(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 …
MatLab scripts quantify the relative osteoblast displacement after caudal fin amputation.
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.
(A) Schematic representation of glucose metabolism. (B) OB gene expression profile of glycolytic enzymes (green), ldha (magenta) and OXPHOS components (blue) at 6 hpa relative to uninjured …
Spreadsheets detailing the results of the metabolic adaptation, specifically the quantification of (C,D) the relative gene expression analysis, (E) the relative metabolite levels, and (N) the average fluorescent levels of 2-NBDG.
(A) Schematic representation of the experimental design used to obtain the transcriptional profile of OBs undergoing dedifferentiation, using bglap:EGFP reporter line. OBs from caudal fin tissue …
(A, B) Relative gene expression of genes related to mitochondrial fission, at (A) 6 hpa and (B) 24 hpa, in comparison to uninjured condition (0 hpa). Statistical analysis corresponds to Paired …
Spreadsheets detailing the results of the mitochondrial dynamics, specifically (A,B) the relative gene expression analysis, and the quantification of (I) the number of mitochondria per cell and (J) the percentage of each mitochondria volume.
(A) Schematic representation of the compounds used to manipulate glucose metabolism. (B) Experimental design used to inhibit the glycolytic influx during fin regeneration. Control and treated fish …
Spreadsheets detailing the results of glycolysis and OXPHOS inhibitory assays, specifically the quantification of (C) the percentage of regenerated fin area after 2DG treatment, (O) the percentage of regenerated fin area after S.O. treatment, and (S) the percentage of regenerated fin area after UK5099 treatment.
(A) Experimental design used to inhibit glycolysis during fin regeneration. Fish are incubated with control vehicle (PBS) or with the glycolytic inhibitor 3PO, every 24 hr from caudal fin amputation …
Spreadsheets detailing the results of alternative glycolysis and OXPHOS inhibitory assays, specifically the quantification of the percentage of the regenerated fin area after (D) 3PO and (H) MB6 treatment.
(A) Schematic representation of pre-OBs formation during regeneration. Pre-OBs arise from OB dedifferentiation and potentially from the joint OP niche. OB dedifferentiation is correlated with …
Spreadsheets detailing the results of the impaired osteoblast dedifferentiation after glycolysis inhibition with 2DG, specifically (C) the relative gene expression analysis, and the quantification of (F) the number of Runx2 +Bglap + and Runx2 +Bglap cells, and (G) the percentage of osteoblasts subtypes (Runx2 +Bglap + and Runx2 +Bglap-).
(A–D) Representative images of bglap:EGFP caudal fins at 24 hpa, treated with (A–B) vehicle (PBS) or (C–D) 2DG. Double white arrows indicate the anterior (A) and posterior (P) axis. White dashed …
MatLab scripts to quantify the relative osteoblast displacement after caudal fin amputation in controls and after 2DG treatment.
Spreadsheets detailing the results of impaired osteoblasts cell cycle re-entry after glycolysis inhibition with 2DG, specifically the quantification of (H) the number of Runx2 +Bglap + and Runx2 +Bglap- EdU + cells, (I) the percentage of Runx2 +Bglap + and Runx2 +Bglap- EdU + cells and (J) the relative gene expression analysis.
(A–F) Representative 24 hpa cryosection images of bglap:EGFP (orange) caudal fins immunostained for PCNA (magenta), EdU (green) and counterstained with DAPI (blue), in fish treated with (A–C) …
Spreadsheets detailing the results of impaired cell cycle re-entry after glycolysis inhibition on individual fin tissues, specifically the quantification of the number of PCNA +and EdU + cells in the (G) epidermis and (H) mesenchyme after 2DG treatment, and (I) the number of PCNA + cells in the epidermis, mesenchyme, and osteoblasts after 3PO treatment.
(A–H) Representative 24 hpa cryosection images of osx:mCherry (orange) caudal fins immunostained for Runx2 (magenta), TUNEL (green) and counterstained with DAPI (blue), in fish treated with (A–D) …
Spreadsheets detailing the results of the impact of glycolysis inhibition, after 2DG treatment, on pre-osteoblasts cell death, specifically the quantification of the number of TUNEL + cells in the (I) epidermis, (J) mesenchyme and (K) pre-osteoblasts, and (L) the percentage of TUNEL +osteoblasts.
(A) Experimental design used to inhibit glycolysis. Fish are administered, via IP injection, with control (PBS) or 2DG every 12 hr, from fin amputation (0 hpa) until 48 hpa. (B) Schematic …
Spreadsheets detailing the results of the formation of osteoblast subtypes and proliferation within the blastema after glycolysis inhibition with 2DG, specifically the quantification of the number of (E) Runx2 +Osx- pre-osteoblasts and (F) Runx2 +Osx + osteoblasts, (G) the percentage of osteoblasts subtypes, (K) the total number of Runx2 +Osx + and Runx2 +Osx- EdU +osteoblast subtypes, and (K) the percentage of proliferative Runx2 +Osx + and Runx2 +Osx- osteoblast subtypes.
(A–H) Representative 48 hpa caudal fin images of osx:mCherry (magenta) and runx2:EGFP (green) double transgenics treated with (A–D) vehicle (PBS) or (E–H) 2DG. Dashed lines delineate regenerated …
Spreadsheets detailing the results of the distribution of osteoblast subtypes in the blastema after glycolysis inhibition with 3PO, specifically the quantification of the number of (K) Runx2 +Osx and (L) Runx2 +Osx + cells.
(A–F) Representative 48 hpa cryosection images of osx:mCherry (orange) caudal fins immunostained for PCNA (magenta), EdU (green) and counterstained with DAPI (blue), in fish treated with (A–C) …
Spreadsheets detailing the results of cell proliferation after glycolysis inhibition with 2DG, specifically the quantification of the number of EdU +and PCNA + cells in the (G) epidermis and (H) mesenchyme.
(A,B) Representative cryosection images of 48 hpa bglap:EGFP (green) caudal fins immunostained for Runx2 (magenta) and counterstained with DAPI (blue), in fish treated with (A, A) PBS and (B, B’) …
Spreadsheets detailing the results of mature osteoblasts accumulation in the stump region after inhibition of glycolysis with 2DG, specifically (C) the quantification of the number of Runx2 +Bglap + cells.
(A, B) Experimental design used for extended inhibition of the glycolytic influx during (A) fin regeneration and in (B) uninjured caudal fins. Control and treated fish are administered, via IP …
Spreadsheets detailing the results of inhibiting glycolysis with 2DG in uninjured caudal fins, specifically the quantification of (L) fin area to width ratio, and (N) the average of bony-ray length to width ratio.
(A) In homeostasis, mature OBs reside in close contact with the bony-ray surface, secreting the collagenous bone matrix. (B) Upon caudal fin amputation, OBs and other cell types in the regenerating …
Statisical analysis with t test (n=5 (A) and 4 (B) biological replicates).
Mean and SD displayed on the graphs. n.s., not significative, *, p-value <0,01.
Spreadsheet detailing the list of significantly differentially expressed genes with p-value lower than the 0.05 threshold and log2 fold change < –1 or >1.
Tables detailing primer sequences for q-PCR assay (a), primary antibodies used for immunofluorescence assays(b), secondary antibodies used for immunofluorescence assays (c) and sample size number and statistical test preformed for each quantitative experimental design (d).