Editor's evaluation: A regeneration-triggered metabolic adaptation is necessary for cell identity transitions and cell cycle re-entry to support blastema formation and bone regeneration (2022)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation The authors provide convincing 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. This important study will be of interest to a broad audience in the fields of regeneration and metabolic regulation of developmental processes. https://doi.org/10.7554/eLife.76987.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The zebrafish (Danio rerio) is a well-established model to study vertebrate regeneration due to its capacity to efficiently regenerate multiple organs and complex tissues, such as the caudal fin (Sehring and Weidinger, 2020; Antos et al., 2016; Marques et al., 2019). Caudal fin regeneration is an epimorphic process dependent on the formation of a blastema, a transient structure composed of proliferative lineage-restricted progenitor cells that originate from mature cells of the uninjured tissue (Kawakami, 2010; Poss et al., 2003). One of the main components of the caudal fin are the segmented bone elements, or bony-rays (Pfefferli and Jaźwińska, 2015; Marí-Beffa and Murciano, 2010), which have been shown to regenerate from new osteoblasts (OBs) arising from dedifferentiation of mature OBs close to the amputation site. Mature OBs acquire the cellular properties of less differentiated cells or pre-osteoblasts (pre-OBs) (Knopf et al., 2011; Sousa et al., 2011; Stewart and Stankunas, 2012; Tu and Johnson, 2011), which proliferate and redifferentiate into mature bone-producing OBs, thereby reconstituting the fin skeletal tissue (Stewart et al., 2014; Brandão et al., 2019; Brown et al., 2009; Wehner et al., 2014). Mammals, in contrast to zebrafish, have a poor ability to regenerate (Xia et al., 2018; Poss, 2010; Tanaka and Reddien, 2011). Mammalian bone, in particular, has an intrinsic capacity to be remodeled throughout life and, to a certain extent, repair after fracture (Salhotra et al., 2020; Raggatt and Partridge, 2010), but it fails to fully regenerate. This is mainly achieved through activation of signalling cascades that culminate in the recruitment and differentiation of osteoprogenitors into OBs, the bone-forming cells. In this scenario, understanding how OBs and OB sources are activated and recruited in zebrafish can greatly contribute to new therapeutic solutions to improve bone formation and restoration in mammals (Yao et al., 2013). Activation of reprogramming events that allow cells to change fate in response to injury appear to be conserved biological processes, which are used to prompt tissue regeneration in several organisms (Tanaka and Reddien, 2011; Tanaka, 2016; Jopling et al., 2011), including in mammals (Seifert and Muneoka, 2018; Johnson et al., 2020). Therefore, it is crucial to identify the early molecular events inducing cell dedifferentiation to promote tissue regeneration. Cell identity and functional state often reflect a specific metabolic profile that depends, for instance, on nutrient and oxygen availability, and on bioenergetics and biomass requirements (Folmes et al., 2012; Ito and Suda, 2014). Metabolic routes serve not only the crucial purpose of converting or use energy to maintain cellular integrity and survival, but also have a pivotal role in restructuring gene expression to determine cell identity and function by influencing cell signalling and epigenetic modulators (Tarazona and Pourquié, 2020; Ly et al., 2020). Glucose metabolism is currently regarded as a powerful instructor of cell fate decisions (Wei et al., 2018; Tatapudy et al., 2017; Ghosh-Choudhary et al., 2020) during development. It is well documented that stem cells and their differentiated progeny have distinct metabolic preferences: stem cells often favour non-oxidative glycolysis, while differentiated non-dividing somatic cells rely on mitochondrial oxidative phosphorylation (OXPHOS) (Shyh-Chang and Daley, 2015; Panopoulos and Izpisua Belmonte, 2011; Tsogtbaatar et al., 2020; Mathieu and Ruohola-Baker, 2017). Importantly, the metabolic signature is not static and can rapidly switch according to the cellular demands, a phenomenon commonly designated as metabolic reprogramming (Folmes et al., 2012; Ghosh-Choudhary et al., 2020). This is particularly relevant under certain environmental conditions when tissue homeostasis is breached, such as during inflammation, and during disease. Both activated T cells and pro-inflammatory macrophages switch metabolic profiles compatible with a prevalence of aerobic glycolysis (Shyer et al., 2020; Viola et al., 2019). The shift to aerobic glycolysis is also observed in cancer cells, where it is referred as the Warburg effect (DeBerardinis et al., 2008; Intlekofer and Finley, 2019; Warburg et al., 1927; Liberti and