Metamorphosis of memory circuits in Drosophila reveals a strategy for evolving a larval brain
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Curated by eLife
Evaluation Summary:
The complete metamorphosis of the higher insects is one of the most fascinating and complex processes in nature: The discrepancy in form and function between larvae, pupa, and adult insects is breathtaking, begging the question of how these forms and functions can so seamlessly follow each other. For the highest-order brain centre of the insects, the mushroom body, the authors provide a masterpiece analysis of this process at the cellular level. Given the breadth and depth of the data that the authors present, the current study will serve as a reference for the field of developmental neuroscience for many years to come; this study is eagerly awaited in the field. Perhaps ever more importantly, the insights into the relationship between evolutionary development and individual development at the cellular level might have a profound and lasting conceptual impact on life and natural sciences.
(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 #3 agreed to share their name with the authors.)
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Abstract
Mushroom bodies (MB) of adult Drosophila have a core of thousands of Kenyon neurons; axons of the early-born g class form a medial lobe and those from later-born α'β' and αβ classes form both medial and vertical lobes. The larva, however, hatches with only γ neurons and forms a vertical lobe 'facsimile' using larval-specific axon branches from its γ neurons. MB input (MBINs) and output (MBONs) neurons divide the Kenyon neuron lobes into discrete computational compartments. The larva has 10 such compartments while the adult has 16. We determined the fates of 28 of the 32 MBONs and MBINs that define the 10 larval compartments. Seven compartments are subsequently incorporated into the adult MB; four of their MBINs die, while 12 MBINs/MBONs remodel to function in adult compartments. The remaining three compartments are larval specific. At metamorphosis their MBIN/MBONs trans-differentiate, leaving the MB for other adult brain circuits. The adult vertical lobes are made de novo using MBONs/MBINs recruited from pools of adult-specific neurons. The combination of cell death, compartment shifting, trans-differentiation, and recruitment of new neurons result in no larval MBIN-MBON connections being maintained through metamorphosis. At this simple level, then, we find no anatomical substrate for a memory trace persisting from larva to adult. The adult phenotype of the trans-differentiating neurons represents their evolutionarily ancestral phenotype while their larval phenotype is a derived adaptation for the larval stage. These cells arise primarily within lineages that also produce permanent MBINs and MBONs, suggesting that larval specifying factors may allow information related to birth-order or sibling identity to be interpreted in a modified manner in the larva to allow these neurons to acquire larval phenotypic modifications. The loss of such factors at metamorphosis then allows these neurons to revert to their ancestral functions in the adult.
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Author Response
Reviewer #1 (Public Review):
It is a strength of the current manuscript that it provides a near-complete picture of how the metamorphosis of a higher brain centre comes about at the cellular level. The visualization of the data and analyses is a weakness.
I do not see any point where the conclusions of the authors need to be doubted, in particular as speculations are expressly defined as such whenever they are presented.
The fact that molecular or genetic analyses of how the described metamorphic processes are organized are not presented should, I think, not compromise enthusiasm about what is provided at the cellular level.
We appreciate the comments and guidance that Reviewer #1 has given us on data presentation. We have tried to simplify figures and make the images larger. For the developmental figures, a …
Author Response
Reviewer #1 (Public Review):
It is a strength of the current manuscript that it provides a near-complete picture of how the metamorphosis of a higher brain centre comes about at the cellular level. The visualization of the data and analyses is a weakness.
I do not see any point where the conclusions of the authors need to be doubted, in particular as speculations are expressly defined as such whenever they are presented.
The fact that molecular or genetic analyses of how the described metamorphic processes are organized are not presented should, I think, not compromise enthusiasm about what is provided at the cellular level.
We appreciate the comments and guidance that Reviewer #1 has given us on data presentation. We have tried to simplify figures and make the images larger. For the developmental figures, a couple of illustrative examples are provided in the main figure with the remainder given in “figure supplements”
Reviewer #2 (Public Review):
This very nice piece of work describes and discusses the developmental progression of larval neurons of the mushroom body into those in the adult Drosophila brain. There are many surprising findings that reveal a number of strategies for how brain development has evolved to serve both the early functions specific to the larval brain and then their eventual roles in the adult brain. I think it is fascinating biology and I was educated while reviewing the paper.
