviewer outlines there is a large concentration of cells in the Dome-Meso-GFP (low expressing), eater-dsRed (low expressing) state. This is an intermediate state for the sigmoid differentiation trajectory. The reviewer suggests two scenarios to explain this. The first scenario is that this is the slowest (and thus rate limiting) step in the sigmoid differentiation trajectory. But, also as the reviewer notes, our tracking of individual cell trajectories doesn't show that cells spend a lot of time in this state. This leaves the second scenario the reviewer outlines, that only a small fraction of the cells that are in the Dome-Meso-GFP (low expressing), eater-dsRed (low expressing) state go on to differentiate (at least in the larval stage). We favor this model, because it is consistent with our observations, mainly that manipulating the sigmoid pathway is not a good way to induce the production of mature blood cells following infection, compared to manipulating the linear pathway. As the reviewer correctly points out the linear pathway provides a powerful way to change the rate of production of mature blood cells, with a few hours of infection the number of cells that are found in the intermediate state for this trajectory (Dome-Meso-GFP (high expressing), eater-DSred (high expressing)) increases 5-6 times. We now mention this specifically in the Discussion in line 532-539.

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3. 

   eLife

   Feb 15, 2023

   eLife assessment

   This important study develops a new technical advancement in ex vivo live imaging of hematopoietic tissues to monitor blood cells in their native microenvironment. The new method for live imaging and tracking is compelling, and the strength and breadth of hematopoietic analysis are convincing. This work provides a very useful new system for immunologists and cell biologists, which will supply new perspectives on the system-level mechanisms of cell differentiation and innate immunity.

   Read the original source
4. 

   eLife

   Feb 15, 2023

   Reviewer #1 (Public Review):

   In this study, Tanentzapf and colleagues have developed a new live-imaging technique for the lymph gland hematopoiesis over 12 hours, which is enough to visualize changes in the cell state or cell division by tracking the same cell. With the new method, the authors successfully cultured the lymph gland for a long period without modifying cell viability or stress responses and detected a continuous cell cycle and division. Moreover, the authors showed that lymph gland progenitors divide when they reach a certain size and regrow upon division, supporting previous findings and providing a new concept in lymph gland biology. The authors moved on to resolve the spatial distribution of progenitor mitosis in 3D and found that progenitors divide in a polarized manner which contributes to the typical shape of lymph …

   Reviewer #1 (Public Review):

   In this study, Tanentzapf and colleagues have developed a new live-imaging technique for the lymph gland hematopoiesis over 12 hours, which is enough to visualize changes in the cell state or cell division by tracking the same cell. With the new method, the authors successfully cultured the lymph gland for a long period without modifying cell viability or stress responses and detected a continuous cell cycle and division. Moreover, the authors showed that lymph gland progenitors divide when they reach a certain size and regrow upon division, supporting previous findings and providing a new concept in lymph gland biology. The authors moved on to resolve the spatial distribution of progenitor mitosis in 3D and found that progenitors divide in a polarized manner which contributes to the typical shape of lymph glands. In addition to developing lymph glands, the authors observed the lymph gland following oral infection and found that progenitors divide less upon infection but significantly increase the number of differentiations at the MZ-CZ boundary. Furthermore, the authors found two different modes of differentiation in the lymph gland: sigmoid and linear, which are altered during infection.

   Studies in the lymph gland hematopoiesis have heavily relied on snapshots of the lymph gland phenotypes although stem-progenitor differentiation is a continuous process. In this regard, the method developed in this study is extremely valuable to the fly community and will help improve the ex vivo culture and analysis techniques of fly organs as well as the lymph gland. The authors rigorously took advantage of numerous measures to validate the new method, including cell death, oxidative stress response, cell viability, and cell cycle, and observed biologically significant phenomena of the correlation between cell size and cell division, cell division polarity, and changes in the mode of differentiation during development or infection. This study provides a useful system for Drosophila immunologists and developmental biologists and will help explore the real-time mechanisms underlying blood development and immune reactions.

