Osteoclast-mediated resorption primes the skeleton for successful integration during axolotl limb regeneration
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Evaluation Summary:
This paper is of interest to skeletal biologists studying skeletal development and tissue regeneration. The study applies a well-established and elegant axolotl limb regeneration model and transgenic reporter strains to reveal the potential role of osteoclast-mediated resorption in limb regeneration.
(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 agreed to share their name with the authors.)
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Abstract
Early events during axolotl limb regeneration include an immune response and the formation of a wound epithelium. These events are linked to a clearance of damaged tissue prior to blastema formation and regeneration of the missing structures. Here, we report the resorption of calcified skeletal tissue as an active, cell-driven, and highly regulated event. This process, carried out by osteoclasts, is essential for a successful integration of the newly formed skeleton. Indeed, the extent of resorption is directly correlated with the integration efficiency, and treatment with zoledronic acid resulted in osteoclast function inhibition and failed tissue integration. Moreover, we identified the wound epithelium as a regulator of skeletal resorption, likely releasing signals involved in recruitment/differentiation of osteoclasts. Finally, we reported a correlation between resorption and blastema formation, particularly, a coordination of resorption with cartilage condensation. In sum, our results identify resorption as a major event upon amputation, playing a critical role in the overall process of skeletal regeneration.
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Author Response
Reviewer #1 (Public Review):
The manuscript shows that bone is resorbed during the early steps of limb regeneration in urodeles, and osteoclasts are required for this process. In case of impaired resorption, integration of newly-formed tissue with the original bone shaft is compromised. The manuscript further shows that wound epithelium is required for bone resorption and suggests that it induces osteoclastogenesis or migration of osteoclasts. Furthermore, the authors showed that the formation of novel skeletal elements is initiated while the resorption of the old one is still actively ongoing.
The study is well designed, conclusions are relatively well supported, and data are presented in a clear way. Two new models of transgenic axolotls have been created. The strongest and most important finding is that partial …
Author Response
Reviewer #1 (Public Review):
The manuscript shows that bone is resorbed during the early steps of limb regeneration in urodeles, and osteoclasts are required for this process. In case of impaired resorption, integration of newly-formed tissue with the original bone shaft is compromised. The manuscript further shows that wound epithelium is required for bone resorption and suggests that it induces osteoclastogenesis or migration of osteoclasts. Furthermore, the authors showed that the formation of novel skeletal elements is initiated while the resorption of the old one is still actively ongoing.
The study is well designed, conclusions are relatively well supported, and data are presented in a clear way. Two new models of transgenic axolotls have been created. The strongest and most important finding is that partial bone resorption is required for tissue reintegration. My main concern is the novelty of this study, which is quite limited in my opinion.
Specifically, resorption of bone stump during limb regeneration has been shown before in various model organisms.
The role of osteoclasts in this process has not been well characterized in urodeles but has been shown during the regeneration of a mouse digit.
It is reasonable to anticipate that similarly, osteoclasts are resorbing bone in salamanders, especially since this is the only cell type known for bone resorption.
Thus, this observation, despite being nicely and thoroughly done, is of limited interest.
The role of wound epithelium in bone histolysis is well demonstrated via skin flap experiments in this manuscript. However, upon skin flap surgery no limb regeneration occurs, implying wound epithelium is a key tissue triggering all the processes of limb regeneration. Accordingly, the absence of bone histolysis in such conditions can be secondary to the absence of any other part of the regenerative process, e.g., blastema formation, macrophage M1 to M2 transition, reinnervation, etc. The proposed link between wound epithelium and osteoclastogenesis (i.e., Sphk1, Ccl4, Mdka) is very superficial and very suggestive.
No functional evidence was provided to confirm these connections. Finally, the authors showed that new bone formation occurs while resorption of the bone stump is still ongoing. This is a nice observation, but again, rather indirect as it is based on the dynamics of bone resorption and bone formation in different animals. Due to high variability among animals, direct evidence, like double staining for osteoclasts and blastema markers would address this point more precisely.
We consider that our work provides evidence, for the first time, that skeletal resorption in early stages of regeneration has a durable impact by affecting tissue integration. We show that this process occurs in a short and conserved time, which provides a window of interest for comparative research with other models, and interventional therapies. To our knowledge, limb regeneration is studied mainly in amphibians, as they are the only established lab model with this ability. Some lizards, geckos and possibly iguanas, have been reported to regrow an appendage albeit lacking the regenerative fidelity amphibians have. In an established regeneration lab model, such as the axolotl, the study of regeneration-induced resorption has been scarce.
