Ferreira F , Raghunathan V , Luxardi G , Zhu K , Zhao M .

P30 EY012576 NEI NIH HHS , R01 EY016134 NEI NIH HHS , R01 EY019101 NEI NIH HHS

	Fig. 1. Extracellular O2 flux dynamically correlates with regeneration. a Regeneration time-lapse and phases of a representative tadpole tail amputated at st. 40–41. Major regeneration structures annotated: wound epithelium (WE) and regeneration bud (R. bud). Photomicrographs are displayed in the same orientation as the whole-organism anteroposterior (A/P), dorsoventral (D/V), and left–right (L/R) axes (top left scheme; applies to subsequent figures). White solid lines: amputation plane (black solid lines in schematic tadpole); scale bars: 1 mm. b Temporal profile of O2 flux in the regeneration bud in MMR 0.1× (control). Negative values are net influx (applies to subsequent figures). Profile is descriptively divided into three parts: S slope; P plateau, and B baseline. c Diagrammatic representation of the temporal dynamic of O2 influx during regeneration. Profile is divided into two phases: regeneration-specific and homeostatic. Magnitudes are not at absolute scale but present the relative dynamics in temporal directions and magnitudes. Statistical analyses were performed by unpaired Student’s t-test (two-tailed p-value). The data are presented as mean ± s.e.m. n biological replicates indicated in brackets. NS non-significant; *p < 0.05; **p < 0.01
	Fig. 2. Extracellular O2 influx predicts regeneration efficiency. a Representative tails at 7 dpa in MMR 0.1× from tadpoles amputated in regenerative (st. 40–41) or refractory period (st. 45–46). White solid lines: amputation plane; scale bar: 1 mm. b Qualitative and quantitative analyses of regeneration efficiency for the different conditions tested. RI regeneration index; a.u. arbitrary units. c O2 flux measured in two different time-points in the bud of regenerative or refractory period tadpoles. Statistical analyses were performed by Fisher’s exact test b, or unpaired Student’s t-test (both two-tailed p-value) c. The data are presented as median ± min to max (with outliers). n biological replicates indicated in brackets. ***p < 0.001
	Fig. 3. Exogenous O2 fuels ROS production that is necessary for regeneration. a–d O2 influx fuels ROS production in regenerative but not likely in refractory period tadpoles. a Representative tails at 7 dpa in vehicle-control and pharmacological treatment from tadpoles amputated at st. 40–41. b Qualitative and quantitative analyses of regeneration efficiency for the different conditions tested. Most tadpoles from O2 flux measurements. RI regeneration index. Stacked bars legend applies to f. O2 flux measured in two different time-points in the bud of vehicle-control and pharmacological treatment from tadpoles amputated in regenerative c or refractory period d. e–g The magnitude of O2 influx is independent of ROS per se. e Representative tails at 7 dpa in vehicle-control and pharmacological treatment from tadpoles amputated at st. 40–41. f Qualitative and quantitative analyses of regeneration efficiency for the different conditions tested. Tadpoles from O2 flux measurements included in quantification. g O2 flux measured in two different time-points in the bud of vehicle-control and pharmacological treatment from tadpoles amputated at st. 40–41. White solid lines: amputation plane; scale bar: 1 mm; a.u. arbitrary units. Statistical analyses were performed by Fisher’s exact test b, f, or unpaired Student’s t-test (both two-tailed p-value) c, d, g. Data are presented as median ± min to max (with outliers). n biological replicates indicated in brackets. NS non-significant; *p < 0.05; **p < 0.01; ***p < 0.001
	Fig. 4. HIF-1α is necessary for and sufficient to induce regeneration. a–d Loss of regeneration by HIF-1α inhibition and temporal requirement for HIF-1α activity during regeneration. a Representative tails at 7 dpa in vehicle-control and pharmacological treatment from tadpoles amputated at st. 40–41. b–d Qualitative and quantitative analyses of regeneration efficiency for the different conditions tested. RI regeneration index. Stacked bars legend in b applies to c, d, and f. e, f Induction of regeneration by HIF-1α stabilization. e Representative tails at 7 dpa in vehicle-control and pharmacological treatment from tadpoles amputated in refractory period. f Qualitative and quantitative analyses of regeneration efficiency for the different conditions tested. White solid lines: amputation plane; scale bar: 1 mm; a.u. arbitrary units. Statistical analyses were performed by Fisher’s exact test (two-tailed p-value). n biological replicates indicated in brackets. NS non-significant; **p < 0.01; ***p < 0.001