   Read the original source
5. 

   eLife

   Feb 15, 2023

   Reviewer #2 (Public Review):

   The authors sought to be able to examine what cellular mechanisms underlie increases in mature blood cell production upon immune challenge. To this end they devised a new in vitro organ culturing system for the lymph gland, the main hematopoetic organ of the fruit fly Drosophila melanogaster; the fly serves as an excellent model for studying fundamental questions in immunology, as it allows live imaging combined with genetic manipulation, and the molecular pathways and cellular functions of its innate immune system are highly conserved with vertebrates.

   The authors provide compelling evidence that the cultured lymph gland shows a similar time scale, dynamics, and capacity for cell division as was observed in vivo, and does not undergo undue oxidative stress in their optimized culture conditions. This …

   Reviewer #2 (Public Review):

   The authors sought to be able to examine what cellular mechanisms underlie increases in mature blood cell production upon immune challenge. To this end they devised a new in vitro organ culturing system for the lymph gland, the main hematopoetic organ of the fruit fly Drosophila melanogaster; the fly serves as an excellent model for studying fundamental questions in immunology, as it allows live imaging combined with genetic manipulation, and the molecular pathways and cellular functions of its innate immune system are highly conserved with vertebrates.

   The authors provide compelling evidence that the cultured lymph gland shows a similar time scale, dynamics, and capacity for cell division as was observed in vivo, and does not undergo undue oxidative stress in their optimized culture conditions. This technique will prove extremely useful to the large community studying the fly lymph gland, and potentially vertebrate immunologists seeking to expand the models they utilize.

   In these cultured glands, the authors identify progenitors undergoing symmetric cell divisions and provide some evidence that is consistent with, but does not prove, that these two cells maintain their proliferative capacity. They detect equivalent levels in the two equally sized daughter cells of dome-Meso-GFP, a marker for JAK-STAT activity; however, this could be due to an equal inheritance of the protein from the mother, not an equivalent maintenance of a proliferative capacity.

   The authors develop a technique to conduct tracking of progenitor cell size over time in the cultured lymph glands and identify a switch increase in growth after division, as well as two orientations of the divisions, with the main one occurring 90% of the time.

   They show that bacterial infection results in a significant decrease in the division of Blood progenitors and the elimination of the minor orientation of division, but no obvious change in the rate of division.

   By imaging two markers, Dome-GFP for the progenitor state and Eater dsRed for the differentiated one, they examine the trajectories by which differentiation occurs in the wild-type lymph gland. They describe two main categories of fate transitions. In one that they call linear, the blood cells express high levels of the differentiation marker along with the progenitor marker before turning off the progenitor marker. The dynamics of how these progenitor cells get to the state of expressing both the differentiation and progenitor marker at high levels is not described. In the other, which they call sigmoidal, cells express only high levels of the progenitor marker, and the differentiation marker increases after or as the progenitor marker decreases. The authors show that upon infection there is a large increase in the amount of the linear type of differentiation.
   But how this change in the type of differentiation upon infection explains the increased amount of differentiation is not clear.

   A potential explanation comes from an aspect of their data that the authors don't comment upon. In their live analysis of lymph glands at a distinct time point in the uninfected state (Fig 7M-N), 95% of the cells they analyze traversing the sigmoidal path are in the intermediate step. This would predict that the cells on this path spend a much longer time stuck in this intermediate state before traversing to the final differentiated one, or that only a small fraction of the cells that become sigmoidal intermediate cell progress onwards to full differentiation. But this does not match the trajectories observed in the real-time analysis for uninfected cultured lymph glands (Fig 7A'-D'). marker. Perhaps their algorithm discarded traces from the live imaging in which the differentiation marker did not come up quickly and was thus not analyzed in the trajectories. If my interpretation of the single time point analysis is true, this would argue that the linear path is actually much faster/more fruitful than the sigmoidal one and this would explain why a higher level of total progenitor differentiation infection is the result of infection-inducing more differentiation by the linear path. Otherwise, I don't understand how their data explains that observation.

   This work provides a very useful new system for Drosophila immunologists and could provide an important new perspective on the systems-level mechanisms that an organism utilizes to enable increased differentiation of immune cells upon infection.