Locasale, 2016; Vander Heiden et al., 2009). Despite the considerable amount of information on the role of metabolism during development and disease (Vander Heiden et al., 2009; Lee et al., 2017; DeBerardinis and Thompson, 2012; Tzika et al., 2018; Bettencourt and Powell, 2017), the link between glucose metabolism and regeneration is still far from understood. Shifts in metabolism leading to a prevalence of glycolysis have already been observed in planarians and amphibians during regeneration (Osuma et al., 2018; Love et al., 2014; Alibardi, 2014; Varela-Rodríguez et al., 2020). More recently, glycolysis was proposed to modulate specific aspects of regeneration in the zebrafish, specifically in the larval tail (Sinclair et al., 2021) and in the heart (Honkoop et al., 2019; Fukuda et al., 2020). Nevertheless, much remains to be elucidated on how metabolism influences changes in cell identity necessary to promote tissue regeneration. Here, we use the adult zebrafish caudal fin to determine the role of energy metabolism in mature OB dedifferentiation and pre-OB recruitment. By performing a series of gene expression and metabolomic studies, we observed that changes in the metabolic signature triggered upon amputation are at the core of the initial set-up of the cellular programmes that control caudal fin regeneration. These data indicate that mature OBs, and possibly other cell lineages in the caudal fin, undergo a metabolic adaptation that increases glycolysis. We further demonstrate that these changes in metabolism are necessary for mature OBs to commit to a specialized genetic program that enables them to dedifferentiate, proliferate and act as progenitor cells, ensuring proper bone regeneration. Taken together, our results demonstrate that metabolic reprogramming is one of the earliest described cellular events that dictate adult caudal fin and bone regeneration. Results Osteoblast dedifferentiation occurs before blastema formation and during the wound healing phase Caudal fin bone regeneration is achieved through activation of cell sources, such as mature OBs, that change their identity to generate new OBs. Under homeostatic conditions, mature OBs reside as quiescent bone-lining cells, but lose their differentiated character when undergoing dedifferentiation (Knopf et al., 2011; Sousa et al., 2011). This process is characterized by the downregulation of mature markers, such as bone gamma-carboxyglutamic acid-containing protein (bglap) at 12 hpa (Knopf et al., 2011; Sousa et al., 2011; Long, 2011), and upregulation of the pre-OB marker, runx2, at 24 hpa (Knopf et al., 2011; Sousa et al., 2011; Salhotra et al., 2020). OBs undergoing dedifferentiation detach from the bony-ray surface via an EMT-like event, migrate toward the stump region, re-enter the cell cycle and form a pool of pre-OB cells that incorporates the blastema (Figure 1A; Knopf et al., 2011; Sousa et al., 2011; Stewart et al., 2014). To determine if OBs show signs of dedifferentiation in early stages of regeneration, we increase the time resolution of the initial OB response to amputation. First, we analyze the relative expression of mature (bglap) and pre-OB (runx2a, runx2b) markers 6 hr post-amputation (hpa) in regenerating caudal fins in relation to uninjured caudal fins (0 hpa). We observed a downregulation of bglap and upregulation of runx2a (Figure 1B) suggesting that, in contrast to previous studies (Knopf et al., 2011; Sousa et al., 2011), mature OBs are already undergoing transcriptional changes consistent with dedifferentiation as early as 6 hpa. We then analyzed the bglap:EGFP transgenic line which, due to the stable GFP signal, allows to follow mature OB for long periods even upon bglap downregulation (Sousa et al., 2011). Using live-imaging approaches, we monitored the two segments below the amputation plane of each bony-rays and performed a time-course analysis of OB migration every 5 hr during the first 25 hpa (Figure 1C and D). Tracking of bglap-positive (Bglap+) OBs revealed that OBs from the segment below amputation (segment 0) become motile at the 5–10 hpa time-interval and in average reach the amputation plane around 20–25 hpa (Figure 1C and D), while Bglap +OB from the second segment (segment-1) remain predominantly immotile (Figure 1C). Subsequently, we assessed when OBs acquire proliferative capacity. Immunofluorescence for PCNA (marker of G1 phase) indicated that the proportion of Bglap +OBs entering the cell cycle progressively increases, specifically from 12 to 24 hpa (Figure 1E). At 24 hpa, almost 80% of Bglap +OBs in the first segment have entered the G1 phase (Figure 1E). To examine when OBs begin to show signs of dedifferentiation toward a pre-OB state, we performed immunofluorescence for Runx2 in bglap:EGFP transgenic fins. In homeostasis conditions (0 hpa), Runx2 is observed in the nucleus of bone-lining mature OB (Runx2 +Bglap + ; Figure 1F, F' and H), which is in accordance with Runx2 being expressed at basal levels in fully differentiated OB (Conaway et al., 2013). At 12 and 24 hpa, we observed a slight decrease in the total number of Runx2 +Bglap + OB population within the first segment below amputation (Figure 1G, G' and H). OBs are not described to undergo apoptosis at this stage (Knopf et al., 2011), suggesting that decrease in the Runx2 +Bglap + OBs could be due to actual loss of the bglap differentiation marker in a portion of dedifferentiated OB. Nevertheless, remaining Runx2 +Bglap + possess higher levels of Runx2 when compared to Runx2 +Bglap + OBs at 0 hpa (Figure 1F–G' arrows), further indicating that they converted into a pre-OB phenotype, in accordance with published data (Mishra et al., 2020). In addition, at 0 hpa a small number of pre-OB (Runx2 +Bglap-) was detected in the bony-rays intersegment/joint (Figure 1F and F' asterisk,I), which may correspond to a population of OB progenitors (OPs) recently identified in this region, refered as joint OP (Ando et al., 2017). At 12 and 24 hpa, we observed an increase in the number of Runx2 +Bglap cells, suggesting that pre-OB arise in the first 12 hpa and their numbers increase until the beginning of blastema formation at 24 hpa (Figure 1F-G' and I). Altogether, these data indicate that mature OBs lose their differentiated character and contribute to the pre-OB pool in a time-window between 12 and 24 hpa. Nevertheless, we cannot exclude that pre-OB can also arise from the joint-associated OB progenitor niche. Thus, we hypothesize that at 24 hpa, multiple OB sources are recruited to contribute to the blastema (Figure 1J). Figure 1 Download asset Open asset Osteoblast dedifferentiation time-window during caudal fin regeneration. (A) Biological traits of OB dedifferentiation process. (B) Relative gene expression of mature (green) and pre-OB (magenta) markers, at 6 hpa relative to 0 hpa. Statistical analysis on graph corresponds to paired t-test with Welch's correction. Mean ± SD are displayed (n=4 biological replicates). (C) Live imaging analysis of OB motility in bglap:EGFP fish (green) during the first 25 hpa, highlighted in the segment bellow amputation (segment 0) and segment –1. Bony-rays are labeled with Alizarin red (magenta). White dashed lines delineate the intersegment region. (D) Quantification of the relative OB displacement in segment 0. Statistical analysis displayed on graph corresponds to Kruskal-Wallis test with Mean ± SD (n=9–18 bony-rays). (E) Percentage of proliferating OBs through immunofluorescence against PCNA in bglap:EGFP fish. Statistical analysis displayed on graph corresponds to Kruskal-Wallis test with Mean ± SD (n=9–13 cryosections). (F-G') Representative cryosection images of bglap:EGFP (green) fins immunostained for Runx2 (magenta) and counterstained with DAPI (blue), in (F, F') uninjured fish and (G, G') at 24 hpa; arrows indicate Runx2 +Bglap + cells and dashed arrows indicate Runx2 +Bglap cells. (H, I) Quantification of (H) Runx2 +Bglap + and (I) Runx2 +Bglap cells during the first 24hpa. Statistical analysis displayed on graph corresponds to Mann-Whitney test with Mean ± SD (n=18–27 cryosections). (J) Cellular sources that contribute for new pre-OBs formation after injury include mature osteoblasts and potentially joint OP. White arrowhead indicates amputation plane and dashed squares represent magnified panels in F' and G'. E: epidermis; b: bone; m: mesenchyme; ns: not significative; *p<0,05; **p<0,01; ***p<0,001; ****p<0,0001. Scale bars represent 100 µm and 30 µm in magnified panels. See Figure 1—source data 1. Figure 1—source code 1 MatLab scripts quantify the relative osteoblast displacement after caudal fin amputation. https://cdn.elifesciences.org/articles/76987/elife-76987-fig1-code1-v1.zip Download elife-76987-fig1-code1-v1.zip Figure 1—source data 1 Spreadsheets detailing the results regarding the characterization of osteoblast dedifferentiation through caudal fin regeneration, specifically (B) the relative gene expression analysis, (E) the percentage of proliferating osteoblast, and (H,I) the quantification of Runx2+Bglap + and Runx2+Bglapcells during the first 24hpa. https://cdn.elifesciences.org/articles/76987/elife-76987-fig1-data1-v1.xlsx Download elife-76987-fig1-data1-v1.xlsx Here, we show that mature OB dedifferentiation is an early response to injury and entails important transcriptional and phenotypic alterations, occurring in a narrow time-window between 6–12 hpa, before blastema induction and during the wound healing phase. Metabolic adaptation toward glycolysis occurs at early stages of fin regeneration prior to blastema formation Having established that the first hours after caudal fin amputation are crucial for the activation of OBs, we proceeded with the identification of the initial regulators of OB