Line 115-116. 'Output from PPL1 compartments direct avoidance behavior, while that from PAM compartments results in attraction'. This is not correct and is actually reversed. The learning rule is depression so that aversive learning reduces the drive to approach pathways whereas appetitive learning reduces the drive to avoidance pathways. This should be corrected and reference made to studies demonstrating learning-directed depression.
Line 222. It provides feed-forward inhibition from y4>2>1. I could be wrong but I'm not aware that there is functional evidence for this glutamatergic neuron being inhibitory. It's currently speculation.
We have noted that this function was proposed by Aso et al.
Line 242. I think it would be nice if the authors focused on extreme changes and showed larger and nicer images. The rest can be summarized but why not pick a few of the best examples to illustrate the strategies they consider in the discussion?
We have reduced the number of neurons shown in the new Figs 5 and 6. Hopefully, the images are now large enough to appreciate. Data for the remaining neurons are now in Figure Supplements for Figs 5 and 6.
Line 249 'became sexually dimorphic'. I may have missed it somewhere but this immediately made me think about the sex of all the images that are shown. Is this explicitly stated somewhere? Was it tracked in all larvae, pupae, and adults?
We now begin the Methods addressing this point. We did an initial screen and found sex-specific differences only in MBIN-b1 and -b2. After this time, we kept no records as to the sex of the fly that was used except for the latter cells.
Reviewer #3 (Public Review):
Truman et al. investigated the contribution and remodeling of individual larval neurons that provide input and output to the Drosophila mushroom body through metamorphosis. Hereto, they used a collection of split-GAL4 lines targeting specific larval mushroom body input and output neurons, in combination with a conditional flip-switch and imaging, to follow the fates of these cells.
Interestingly, most of these larval neurons survive metamorphosis and persist in the adult brain and only a small percentage of neurons die. The authors also elegantly show that a substantial number of neurons actually trans-differentiate and exert a different role in the larval brain, compared to their final adult functionality (similar to their role in hemimetabolous insects). This process is relatively understudied in neuroscience and of great interest.
Using the ventral nerve cord as a proxy, the authors claim that the larval state of the neuron would be their derived state, while their adult identity is ancestral. While the authors did not show this directly for the mushroom body neurons under study, it is a very compelling hypothesis. However, writing the manuscript from this perspective and not from the perspective of the neuron (which first goes through a larval state, metamorphosis, and finally adult state), results in confusing language and I would suggest the authors adjust the manuscript to the 'lifeline' of the neuron.
We have tried to be more “linear” in our presentation. This should make the text less confusing.
In general, this manuscript does not explain how the larval brain has evolved as the title suggests but instead describes how the larval brain is remodeled during metamorphosis. It thus generates perspectives on the evolution of metamorphosis, rather than the larval state. Additionally, this manuscript would benefit from major rearrangements in both text and figures for the story to be better comprehended.
We think that the end of the Discussion does relate to how a larval brain evolves. The evolution of the larval brain is faced with constraints related to the shortened period of embryonic development and the highly conserved temporal and spatial mechanisms that insects use to generate their neuronal phenotypes. These constraints result in a potential mismatch between the neurons that are needed and those that are actually made (revealed by the adult phenotypes of these neurons). The larva then turns to trans-differentiation to temporarily transform unneeded (or dead) neurons into the missing cell types to build its larval circuits.
We think that these ideas provide some new insights into how a larval brain may have evolved and that our title is appropriate.
The introduction is very focused on the temporal patterning of the insect nervous system, while none of the data collected incorporate this temporal code. Temporal patterning comes back in the discussion but is purely speculative.
The Speculation about the importance of temporal patterning is now brought in late in the Discussion in reference to Figure 12
Furthermore, the second part of the introduction describes one strategy for remodeling and why that strategy is not likely but does not present an alternative hypothesis. The first section of the results might serve as a better introduction to the paper instead, as it places the results of the paper better and concludes with the main findings. The accompanying Figure 1 would also benefit from a schematic overview of the larval and adult mushroom bodies as presented in Fig. 2A (left).