   Read the original source
6. 

   eLife

   Feb 15, 2023

   Reviewer #3 (Public Review):

   In this study, the authors sought to develop an ex vivo organ culture system that would allow for long-term (>12 hours) live imaging of the lymph gland (LG), the hematopoietic organ in Drosophila, in order to gain insights into the process of differentiation during hematopoiesis. The authors successfully built such a system through trial and error and showed that the LG could survive for over 12 hours and that it recapitulated many of the aspects seen in in vivo LGs.

   The authors also developed sophisticated quantitative image analysis tools that allowed them to identify new modes of differentiation that may help explain the cellular heterogeneity previously seen by other groups. Furthermore, they were able to follow mitosis in real-time and showed evidence that not only can progenitors undergo symmetric cell …

   Reviewer #3 (Public Review):

   In this study, the authors sought to develop an ex vivo organ culture system that would allow for long-term (>12 hours) live imaging of the lymph gland (LG), the hematopoietic organ in Drosophila, in order to gain insights into the process of differentiation during hematopoiesis. The authors successfully built such a system through trial and error and showed that the LG could survive for over 12 hours and that it recapitulated many of the aspects seen in in vivo LGs.

   The authors also developed sophisticated quantitative image analysis tools that allowed them to identify new modes of differentiation that may help explain the cellular heterogeneity previously seen by other groups. Furthermore, they were able to follow mitosis in real-time and showed evidence that not only can progenitors undergo symmetric cell division but that mitosis shows some orientation bias which may help explain the overall structure of the organ. The authors went on to show that upon infection, modes of differentiation and mitosis orientation seem to shift, but they did not provide any mechanistic insight into how this may occur or whether these shifts would impact the final cell fate or function of the mature hemocytes. Nevertheless, the identification and description of these patterns are in itself helpful and informative and provide a basis for future studies delving into these mechanistic questions.

   The major strengths of the methods include the advancement in live-imaging technology and the development of quantitative image analysis tools. Weaknesses of the results include small sample sizes (and relatively high p-values), which limit the strength and breadth of some conclusions. This is to be expected as there is a trade-off between long-term live imaging of individual samples and sample number, nevertheless, it represents a minor weakness. Overall this weakness is overshadowed by the strength of the advancements afforded by live imaging and following in real-time the process of differentiation and mitosis. Furthermore, the quantitative analysis tools developed and used in this study can be applied across multiple subfields and represents an important step forward in the field.

   The evidence presented here is generally solid and the results tend to support their conclusions although some specific conclusions are supported by data with no p-values noted or relatively high p-values and low correlation coefficients, and so should be interpreted with this in mind.

   This study represents a compelling and convincing theoretical and technical advance in efforts to understand hematopoiesis in flies. This is a powerful and versatile system that will allow for not only genetic manipulation of the LG but also of the tissues co-cultured with the LG to elucidate the mechanisms that control various signaling pathways during homeostasis. In fact, which additional tissues (like the fat body and brain) that had to be included in the co-culture system in order for the LG to survive recapitulate what past studies have shown about where key signals come from that help maintain homeostasis in the LG.

   One caveat of the work is that because the authors used Eater-DsRed to follow differentiation, these modes may only apply to the formation of plasmatocytes and not necessarily crystal cells, which the authors noted do not tend to go through an Eater-DsRed intermediate state. Future work using this live-imaging system and image analysis tools to study the formation of the various mature cell types in flies will be a valuable addition to the field.

   The methods developed here will be highly useful to both the specific subfield and to the general scientific community and will likely spark new insights into the process of hematopoiesis when combined with different markers and genetic manipulations, as outlined by the authors in the discussion. Future studies that explore whether the different modes of differentiation identified here ultimately result in divergent cell fates for the mature hemocytes will be important for understanding the significance of the findings more generally. But the identification of changes in the ratios and rates of the modes of differentiation upon infection with E.coli suggests functional ramifications of the different modes. It will be interesting to see if other types of infection or systemic stresses cause similar or different changes in differentiation modes.

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7. 

   Version published to 10.1101/2022.10.06.511224v1 on bioRxiv

   Oct 7, 2022