dedifferentiation. We isolated OBs from the first bony-ray segment below amputation at 0 hpa, our control population and closest to a homeostatic state, and at 6 hpa, when most dedifferentiation features are not detected yet, and performed a genome-wide transcriptomic analysis (zebrafish 8x60 K ArrayXS technology) (Figure 2—figure supplement 1A). OBs were isolated by FACS using the bglap:EGFP transgenic line (Figure 2—figure supplement 1B). By comparing the expression profiles of mature OBs (0 hpa) and dedifferentiating OBs (6 hpa), we evaluated whether our set of differentially expressed (DE) genes was associated with a specific biological process or signalling pathway, particularly relevant for OB dedifferentiation. We observed that OBs show a dynamic transcriptional response at 6 hpa in comparison to OBs from uninjured conditions with almost 2200 differentially expressed genes, from which 1040 were downregulated and 1130 were upregulated (Figure 2—figure supplement 1C,D). These data further demonstrates that the dedifferentiation machinery is triggered very early during regeneration (for more details about the set of DE genes see Supplementary file 1). Importantly, a large set of genes related to energy metabolism was also dramatically altered (Supplementary file 1). Depending on the energy and biomass demands, cells can uptake glucose and, through glycolysis, use it to produce pyruvate, which can serve as a substrate to: generate acetyl-CoA and fuel mitochondrial OXPHOS, obtaining a high energy yield; or to produce lactate, allowing diversion of metabolic intermediates from glycolysis toward various biosynthetic pathways (Lunt and Vander Heiden, 2011; Rabinowitz and Enerbäck, 2020; Figure 2A). From our transcriptome data set of DE, we observed that at 6 hpa OBs upregulate the expression of major glycolytic enzymes, such as hexokinase 1 (hk1) and phosphofructokinase (pfkp), whereas OXPHOS components remain mostly unchanged (Figure 2B, Supplementary file 1). Most importantly, we detected a significant increase in lactate dehydrogenase (ldha) expression, suggesting an increase in lactate production, possibly by diverting pyruvate from mitochondrial oxidation. (Figure 2B, Supplementary file 1). These data suggest that dedifferentiating OBs change their metabolic signature, adopting a metabolic program that increases glycolysis. Since OBs only account for around 1–3% of the total cell number in a bony-ray segment (Figure 2—figure supplement 1B), we evaluated whether these changes in gene expression were specific to OB undergoing dedifferentiation or are also observed as a general behavior induced upon injury. For that, we collected the first segment below the amputation and analyzed by qPCR the expression profile of key glycolytic and OXPHOS genes in regenerating caudal fins from 6 hpa and 24 hpa and compared to control uninjured caudal fins (0 hpa). At 6 hpa, when cells start to dedifferentiate, most glycolytic enzymes were highly upregulated, including hk1, hk2, pfkpa, and pyruvate kinase (pkma) (Figure 2C). Importantly, as observed during OB dedifferentiation, ldha expression was increased and pyruvate dehydrogenase a 1b (pdha1b) was downregulated. pdha1b is part of the Pdh1 complex, which catalyzes irreversibly the conversion of pyruvate to acetyl-CoA and progression to OXPHOS, further corroborating that at this stage pyruvate is being shunt from the mitochondria and stimulating glycolysis (Figure 2C). for the OXPHOS components most unchanged or this increase in expression was not as as the one observed for the glycolytic (Figure 2C). at 24 hpa, when most cells have dedifferentiated and the blastema to be we observed that most glycolytic enzymes were still upregulated, not as as at 6 hpa and ldha expression to be upregulated (Figure At this we also observed an increase in expression of OXPHOS components (Figure suggesting that blastema formation may also mitochondrial glucose oxidation. upregulation of the genes associated with glycolysis and lactate that the OB dedifferentiation process and possibly the early response to amputation are characterized by a metabolic adaptation toward glycolysis. Figure with see Download asset Open asset Metabolic adaptation is triggered during zebrafish caudal fin regeneration. (A) of glucose (B) OB gene expression profile of glycolytic enzymes ldha (magenta) and OXPHOS components at 6 hpa relative to uninjured conditions (0 hpa) from the OB The the and on a Statistical analysis with test and Welch's biological Mean ± SD are Relative gene expression of glycolytic enzymes ldha (magenta) and components (blue), in the fin at (C) 6 hpa and at (D) 24 hpa in comparison to uninjured conditions (0 hpa). Statistical analysis with paired test (C) and (D) biological replicates). (E) at 6 hpa and 24 hpa in relation to uninjured conditions (0 hpa), in the fin Statistical analysis with Mann-Whitney