This has been revised in the spirit of these comments
In the second results section, the authors show the post-metamorphic fates of mushroom body input and output neurons and introduce the concept of trans-differentiation. Readers might benefit from a short explanation of this process. I also encourage the authors to revisit this part of the text since it gives the impression that the neurons themselves undergo active migration (instead of axon remodeling).
We have tried to make it clear that there is no cell migration. Rather there is retraction/fragmentation of larval arbors followed by outgrowth to new, adult targets
The discussion starts with a very comprehensive overview of the different strategies that neurons could use during metamorphosis (here too, re-writing the text from the neurons' perspective would increase the reflection of what actually happens to them).
The Discussion now begins by dealing with gross changes in the MB, with reference to the compartments and eventually moves to changes in individual cells. We have reduced our discussion of the metamorphic strategies of cells and no longer have Fig 8A
The discussion covers multiple topics concerning trans-differentiation, metamorphosis, memory, and evolution and is often disconnected from the results. It could be significantly shortened to discuss the results of the paper and place them in current literature. Generally, the figures supporting the discussion are hard to comprehend and often do not reflect what the text is saying they are showing.
The Discussion is still long, but, hopefully, our organization now makes it much easier to read and comprehend.
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Evaluation Summary:
The complete metamorphosis of the higher insects is one of the most fascinating and complex processes in nature: The discrepancy in form and function between larvae, pupa, and adult insects is breathtaking, begging the question of how these forms and functions can so seamlessly follow each other. For the highest-order brain centre of the insects, the mushroom body, the authors provide a masterpiece analysis of this process at the cellular level. Given the breadth and depth of the data that the authors present, the current study will serve as a reference for the field of developmental neuroscience for many years to come; this study is eagerly awaited in the field. Perhaps ever more importantly, the insights into the relationship between evolutionary development and individual development at the cellular level might …
Evaluation Summary:
The complete metamorphosis of the higher insects is one of the most fascinating and complex processes in nature: The discrepancy in form and function between larvae, pupa, and adult insects is breathtaking, begging the question of how these forms and functions can so seamlessly follow each other. For the highest-order brain centre of the insects, the mushroom body, the authors provide a masterpiece analysis of this process at the cellular level. Given the breadth and depth of the data that the authors present, the current study will serve as a reference for the field of developmental neuroscience for many years to come; this study is eagerly awaited in the field. Perhaps ever more importantly, the insights into the relationship between evolutionary development and individual development at the cellular level might have a profound and lasting conceptual impact on life and natural sciences.
(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 #3 agreed to share their name with the authors.)
-
Reviewer #1 (Public Review):
It is a strength of the current manuscript that it provides a near-complete picture of how the metamorphosis of a higher brain centre comes about at the cellular level. The visualization of the data and analyses is a weakness.
I do not see any point where the conclusions of the authors need to be doubted, in particular as speculations are expressly defined as such whenever they are presented.
The fact that molecular or genetic analyses of how the described metamorphic processes are organized are not presented should, I think, not compromise enthusiasm about what is provided at the cellular level.
-
Reviewer #2 (Public Review):
This very nice piece of work describes and discusses the developmental progression of larval neurons of the mushroom body into those in the adult Drosophila brain. There are many surprising findings that reveal a number of strategies for how brain development has evolved to serve both the early functions specific to the larval brain and then their eventual roles in the adult brain. I think it is fascinating biology and I was educated while reviewing the paper.
Line 115-116. 'Output from PPL1 compartments direct avoidance behavior, while that from PAM compartments results in attraction'. This is not correct and is actually reversed. The learning rule is depression so that aversive learning reduces the drive to approach pathways whereas appetitive learning reduces the drive to avoidance pathways. This should …
Reviewer #2 (Public Review):
This very nice piece of work describes and discusses the developmental progression of larval neurons of the mushroom body into those in the adult Drosophila brain. There are many surprising findings that reveal a number of strategies for how brain development has evolved to serve both the early functions specific to the larval brain and then their eventual roles in the adult brain. I think it is fascinating biology and I was educated while reviewing the paper.