During murine digit tip, osteoclasts are recruited to the amputation site and resorb the bone in a similar time frame as we show here in the axolotl. Ablating osteoclasts delays the regeneration time, however, no study has been conducted on the impact of tissue integration. Additionally, a key difference between mouse digit and adult axolotl limb regeneration is that the new skeletal elements are built fundamentally different: direct ossification (bone on top of bone) in mouse, versus endochondral ossification (cartilage on top of osteo-cartilage elements) in the axolotl limb. The tissue integration of the latter may present different challenges worth exploring to understand its regulation. What this work adds, is a characterization of the temporal and cellular dynamic of regeneration-induced resorption, the interaction of osteoclasts with skeletal cells and lastly, the impact on tissue integration.
Based on previous studies in mammals, it is reasonable to anticipate the presence and role of osteoclasts in salamanders. However, the growing body of work in the field, as well as our own work in the axolotl, have shown that extrapolations of mammalian skeletal biology to other species come with their risks.
We agree that the role of the wound epithelium (WE) in skeletal histolysis will require further and extensive work. The evidence shown here, provides a glimpse of the complex response and crosstalk of the WE with the tissue underneath, and we hypothesize this response is tailored to the tissue composition exposed during the injury.
Finally, following the reviewer’s advice, we have conducted new experiments to prove the temporal connection between skeletal resorption and regeneration, showing that these processes occur simultaneously.
Reviewer #3 (Public Review):
This study outlines the role of osteoclast-mediated resorption in integrating the skeletal elements during limb regeneration, using axolotls that can regenerate the entire limb upon amputation. Using calcium-binding vital dyes (calcein and alizarin red), the authors first demonstrated that a large portion of amputated skeletal elements is resorbed prior to blastema formation. They further show that 1) inhibiting bone resorption by zoledronic acid impairs proper integration of the pre-existing and regenerating skeletal elements, 2) removing the wound epithelium using the full skin flap surgery inhibits bone resorption, and 3) bone resorption and blastema formation are correlated. The authors reached the major conclusion that bone resorption is essential for successful skeletal regeneration. Notably, this study applies a well-established and elegant axolotl limb regeneration model and transgenic reporter strains to reveal the potential roles of resorption in limb regeneration.
Strengths:
- The authors utilized a well-established axolotl limb regeneration model and applied elegant vital mineral dyes and transgenic reporter lines for sequential in vivo imaging. The authors also provided quantitative assessment by examining multiple animals, particularly in the early sections, ensuring the rigor and the reproducibility of the study.
- The authors further performed important interventions that can impinge upon successful limb regeneration, including inhibition of bone resorption by zoledronic acid and impairment of the wound epithelium by full skin flap surgery. These procedures gave rise to useful insights into the relationship between bone resorption and successful limb regeneration.
- The imaging presented in this manuscript is of exceptionally high quality.
Weaknesses:
- Despite the high quality of the work, many analyses in this study are incomplete, making it insufficient to support the major conclusion. For example, in Figure 4, the authors did not provide any quantitative assessment to show how zol affects the integration of the skeletal elements (angulation?), which seems to be essential for supporting the conclusion. Likewise in Figure 7, the analyses of EdU+ cells and Sox9 reporter expression were not included in zol-treated animals. Similarly in Figure 5, quantification of osteoclasts was not performed with the full skin flap surgery group. Analyses of only normally regenerated animals are not sufficient to support many of the conclusions.
- The phenotype of zol-treated animals in limb regeneration is somewhat disappointing. Although zol-treated animals show decreased blastema formation and unresorbed pre-existing skeletal elements, limb regeneration still occurs and the only phenotype is a relatively minor defect in skeletal integration. It is possible that zol-induced defect in blastema formation is not directly linked to the failure of integration at a later stage. I find this “weakness” a bit subjective.
- As an integration failure of the newly formed skeleton still occurs in untreated animals, it is not entirely clear how the authors can attribute this defect to a lack of bone resorption. More quantitative analyses would be necessary to demonstrate the correlation between zol treatment and lack of integration.
Taking into consideration the reviewer’s concerns, we have improved our analysis of integration phenotype. The assessment of integration success was carried out using a score matrix and with it, we correlated the extent of resorption with integration efficiency more accurately. We believe our results provide sufficient evidence to support this correlation.
When we first saw the phenotype of zol-treated animals, we were far from disappointed, we were actually intrigued that we could observe a significant failure in tissue integration after removing the function of osteoclasts in an early phase of regeneration. All or nothing results are exciting, subtle results on the other hand, could prove more informative, and we think this is the case here. Our treatment does not inhibit regeneration, but disrupts tissue integration, opening another fascinating aspect of regeneration: how old tissue is capable of functionally integrate newly-formed tissue?