test (n=4 biological replicates). Live imaging of uptake (green) in fish (magenta) at 0 hpa and 24 hpa. indicate uptake of in the intersegment White dashed line the Scale of uptake in close and to the amputation at 0 hpa and 24 hpa. indicate of uptake in the of uptake at 0 hpa and 24 hpa. Statistical analysis on graph corresponds to Mann-Whitney Mean ± SD are displayed and bony-rays). ns: not significative; See Figure data 1. Figure data 1 Spreadsheets detailing the results of the metabolic specifically the quantification of the relative gene expression analysis, (E) the relative and the average levels of Download Nevertheless, gene expression data may not reflect an actual activation of a specific metabolic Therefore, to these we used to quantify specific and determine the energy metabolism For that, we the fin stump and analyzed the amount of lactate, and at and 24 hpa. with our transcriptome we observed an increase in glucose and lactate at 6 and 24 hpa in relation to control fins (0 hpa), which is consistent with an in the glycolytic (Figure We also observed a increase of and at 24 hpa but not at 6 hpa (Figure and act as important for protein and necessary to cellular integrity and suggesting an increase in et al., et al., 2020). We also monitored glucose uptake during regeneration using a glucose In control fins (0 hpa), we detected glucose uptake in the intersegment (Figure where a population of OP have been identified (Ando et al., 2017), which may that under homeostatic conditions, these cells higher levels of glucose to maintain the progenitor niche. At 24 hpa, we observed a significant increase of glucose uptake in the blastema and in the first segment below the amputation (Figure and metabolic changes are associated to mitochondria and which control mitochondria and et al., 2013). between and allows mitochondria to acquire the structure to specific cellular and OXPHOS for energy in differentiated cells by of the mitochondrial characterized by long mitochondria In and mitochondria and is associated with and with cells that rely on a glycolytic metabolism et al., 2015; et al., 2018; et al., the increase in the glycolytic we to how mitochondria were during regeneration. By performing a qPCR analysis at and 24 hpa, we observed an increase in expression of genes associated with mitochondrial and at 6 hpa but not at 24 hpa, when compared to uninjured (0 hpa) (Figure 2—figure supplement By the number of mitochondria using the line which the mitochondria et al., we an increase in the number of mitochondria cell at 6 and 24 hpa, in relation to uninjured control (Figure 2—figure supplement To if were also changes in mitochondria that could correspond to an actual increase in we mitochondria and them into between and but their and can et al., and In accordance with an increase in mitochondria we observed a significant increase in percentage of mitochondria at 6 hpa, while at 24 hpa the mitochondria profile is to the 0 hpa (Figure 2—figure supplement Taken together, these results are in consistent with an increase in mitochondrial triggered by amputation and further the that the early stages of regeneration an upregulation of glycolytic which is by a program of mitochondria Thus, metabolic adaptation glycolysis to an response to tightly in of time and transcriptome and studies indicate a specific metabolic signature as part of an early response to amputation by OBs and by the remaining caudal fin Overall, cells respond to injury by the glycolytic lactate and mitochondrial preceding dedifferentiation and blastema formation. These results the that amputation induces a reprogramming of the metabolic which may be part of a general program that the regenerative and lactate blastema formation during caudal fin regeneration To the functional relevance of the energy we to start with a analysis in which we specific of energy metabolism with and its for blastema formation (Figure The blastema is a and of epimorphic regeneration and is fully within hpa (Kawakami, 2010; Poss et al., and Jaźwińska, we glycolysis using an established glucose (Sinclair et al., et al., that with glucose for the first of glycolysis, the from was in time after amputation to stages of blastema for a before blastema formation (0 hpa and or for a which the blastema phase and were at hpa to determine the effect of on blastema by the total fin and the percentage of in relation to control caudal fin (Figure We observed that the higher the of the the the effect on blastema in comparison to control fins (Figure and a of at 0 hpa already significant when compared to control fins (Figure of between and hpa a effect on the (Figure and of the glycolytic for the first hpa led to a complete abrogation of blastema (Figure and the glycolysis of which the glycolytic of et al., Figure also a general of caudal fin regeneration, as observed at hpa in relation to