A regeneration-triggered metabolic adaptation is necessary for cell identity transitions and cell cycle re-entry to support blastema formation and bone regeneration
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Evaluation Summary:
The authors provide compelling evidence to show that injury induces activation of glycolysis during zebrafish adult tail fin regeneration. This early activation is crucial for osteoblast dedifferentiation and proliferation, which are required for blastema formation and tail fin regeneration. However, additional data are required to support the claim of a "metabolic switch" from oxidative phosphorylation to glycolysis. This study will be of interest to a broad audience in the fields of regeneration and metabolic regulation of developmental processes.
(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 #2 agreed to share their name with the authors.)
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Abstract
Regeneration depends on the ability of mature cells at the injury site to respond to injury, generating tissue-specific progenitors that incorporate the blastema and proliferate to reconstitute the original organ architecture. The metabolic microenvironment has been tightly connected to cell function and identity during development and tumorigenesis. Yet, the link between metabolism and cell identity at the mechanistic level in a regenerative context remains unclear. The adult zebrafish caudal fin, and bone cells specifically, have been crucial for the understanding of mature cell contribution to tissue regeneration. Here, we use this model to explore the relevance of glucose metabolism for the cell fate transitions preceding new osteoblast formation and blastema assembly. We show that injury triggers a modulation in the metabolic profile at early stages of regeneration to enhance glycolysis at the expense of mitochondrial oxidation. This metabolic adaptation mediates transcriptional changes that make mature osteoblast amenable to be reprogramed into pre-osteoblasts and induces cell cycle re-entry and progression. Manipulation of the metabolic profile led to severe reduction of the pre-osteoblast pool, diminishing their capacity to generate new osteoblasts, and to a complete abrogation of blastema formation. Overall, our data indicate that metabolic alterations have a powerful instructive role in regulating genetic programs that dictate fate decisions and stimulate proliferation, thereby providing a deeper understanding on the mechanisms regulating blastema formation and bone regeneration.
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Author Response
Reviewer #2 (Public Review):
In the report by Brandao A. et al the authors used a zebrafish adult tail fin regeneration model to elucidate the role of metabolic adaptation in cell fate transition and cell proliferation during regeneration with a focus on bone regeneration. Firstly, the authors used transgenic reporter bglap:GFP to label mature osteoblasts and co-immunostaining with a pre-osteoblast marker runx2 to show that within 6 hours post amputation, osteoblasts show signs of dedifferentiation giving rise to pre-osteoblasts that re-enter the cell cycle between 12 - 24 hpa. The authors then use evidence from gene expression changes, metabolomic analysis, pharmacological perturbation experiments, cell proliferation analysis and histological detection of lineage markers to demonstrate that an immediate metabolic …
Author Response
Reviewer #2 (Public Review):
In the report by Brandao A. et al the authors used a zebrafish adult tail fin regeneration model to elucidate the role of metabolic adaptation in cell fate transition and cell proliferation during regeneration with a focus on bone regeneration. Firstly, the authors used transgenic reporter bglap:GFP to label mature osteoblasts and co-immunostaining with a pre-osteoblast marker runx2 to show that within 6 hours post amputation, osteoblasts show signs of dedifferentiation giving rise to pre-osteoblasts that re-enter the cell cycle between 12 - 24 hpa. The authors then use evidence from gene expression changes, metabolomic analysis, pharmacological perturbation experiments, cell proliferation analysis and histological detection of lineage markers to demonstrate that an immediate metabolic switch from OXPHOS to glycolysis precedes blastema formation in amputated tail fin stump. Importantly, blocking glycolysis with 2-DG suppressed mature osteoblast dedifferentiation and proliferation as well as blastema formation which resulted in failure of tail fin regeneration. In summary, this study has shown that a rapid metabolic switch from OXPHOS to glycolysis immediately after tissue damage is important for subsequent bone regeneration, and more specifically the authors provide evidence to show that glycolysis is required for both dedifferentiation and for cell proliferation, both processes are crucial for appropriate blastema formation.
This study established that metabolic switch is an early response to tissue damage and metabolic adaption is key for cellular responses during bone regeneration. Conclusions of this study are well supported by data provided. There are some details of data mentioned in the text should be clarified.
The authors used Microarray transcriptome analysis to demonstrate dynamic gene expression response in 6hpa OBs compared to 0hpa OBs and stated in page 6 line 154 - 159 that at 6hpa, OBs undergo dramatic gene expression changes with 2200 differentially expressed genes, and a set of genes related to energy metabolism was also dramatically altered. However, in supplement figure 1 there was no mention of which genes related to energy metabolism are altered, there is no real data as to what kind of gene expression changes are happening in 6hpa OBs, because DE gene list is not provided. Do the gene expression changes reflect partial dedifferentiation of OBs? The authors should provide more details of their microarray analysis, or at least provide the DE gene list.
We are thankful for the reviewer positive and constructive comments regarding our experimental work and the feedback for the improvement of the manuscript. We just want to point out to the reviewer that the data on Fig 2B relates to major glycolytic enzymes and OXPHOS components retrieved from the Osteoblast ArrayXS and that, as it was mentioned in the methods section (Lines 714-715), we submitted to NCBI Gene Expression Omnibus archive the transcriptome datasets analysed on this study (accession number GSE194385).