The integration phenotypes observed in the un-resorbed limbs does not resemble anything reported in the field so far. Moreover, the range of phenotypes observed led us to better determine its correlation with resorption. Importantly, the presence of integration failures in untreated animals allowed us to look into ECM organization at this old-new tissue interphase, while highlighting the normal occurrence of imperfect regeneration in the axolotl limb.
Finally, we have included new results to complement the conclusions presented at the end of our work. Albeit we observed differences in blastema size in zol-treated animals, we did not observe difference in the amount of EdU+ cells, which reveals that the skeleton cannot be used as a reference for assessing blastema location. This conclusion is complemented with our in vivo assays in which we observed condensation of cartilage despite resorption still occurring. We consider our conclusions to be justified and supported by the assays presented in our work.
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Evaluation Summary:
This paper is of interest to skeletal biologists studying skeletal development and tissue regeneration. The study applies a well-established and elegant axolotl limb regeneration model and transgenic reporter strains to reveal the potential role of osteoclast-mediated resorption in limb regeneration.
(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 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The manuscript shows that bone is resorbed during the early steps of limb regeneration in urodeles, and osteoclasts are required for this process. In case of impaired resorption, integration of newly-formed tissue with the original bone shaft is compromised. The manuscript further shows that wound epithelium is required for bone resorption and suggests that it induces osteoclastogenesis or migration of osteoclasts. Furthermore, the authors showed that the formation of novel skeletal elements is initiated while the resorption of the old one is still actively ongoing.
The study is well designed, conclusions are relatively well supported, and data are presented in a clear way. Two new models of transgenic axolotls have been created. The strongest and most important finding is that partial bone resorption is …
Reviewer #1 (Public Review):
The manuscript shows that bone is resorbed during the early steps of limb regeneration in urodeles, and osteoclasts are required for this process. In case of impaired resorption, integration of newly-formed tissue with the original bone shaft is compromised. The manuscript further shows that wound epithelium is required for bone resorption and suggests that it induces osteoclastogenesis or migration of osteoclasts. Furthermore, the authors showed that the formation of novel skeletal elements is initiated while the resorption of the old one is still actively ongoing.
The study is well designed, conclusions are relatively well supported, and data are presented in a clear way. Two new models of transgenic axolotls have been created. The strongest and most important finding is that partial bone resorption is required for tissue reintegration. My main concern is the novelty of this study, which is quite limited in my opinion. Specifically, resorption of bone stump during limb regeneration has been shown before in various model organisms. The role of osteoclasts in this process has not been well characterized in urodeles but has been shown during the regeneration of a mouse digit. It is reasonable to anticipate that similarly, osteoclasts are resorbing bone in salamanders, especially since this is the only cell type known for bone resorption. Thus, this observation, despite being nicely and thoroughly done, is of limited interest. The role of wound epithelium in bone histolysis is well demonstrated via skin flap experiments in this manuscript. However, upon skin flap surgery no limb regeneration occurs, implying wound epithelium is a key tissue triggering all the processes of limb regeneration. Accordingly, the absence of bone histolysis in such conditions can be secondary to the absence of any other part of the regenerative process, e.g., blastema formation, macrophage M1 to M2 transition, reinnervation, etc. The proposed link between wound epithelium and osteoclastogenesis (i.e., Sphk1, Ccl4, Mdka) is very superficial and very suggestive. No functional evidence was provided to confirm these connections. Finally, the authors showed that new bone formation occurs while resorption of the bone stump is still ongoing. This is a nice observation, but again, rather indirect as it is based on the dynamics of bone resorption and bone formation in different animals. Due to high variability among animals, direct evidence, like double staining for osteoclasts and blastema markers would address this point more precisely.
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Reviewer #2 (Public Review):
The authors set out to demonstrate two major points in this manuscript: 1) that osteoclasts are responsible for resorbing bones following amputation; and 2) that the wound epidermis plays an important/essential role in activating osteoclasts/resorption.
The authors used multiple techniques to demonstrate that osteoclasts are responsible for bone resorption following amputation in axolotls. Overall this manuscript presents more strengths than weaknesses. The major strength is the fact that they use transgenic animals with osteoclast-specific reporters (a big plus) in addition to using specialized surgical procedures and also pharmacological treatments to target osteoclasts' function. In addition, they also take advantage of RNAseq databases to show that osteoclast modulating genes are expressed in the wound …
Reviewer #2 (Public Review):
The authors set out to demonstrate two major points in this manuscript: 1) that osteoclasts are responsible for resorbing bones following amputation; and 2) that the wound epidermis plays an important/essential role in activating osteoclasts/resorption.
