A conserved strategy for inducing appendage regeneration in moon jellyfish, Drosophila, and mice
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Evaluation Summary:
This paper argues that simple nutritional interventions (L-leucine / insulin / sucrose) can trigger appendage regeneration in species various that do not regenerate appendages in normal conditions. Although the data on Drosophila are not fully convincing and further evidence is needed for this species, in the jellyfish Aurelia and in mice, the results are stunning and provide novel model systems for inducing appendage regeneration in animals and for studying the mechanisms underlying regeneration. These results strengthen an old idea that animals may have an intrinsic capacity to regenerate, which could be revealed by simple (e.g. nutritional) interventions. This paper will be of interest to readers in the field of signaling in regeneration and also in regenerative medicine.
(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 #3 agreed to share their name with the authors.)
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Abstract
Can limb regeneration be induced? Few have pursued this question, and an evolutionarily conserved strategy has yet to emerge. This study reports a strategy for inducing regenerative response in appendages, which works across three species that span the animal phylogeny. In Cnidaria, the frequency of appendage regeneration in the moon jellyfish Aurelia was increased by feeding with the amino acid L-leucine and the growth hormone insulin. In insects, the same strategy induced tibia regeneration in adult Drosophila . Finally, in mammals, L-leucine and sucrose administration induced digit regeneration in adult mice, including dramatically from mid-phalangeal amputation. The conserved effect of L-leucine and insulin/sugar suggests a key role for energetic parameters in regeneration induction. The simplicity by which nutrient supplementation can induce appendage regeneration provides a testable hypothesis across animals.
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Author Response:
Reviewer #1:
The paper is very well written and the results are well presented. I only have minor comments.
The introduction insists on the idea that the ability to regenerate might be ancestral (line 45) but convergent evolution is an extremely common phenomenon. The hypothesis of convergent evolution cannot be excluded here. In any case, whether convergence or ancestrality, one can ask whether the mechanisms underlying organ regeneration are the same in various taxa.
We thank Reviewer 1 for all the helpful suggestions. We will make it clearer in the revised Introduction that the notion convergence still cannot be fully excluded.
Reviewer #2:
Weaknesses:
The work presented on Drosophila is intriguing because the adult legs of flies were not thought to be capable of any regeneration. One of the major …
Author Response:
Reviewer #1:
The paper is very well written and the results are well presented. I only have minor comments.
The introduction insists on the idea that the ability to regenerate might be ancestral (line 45) but convergent evolution is an extremely common phenomenon. The hypothesis of convergent evolution cannot be excluded here. In any case, whether convergence or ancestrality, one can ask whether the mechanisms underlying organ regeneration are the same in various taxa.
We thank Reviewer 1 for all the helpful suggestions. We will make it clearer in the revised Introduction that the notion convergence still cannot be fully excluded.
Reviewer #2:
Weaknesses:
The work presented on Drosophila is intriguing because the adult legs of flies were not thought to be capable of any regeneration. One of the major constraints is that growth in arthropods is limited by the hard exoskeleton (cuticle) surrounding the body. Periodic molting allows these animals to grow in a stepwise fashion (shedding the old cuticle and forming a new one), but adult flies do not molt, so it is unclear how an adult regenerating leg would break that constraint. Abrams et al. report that a small proportion (~1%) of amputated legs regrow part of the limb when the flies are kept on a medium supplemented with leucine, glutamine and human insulin. The number of legs in which this has been observed is small and the extent of regeneration is variable and not well documented in relation to the site of amputation (which is unmarked). A more detailed documentation of the regrowth would be needed to validate the authors conclusions.
We thank Reviewer 2 for the more detailed suggestions in the full review. In the present data, our conclusion is enabled by the non-ambiguous phenotype: there are fully regrown tibias in the treated population, and there are none in the (now) over >1000 control flies examined. In the revised manuscript, we will include as the Reviewer suggested new extensive documentation to show single-fly tracking.