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Evaluation Summary:
The authors provide compelling evidence to show that injury induces activation of glycolysis during zebrafish adult tail fin regeneration. This early activation is crucial for osteoblast dedifferentiation and proliferation, which are required for blastema formation and tail fin regeneration. However, additional data are required to support the claim of a "metabolic switch" from oxidative phosphorylation to glycolysis. This study will be of interest to a broad audience in the fields of regeneration and metabolic regulation of developmental processes.
(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 #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Zebrafish fin serves as an ideal model of tissue regeneration study. Injury triggers dedifferentiation of preexisting differentiated cells to reprogram to the cell type-specific progenitors, which form blastema and replenish the lost tissues. Brandao et al. employed zebrafish reporter lines, imaging, metabolomics, pharmaceutical assays and fin regeneration assays to investigate the relationship between metabolic reprogramming and tissue regeneration. In particular, the authors target osteoblasts (OBs) as the main cell type in fin tissues. Fin amputation injury triggers a metabolic shift to glycolysis prior to blastema formation, which is confirmed by metabolomics via LC-MS analysis. This metabolic shift likely occurs via transcriptional changes of glycolytic enzymes. Inhibition of glycolysis by treating …
Reviewer #1 (Public Review):
Zebrafish fin serves as an ideal model of tissue regeneration study. Injury triggers dedifferentiation of preexisting differentiated cells to reprogram to the cell type-specific progenitors, which form blastema and replenish the lost tissues. Brandao et al. employed zebrafish reporter lines, imaging, metabolomics, pharmaceutical assays and fin regeneration assays to investigate the relationship between metabolic reprogramming and tissue regeneration. In particular, the authors target osteoblasts (OBs) as the main cell type in fin tissues. Fin amputation injury triggers a metabolic shift to glycolysis prior to blastema formation, which is confirmed by metabolomics via LC-MS analysis. This metabolic shift likely occurs via transcriptional changes of glycolytic enzymes. Inhibition of glycolysis by treating several drugs impairs OB dedifferentiation and proliferation, resulting in defective blastema formation and fin regeneration. The molecular and cellular data are of high quality and the manuscript is well-written. However, there are two major weaknesses. First, although the negative impact of glycolysis inhibition is nicely demonstrated, the positive impact of glycolysis activation or oxidative phosphorylation (OXPHOS) inhibition is not proved. Second, the authors likely misunderstand blastema compartmentalization as distal blastema (DB), proximal blastema (PB), and patterning zone (PZ) are incorrectly defined. Overall, this is an interesting and significant work implicating metabolic reprogramming and regeneration but a number of points need to be addressed.
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Reviewer #2 (Public Review):
In the report by Brandao A. et al the authors used a zebrafish adult tail fin regeneration model to elucidate the role of metabolic adaptation in cell fate transition and cell proliferation during regeneration with a focus on bone regeneration. Firstly, the authors used transgenic reporter bglap:GFP to label mature osteoblasts and co-immunostaining with a pre-osteoblast marker runx2 to show that within 6 hours post amputation, osteoblasts show signs of dedifferentiation giving rise to pre-osteoblasts that re-enter the cell cycle between 12 - 24 hpa. The authors then use evidence from gene expression changes, metabolomic analysis, pharmacological perturbation experiments, cell proliferation analysis and histological detection of lineage markers to demonstrate that an immediate metabolic switch from OXPHOS to …
Reviewer #2 (Public Review):
In the report by Brandao A. et al the authors used a zebrafish adult tail fin regeneration model to elucidate the role of metabolic adaptation in cell fate transition and cell proliferation during regeneration with a focus on bone regeneration. Firstly, the authors used transgenic reporter bglap:GFP to label mature osteoblasts and co-immunostaining with a pre-osteoblast marker runx2 to show that within 6 hours post amputation, osteoblasts show signs of dedifferentiation giving rise to pre-osteoblasts that re-enter the cell cycle between 12 - 24 hpa. The authors then use evidence from gene expression changes, metabolomic analysis, pharmacological perturbation experiments, cell proliferation analysis and histological detection of lineage markers to demonstrate that an immediate metabolic switch from OXPHOS to glycolysis precedes blastema formation in amputated tail fin stump. Importantly, blocking glycolysis with 2-DG suppressed mature osteoblast dedifferentiation and proliferation as well as blastema formation which resulted in failure of tail fin regeneration. In summary, this study has shown that a rapid metabolic switch from OXPHOS to glycolysis immediately after tissue damage is important for subsequent bone regeneration, and more specifically the authors provide evidence to show that glycolysis is required for both dedifferentiation and for cell proliferation, both processes are crucial for appropriate blastema formation.
This study established that metabolic switch is an early response to tissue damage and metabolic adaption is key for cellular responses during bone regeneration. The conclusions of this study are well supported by data provided. There are some details of data mentioned in the text that should be clarified.
The authors used Microarray transcriptome analysis to demonstrate dynamic gene expression response in 6hpa OBs compared to 0hpa OBs and stated on page 6 line 154 - 159 that at 6hpa, OBs undergo dramatic gene expression changes with 2200 differentially expressed genes, and a set of genes related to energy metabolism was also dramatically altered. However, in supplement figure 1 there was no mention of which genes related to energy metabolism are altered, there is no real data as to what kind of gene expression changes are happening in 6hpa OBs, because DE gene list is not provided. Do the gene expression changes reflect partial dedifferentiation of OBs? The authors should provide more details of their microarray analysis, or at least provide the DE gene list.
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