The authors used multiple techniques to demonstrate that osteoclasts are responsible for bone resorption following amputation in axolotls. Overall this manuscript presents more strengths than weaknesses. The major strength is the fact that they use transgenic animals with osteoclast-specific reporters (a big plus) in addition to using specialized surgical procedures and also pharmacological treatments to target osteoclasts' function. In addition, they also take advantage of RNAseq databases to show that osteoclast modulating genes are expressed in the wound epithelium at the proper time. The results are therefore very convincing that indeed osteoclasts are resorbing bones and that the wound epithelium is modulating this activity in great part.
The aim of this manuscript to show that osteoclasts are essential for bone resorption and that this process is in great part under the control of the wound epithelium during axolotl limb regeneration has been demonstrated. The results support the conclusions.
If we are ever to develop therapies to promote regeneration in humans it is essential to understand every aspect of regeneration in animals such as axolotls which can regenerate highly complex structures to near perfection. Bone remodeling is an essential part of limb regeneration and this is the first study to show that osteoclasts are responsible for this process in axolotls. This is very useful for the community at large.
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Reviewer #3 (Public Review):
This study outlines the role of osteoclast-mediated resorption in integrating the skeletal elements during limb regeneration, using axolotls that can regenerate the entire limb upon amputation. Using calcium-binding vital dyes (calcein and alizarin red), the authors first demonstrated that a large portion of amputated skeletal elements is resorbed prior to blastema formation. They further show that 1) inhibiting bone resorption by zoledronic acid impairs proper integration of the pre-existing and regenerating skeletal elements, 2) removing the wound epithelium using the full skin flap surgery inhibits bone resorption, and 3) bone resorption and blastema formation are correlated. The authors reached the major conclusion that bone resorption is essential for successful skeletal regeneration. Notably, this …
Reviewer #3 (Public Review):
This study outlines the role of osteoclast-mediated resorption in integrating the skeletal elements during limb regeneration, using axolotls that can regenerate the entire limb upon amputation. Using calcium-binding vital dyes (calcein and alizarin red), the authors first demonstrated that a large portion of amputated skeletal elements is resorbed prior to blastema formation. They further show that 1) inhibiting bone resorption by zoledronic acid impairs proper integration of the pre-existing and regenerating skeletal elements, 2) removing the wound epithelium using the full skin flap surgery inhibits bone resorption, and 3) bone resorption and blastema formation are correlated. The authors reached the major conclusion that bone resorption is essential for successful skeletal regeneration. Notably, this study applies a well-established and elegant axolotl limb regeneration model and transgenic reporter strains to reveal the potential roles of resorption in limb regeneration.
Strengths:
1. The authors utilized a well-established axolotl limb regeneration model and applied elegant vital mineral dyes and transgenic reporter lines for sequential in vivo imaging. The authors also provided quantitative assessment by examining multiple animals, particularly in the early sections, ensuring the rigor and the reproducibility of the study.
2. The authors further performed important interventions that can impinge upon successful limb regeneration, including inhibition of bone resorption by zoledronic acid and impairment of the wound epithelium by full skin flap surgery. These procedures gave rise to useful insights into the relationship between bone resorption and successful limb regeneration.
3. The imaging presented in this manuscript is of exceptionally high quality.Weaknesses:
1. Despite the high quality of the work, many analyses in this study are incomplete, making it insufficient to support the major conclusion. For example, in Figure 4, the authors did not provide any quantitative assessment to show how zol affects the integration of the skeletal elements (angulation?), which seems to be essential for supporting the conclusion. Likewise in Figure 7, the analyses of EdU+ cells and Sox9 reporter expression were not included in zol-treated animals. Similarly in Figure 5, quantification of osteoclasts was not performed with the full skin flap surgery group. Analyses of only normally regenerated animals are not sufficient to support many of the conclusions.
2. The phenotype of zol-treated animals in limb regeneration is somewhat disappointing. Although zol-treated animals show decreased blastema formation and unresorbed pre-existing skeletal elements, limb regeneration still occurs and the only phenotype is a relatively minor defect in skeletal integration. It is possible that zol-induced defect in blastema formation is not directly linked to the failure of integration at a later stage.
3. As an integration failure of the newly formed skeleton still occurs in untreated animals, it is not entirely clear how the authors can attribute this defect to a lack of bone resorption. More quantitative analyses would be necessary to demonstrate the correlation between zol treatment and lack of integration. -