The work on mice focuses on the regeneration of digit tips, a relatively well-studied example of limited regeneration in these rodents. Mice are known to be able to regenerate the tips of their digits when these are amputated near the distal end, but cuts proximal to the base of the nail fail to regenerate. The authors focus on regeneration of digits amputated near this boundary. They report that animals whose drinking water is supplemented with leucine, glutamine and sucrose are more likely to regenerate part of their digit tips when amputated at the base of the nail. These data are intriguing, but the number of observations is limited (few digits with patterned regeneration) and variation in the site amputation does not make it easy to draw firm conclusions on the extent of regeneration compared with controls.
We perform digit amputation proximal to the established non-regenerating boundary (see red line in Figure 6c-d), and far from the regenerating boundary (see blue line in Figure 6c-d). Moreover, as a control, for every digit amputated, the part removed was fixed, stained, and documented to enable precise definition of the amputation plane. The sample sizes in the study (20 control digits, 48 treated digits) enable statistical power, and are comparable to experiments in adult mouse digit (e.g., n~15 digits PMID: 24209617, n~20 digits PMID: 28975034).
Overall, the authors propose that similar nutritional interventions have similar effects in 'inducing' regeneration in widely divergent animals, revealing a widespread intrinsic capacity of animals to regenerate. The claim that these treatments 'induce' regeneration seems exaggerated, given that appendage regeneration in Aurelia and in mouse digits can occur to a variable extent in untreated animals. These treatments appear to shift the probability and the extent of regeneration. The data on Drosophila legs are more surprising and deserve further analysis.
We have been careful in the manuscript to use the phrase “promote regeneration” to describe the findings in Aurelia because the spontaneous partial regeneration observed in the natural habitat. In the mouse digit, however, the boundaries of regenerating and non-regenerating cuts have been clearly established (by multiple studies, e.g., PMID: 28493324, PMID: 7100922, PMID: 18234177, PMID: 17147657), which enables the question of how to induce regeneration from proximal cuts posed and pursued (e.g., PMID: 30723209, PMID: 20110320). We believe therefore in this case, our choice of wording is validated by the scientific context and precedents in the mouse digit field.
The idea that the same nutritional interventions may have similar effects on regeneration in diverse animals is intriguing. A minor caveat: the nutritional interventions tested in each species were not identical; in Aurelia high-nutrient, insulin and leucine treatments were tested separately, in Drosophila leucine and insulin were combined, in the mouse leucine and sucrose were combined. Future work could determine which components in these treatments (nutritional, metabolic or hormonal) are responsible for the observed responses in each species.
We agree with the Reviewer. We will make it clearer in the revised Discussion the differences in the molecule administration across species, and that further studies will determine the specific underlying mechanisms, in spite of which, we could excitingly move across species in a predictive manner.
Reviewer #3:
Weaknesses:
The evolutionary statement underlying the entire study is not fully accurate. While it is true that many animal phyla include species that can regenerate and also that some studies start to identify common molecular components in regeneration, the question of regeneration being ancestral or not is still debated. It is indeed highly tempting to consider regeneration as ancestral but this is not proven yet (see Lai and Aboobaker 2017) and the possibility of convergence have to be considered too. In addition, appendage regeneration versus whole body regeneration versus structure or organ regeneration may not rely on similar mechanisms.
We thank Reviewer 3 for the helpful feedback in the detailed review. We are making it clearer in the revised Introduction that the notion of convergence still cannot be fully excluded since convergences can be very common.
The strategy put in place for identifying putative triggers is questionable and more information about, (i) the reasons to select and test such parameters, (ii) what the different drugs are doing in theory (components of signaling pathways targeted, for ex), (iii) how exactly the various tests have been done, frequency of scoring, different concentration tested, number of individuals per condition, frequency of drug administration etc etc are missing. It appears really surprising that no modulators of signaling pathways (notably the wnt b catenin, known to be involved in many developmental and regeneration contexts, especially in cnidarians) are involved in any sort in such process.