Line 115-116. 'Output from PPL1 compartments direct avoidance behavior, while that from PAM compartments results in attraction'. This is not correct and is actually reversed. The learning rule is depression so that aversive learning reduces the drive to approach pathways whereas appetitive learning reduces the drive to avoidance pathways. This should be corrected and reference made to studies demonstrating learning-directed depression.
Line 222. It provides feed-forward inhibition from y4>2>1. I could be wrong but I'm not aware that there is functional evidence for this glutamatergic neuron being inhibitory. It's currently speculation.
Line 242. I think it would be nice if the authors focused on extreme changes and showed larger and nicer images. The rest can be summarized but why not pick a few of the best examples to illustrate the strategies they consider in the discussion?
Line 249 'became sexually dimorphic'. I may have missed it somewhere but this immediately made me think about the sex of all the images that are shown. Is this explicitly stated somewhere? Was it tracked in all larvae, pupae, and adults?
-
Reviewer #3 (Public Review):
Truman et al. investigated the contribution and remodeling of individual larval neurons that provide input and output to the Drosophila mushroom body through metamorphosis. Hereto, they used a collection of split-GAL4 lines targeting specific larval mushroom body input and output neurons, in combination with a conditional flip-switch and imaging, to follow the fates of these cells.
Interestingly, most of these larval neurons survive metamorphosis and persist in the adult brain and only a small percentage of neurons die. The authors also elegantly show that a substantial number of neurons actually trans-differentiate and exert a different role in the larval brain, compared to their final adult functionality (similar to their role in hemimetabolous insects). This process is relatively understudied in …
Reviewer #3 (Public Review):
Truman et al. investigated the contribution and remodeling of individual larval neurons that provide input and output to the Drosophila mushroom body through metamorphosis. Hereto, they used a collection of split-GAL4 lines targeting specific larval mushroom body input and output neurons, in combination with a conditional flip-switch and imaging, to follow the fates of these cells.
Interestingly, most of these larval neurons survive metamorphosis and persist in the adult brain and only a small percentage of neurons die. The authors also elegantly show that a substantial number of neurons actually trans-differentiate and exert a different role in the larval brain, compared to their final adult functionality (similar to their role in hemimetabolous insects). This process is relatively understudied in neuroscience and of great interest.
Using the ventral nerve cord as a proxy, the authors claim that the larval state of the neuron would be their derived state, while their adult identity is ancestral. While the authors did not show this directly for the mushroom body neurons under study, it is a very compelling hypothesis. However, writing the manuscript from this perspective and not from the perspective of the neuron (which first goes through a larval state, metamorphosis, and finally adult state), results in confusing language and I would suggest the authors adjust the manuscript to the 'lifeline' of the neuron.
In general, this manuscript does not explain how the larval brain has evolved as the title suggests but instead describes how the larval brain is remodeled during metamorphosis. It thus generates perspectives on the evolution of metamorphosis, rather than the larval state. Additionally, this manuscript would benefit from major rearrangements in both text and figures for the story to be better comprehended.
The introduction is very focused on the temporal patterning of the insect nervous system, while none of the data collected incorporate this temporal code. Temporal patterning comes back in the discussion but is purely speculative.
Furthermore, the second part of the introduction describes one strategy for remodeling and why that strategy is not likely but does not present an alternative hypothesis. The first section of the results might serve as a better introduction to the paper instead, as it places the results of the paper better and concludes with the main findings. The accompanying Figure 1 would also benefit from a schematic overview of the larval and adult mushroom bodies as presented in Fig. 2A (left).
In the second results section, the authors show the post-metamorphic fates of mushroom body input and output neurons and introduce the concept of trans-differentiation. Readers might benefit from a short explanation of this process. I also encourage the authors to revisit this part of the text since it gives the impression that the neurons themselves undergo active migration (instead of axon remodeling).
The discussion starts with a very comprehensive overview of the different strategies that neurons could use during metamorphosis (here too, re-writing the text from the neurons' perspective would increase the reflection of what actually happens to them).
The discussion covers multiple topics concerning trans-differentiation, metamorphosis, memory, and evolution and is often disconnected from the results. It could be significantly shortened to discuss the results of the paper and place them in current literature. Generally, the figures supporting the discussion are hard to comprehend and often do not reflect what the text is saying they are showing.
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