	Fig. 5. Hypoxia and HIF-1α stabilization correlate with regeneration efficiency. a–c Hypoxia is significantly affected by depleted ROS and dramatically affected in refractory period. a Representative flow cytograms of DMSO 0.1% st. 40–41 vs. DPI 1 μM st. 40–41 (top panel) and DMSO 0.1% st. 40–41 vs. DMSO 0.1% st. 45–46 (bottom panel) at 1 hpa. MFI mean fluorescence intensity. b Semi-quantitative analysis of hypoxia penetrance normalized to the vehicle-control. Horizontal axis labels in b also apply to e. c Hypoxia immunofluorescence imaging in vehicle-control and pharmacological treatment from tadpoles amputated in regenerative (st. 40–41) and refractory (st. 45–46) periods at 1 hpa. Independent experiments gave consistent readouts. Bottom panels: high magnification of correspondent rectangles in middle panels. Green arrowhead: high hypoxia in the wound epithelium (middle panels) and prospective regeneration bud (bottom panels); orange arrowhead: low hypoxia in the prospective regeneration bud (bottom panels); red arrowhead: no hypoxia in the wound epithelium (middle panels) and prospective regeneration bud (bottom panels); white dotted line: posterior tail outline. d–f HIF-1α stability levels are significantly affected by depleted ROS and dramatically affected in refractory period. d Representative western blot in all conditions at 1 hpa. e Semi-quantitative analysis of HIF-1α stability levels normalized to the vehicle-control. f HIF-1α immunofluorescence imaging in the regenerative condition at 1 hpa. Independent experiments gave consistent readouts. Yellow arrowhead: high HIF-1α stability in the prospective regeneration bud; white arrowhead: high HIF-1α stability in the wound epithelium. a.u. arbitrary units; scale bars: 100 μm. Statistical analyses were performed by paired Student’s t-test (two-tailed p-value). n flows of 20 specimens each, or n blots of 30 specimens each indicated in brackets. NS non-significant; *p < 0.05; **p < 0.01
	Fig. 6. ROS do not directly stabilize HIF-1α to modulate regeneration. a, b Stabilization of HIF-1α does not rescue DPI-impaired regeneration. a Representative tails at 7 dpa in vehicle-control and pharmacological treatments from tadpoles amputated at st. 40–41. b Qualitative and quantitative analyses of regeneration efficiency for the different conditions tested. White solid lines: amputation plane; scale bar: 1 mm. RI regeneration index; a.u. arbitrary units. Statistical analyses were performed by Fisher’s exact test (two-tailed p-value). n biological replicates indicated in brackets. NS non-significant; ***p < 0.001
	Fig. 7. HSP90 is a putative and JI reversal is a de facto downstream targets of HIF-1α. a–d Loss of regeneration by HSP90 inhibition and temporal requirement for HSP90 activity during regeneration. a Representative tails at 7 dpa in control and pharmacological treatment from tadpoles amputated at st. 40–41. b–d Qualitative and quantitative analyses of regeneration efficiency for the different conditions tested. White solid lines: amputation plane; scale bar: 1 mm. RI regeneration index; a.u. arbitrary units. Stacked bars legend in b applies to c and d. e HIF-1α regulates the JI reversal hallmark. JI in regeneration bud measured in two different time-points in vehicle-control and echinomycin-treated tadpoles amputated at st. 40–41. Statistical analyses were performed by Fisher’s exact test b–d; e #, vs. JI reversals), or unpaired Student’s t-test (both two-tailed p-value) (e: *, vs. JI magnitude). The data are presented as mean ± s.e.m. n biological replicates indicated in brackets. NS non-significant; */#p < 0.05; ***/###p < 0.001
	Fig. 8. Stepwise model integrating redox state activities during regeneration. Instantaneous injury-induced O2 influx fuels ROS production and together tune a permissive pO2 microenvironment—hypoxia—for HIF-1α stabilization. Early time-window up to 6 hpa is focused, because is when the magnitude of O2 influx correlates with regeneration and when required ROS and HIF-1α activities occur. ROS per se do not feedback with the magnitude of O2 influx. ROS do not directly stabilize HIF-1α but do so indirectly by regulating hypoxia in the bud owing to local O2 consumption and O2 influx demand (Supplementary Fig. 19). We infer that intracellular HSP90 is at least partially required for early hypoxia-induced stabilization of HIF-1α, resulting in the secretion of eHSP90α. 6 hpa is also the time-point of the hallmark JI reversal, an accurate predictor of regeneration efficiency that mediates redox-modulated regeneration. HIF-1α regulates JI reversal, pointing to an integration of HIF-1α with the bioelectric state, in series or in parallel with ROS. Mechanistically, HIF-1α modulates regeneration via HSP90/eHSP90α and JI reversal presumptive effect on cell migration migration to form the wound epithelium and/or regeneration bud. iHSP90: intracellular HSP90; eHSP90α: extracellular HSP90α. Solid line arrows: demonstrated; dotted line arrows: hypothesized/probable. # mechanism schematized in Supplementary Fig. 19; * demonstrated in ref. 8

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