We will include in the revised manuscript more extensive details about the screen parameters and procedures. Briefly here, similar experimental designs as those described for the main experiments were used. With regard to the Wnt pathway, we tried two inhibitors against GSK3beta, two inhibitors against tankyrase, and a Wnt ligand itself—none so far has shown effects that motivated further follow-up. Nonetheless, we will emphasize more explicitly in the revision that the negative result conclusion can only be specific to the conditions and the modulators used, and that this has by no means ruled out the involvement of developmental pathways in all capacities.
Concerning the results with cnidarians, two aspects puzzle me: the high variability in the regenerative response between batches and the way the amputations were done in ephyra. In the context of appendage regeneration, what justify to perform an amputation that remove almost the half body of the ephyra and not just one arm. Basic information about regeneration occurrence in the context of amputation of only one arm are missing.
We find the variability intriguing too. Some of the variability may arise from technical variations or stochasticity inherent in biological processes. There may also be key physiological parameters yet to be identified; We are continuing screening in the lab to search for factors that may synergize with leucine and insulin, and quantify differences between regenerating vs non-regenerating individuals. Finally, the variability may support the idea that the regeneration we are seeing has been evolutionarily inactivated, and may therefore have drifted over tens or hundreds of years and consequently does not exhibit the robustness we are used to seeing in wild-type processes.
With regard to the amputation scheme, we tested various amputation schemes and observed similar results (this will be included in the revised manuscript). The amputation scheme chosen was the fastest to do, which facilitates testing thousands of ephyrae. We will add this rationale in the revised Methods. With the amputation scheme chosen, arm regeneration is the most dramatic outcome. However, the reviewer is correct that the body is also partially regenerated, which suggests to us that L-leucine and sugar/insulin may have regenerative effect beyond the context of appendage. We include this discussion in the revised manuscript.
More importantly, the results shown, sometimes in extremely low proportions compared to the controls (ie in the case of drosophila), are not supported by other approaches. It would be really important to have some clues about the molecular mechanisms underlying such process and its induction.
We agree with the Reviewer that mechanistic investigation into the underlying molecular mechanism is the next immediate challenge, and is underway in the lab. In the scope of the present work, the focus is on seeing if regeneration can be induced at all and the comparative presence of such latent ability. This was itself an enormous effort, but one that enables laying the groundwork for directing mechanistics investigations and broader pursuit of promoting regeneration across animals.
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Evaluation Summary:
This paper argues that simple nutritional interventions (L-leucine / insulin / sucrose) can trigger appendage regeneration in species various that do not regenerate appendages in normal conditions. Although the data on Drosophila are not fully convincing and further evidence is needed for this species, in the jellyfish Aurelia and in mice, the results are stunning and provide novel model systems for inducing appendage regeneration in animals and for studying the mechanisms underlying regeneration. These results strengthen an old idea that animals may have an intrinsic capacity to regenerate, which could be revealed by simple (e.g. nutritional) interventions. This paper will be of interest to readers in the field of signaling in regeneration and also in regenerative medicine.
(This preprint has been reviewed by eLife. …
Evaluation Summary:
This paper argues that simple nutritional interventions (L-leucine / insulin / sucrose) can trigger appendage regeneration in species various that do not regenerate appendages in normal conditions. Although the data on Drosophila are not fully convincing and further evidence is needed for this species, in the jellyfish Aurelia and in mice, the results are stunning and provide novel model systems for inducing appendage regeneration in animals and for studying the mechanisms underlying regeneration. These results strengthen an old idea that animals may have an intrinsic capacity to regenerate, which could be revealed by simple (e.g. nutritional) interventions. This paper will be of interest to readers in the field of signaling in regeneration and also in regenerative medicine.
(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 #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The paper is very well written and the results are well presented. I only have minor comments.
The introduction insists on the idea that the ability to regenerate might be ancestral (line 45) but convergent evolution is an extremely common phenomenon. The hypothesis of convergent evolution cannot be excluded here. In any case, whether convergence or ancestrality, one can ask whether the mechanisms underlying organ regeneration are the same in various taxa.
-
Reviewer #2 (Public Review):
Abrams et al. show that under certain conditions (high nutrient level, insulin intake, leucine intake and hypoxia) the frequency of arm regeneration in the cnidarian Aurelia aurita increases compared to control ('low food') conditions. The authors argue that similar effects on appendage regeneration can also be found in widely divergent organisms (fruit flies and mice). They suggest that all animals may have an intrinsic capacity to regenerate, which could be revealed by simple (e.g. nutritional) interventions. The idea that many animals possess latent regenerative abilities which could be activated by simple genetic interventions (e.g. by activating specific genes or signalling pathways) has attracted significant interest in the field of regenerative biology, as it underpins hopes that a wide range of …
Reviewer #2 (Public Review):
Abrams et al. show that under certain conditions (high nutrient level, insulin intake, leucine intake and hypoxia) the frequency of arm regeneration in the cnidarian Aurelia aurita increases compared to control ('low food') conditions. The authors argue that similar effects on appendage regeneration can also be found in widely divergent organisms (fruit flies and mice). They suggest that all animals may have an intrinsic capacity to regenerate, which could be revealed by simple (e.g. nutritional) interventions. The idea that many animals possess latent regenerative abilities which could be activated by simple genetic interventions (e.g. by activating specific genes or signalling pathways) has attracted significant interest in the field of regenerative biology, as it underpins hopes that a wide range of regenerative therapies might even be possible in humans. Abrams et al. provide an example of how simple, non-genetic interventions could also influence the extent of regeneration.
Strengths:
The authors nicely describe the regenerative capacity of the Aurelia aurita juvenile. They show that after large amputation (removal of 3 of 8 arms), a few individuals in their natural habitat are able to regrow partially one arm. Taking advantage of this inherent ability, they screen numerous factors in order to identify ones that enhance this response. Their screen includes factors that have been identified as modulators of signalling pathways, metabolism, the immune system and stress responses in other species. Out of >40 factors or conditions tested, Abrams et al. identify four - high nutrient level, insulin intake, leucine intake and hypoxia - that increase the frequency of arm regeneration in Aurelia. The authors show that the regenerated arms can be functional, as they regrow nerves, muscles and contract simultaneously with the remaining arms.
Having identified factors that enhance arm regeneration in Aurelia, the authors test whether similar conditions could influence the extent of regeneration in two widely divergent systems: the legs of the adult fruit fly Drosophila, which are thought to completely lack the ability to regenerate, and the digit tips of the mouse, which have a limited regenerative ability. These bold tests yield some intriguing preliminary results.
Weaknesses:
The work presented on Drosophila is intriguing because the adult legs of flies were not thought to be capable of any regeneration. One of the major constraints is that growth in arthropods is limited by the hard exoskeleton (cuticle) surrounding the body. Periodic molting allows these animals to grow in a stepwise fashion (shedding the old cuticle and forming a new one), but adult flies do not molt, so it is unclear how an adult regenerating leg would break that constraint. Abrams et al. report that a small proportion (~1%) of amputated legs regrow part of the limb when the flies are kept on a medium supplemented with leucine, glutamine and human insulin. The number of legs in which this has been observed is small and the extent of regeneration is variable and not well documented in relation to the site of amputation (which is unmarked). A more detailed documentation of the regrowth would be needed to validate the authors conclusions.
The work on mice focuses on the regeneration of digit tips, a relatively well-studied example of limited regeneration in these rodents. Mice are known to be able to regenerate the tips of their digits when these are amputated near the distal end, but cuts proximal to the base of the nail fail to regenerate. The authors focus on regeneration of digits amputated near this boundary. They report that animals whose drinking water is supplemented with leucine, glutamine and sucrose are more likely to regenerate part of their digit tips when amputated at the base of the nail. These data are intriguing, but the number of observations is limited (few digits with patterned regeneration) and variation in the site amputation does not make it easy to draw firm conclusions on the extent of regeneration compared with controls.
Overall, the authors propose that similar nutritional interventions have similar effects in 'inducing' regeneration in widely divergent animals, revealing a widespread intrinsic capacity of animals to regenerate. The claim that these treatments 'induce' regeneration seems exaggerated, given that appendage regeneration in Aurelia and in mouse digits can occur to a variable extent in untreated animals. These treatments appear to shift the probability and the extent of regeneration. The data on Drosophila legs are more surprising and deserve further analysis.
The idea that the same nutritional interventions may have similar effects on regeneration in diverse animals is intriguing. A minor caveat: the nutritional interventions tested in each species were not identical; in Aurelia high-nutrient, insulin and leucine treatments were tested separately, in Drosophila leucine and insulin were combined, in the mouse leucine and sucrose were combined. Future work could determine which components in these treatments (nutritional, metabolic or hormonal) are responsible for the observed responses in each species.
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Reviewer #3 (Public Review):
In this article, Abrams et al. aim at identifying a potential conserved strategy for inducing appendage regeneration in Metazoa. Appendage regeneration is a process shared by many animal lineages, raising the possibility of a common strategy at play. Using a large-scale screening of molecular and physical putative modulators of regeneration, they identify the amino acid L-leucine and the growth hormone insulin/sucrose as molecules potentially able to trigger appendages regeneration in three species, the cnidarian Aurelia, the ecdyzosoan Drosophila and the mammal Mus musculus. The question addressed in this paper is really interesting and some results, especially in Drosophila and Mus musculus, are intriguing but need to be confirmed and extended.
Strengths:
One main strength of this study is the use of three …
Reviewer #3 (Public Review):
In this article, Abrams et al. aim at identifying a potential conserved strategy for inducing appendage regeneration in Metazoa. Appendage regeneration is a process shared by many animal lineages, raising the possibility of a common strategy at play. Using a large-scale screening of molecular and physical putative modulators of regeneration, they identify the amino acid L-leucine and the growth hormone insulin/sucrose as molecules potentially able to trigger appendages regeneration in three species, the cnidarian Aurelia, the ecdyzosoan Drosophila and the mammal Mus musculus. The question addressed in this paper is really interesting and some results, especially in Drosophila and Mus musculus, are intriguing but need to be confirmed and extended.
Strengths:
One main strength of this study is the use of three different models to approach a biological question in a comparative manner. Such a strategy is still rare and the authors are commended for it.
An impressive number of various molecular and physical putative modulators was screened in Aurelia.
While not being a specialist, statistics appeared to be performed in a correct manner and the number of individuals used in the different experiments is appropriate.
Results showing tibia regeneration in Drosophila and digit regeneration in adult mice are impressive.
Weaknesses:
The evolutionary statement underlying the entire study is not fully accurate. While it is true that many animal phyla include species that can regenerate and also that some studies start to identify common molecular components in regeneration, the question of regeneration being ancestral or not is still debated. It is indeed highly tempting to consider regeneration as ancestral but this is not proven yet (see Lai and Aboobaker 2017) and the possibility of convergence have to be considered too. In addition, appendage regeneration versus whole body regeneration versus structure or organ regeneration may not rely on similar mechanisms.
The strategy put in place for identifying putative triggers is questionable and more information about, (i) the reasons to select and test such parameters, (ii) what the different drugs are doing in theory (components of signaling pathways targeted, for ex), (iii) how exactly the various tests have been done, frequency of scoring, different concentration tested, number of individuals per condition, frequency of drug administration etc etc are missing. It appears really surprising that no modulators of signaling pathways (notably the wnt b catenin, known to be involved in many developmental and regeneration contexts, especially in cnidarians) are involved in any sort in such process.
Concerning the results with cnidarians, two aspects puzzle me: the high variability in the regenerative response between batches and the way the amputations were done in ephyra. In the context of appendage regeneration, what justify to perform an amputation that remove almost the half body of the ephyra and not just one arm. Basic information about regeneration occurrence in the context of amputation of only one arm are missing.
More importantly, the results shown, sometimes in extremely low proportions compared to the controls (ie in the case of drosophila), are not supported by other approaches. It would be really important to have some clues about the molecular mechanisms underlying such process and its induction.
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