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Fig. 1. Tadpoles lose the ability to regenerate tails at stage 45. (A) For regeneration experiments and probe measurements approximately 50% of the tail was amputated. (B) This montage compares a single tail cut at stage 40 with another cut at stage 45. The stage 40 tail begins to re-grow about 5 days after cutting, and is almost fully regenerated at 12 days. The stage 45 tail does not re-grow. Scale bar 1 mm. (C) Percentage regeneration was dramatically reduced at stage 45. More than three quarters of stage 40 tails regenerated (77.6%) whereas only 5.2% of stage 45 tails re-grew. Data from 4 different spawnings (batches) of tadpoles; total numbers: 88 (stage 40), 76 (stage 45); âŽP < 0.001.
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Fig. 2. Regenerating tails show active nerve and blood vessel growth. (A) Fluorescent imaging of spinal cord regeneration. GAP43 (growth associated protein 43) specifically labels new growth of nerve. GAP43 labeling 6 days after tail cutting reveals regeneration of spinal cord (arrow) at stage 40 but not stage 45. (B) Labeling of sprouting blood vessels by fluorescent dye injection. DiI-Ac-LDL was injected into the beating hearts of anesthetized tadpoles 7 days after tail amputation. Dye in the bloodstream is taken up by vascular endothelial cells, labeling sprouting blood vessels in regenerating tails (arrow). Scale bars 0.5 mm.
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Fig. 3. Electric currents at stumps correlate with regeneration. Currents were measured at different positions on the cut tail stump. Consistently, the currents at all positions correlated with regeneration. (A) Photograph and schematic drawing showing the five measuring positions. They correspond to: dorsal fin muscle (a), spinal cord (b), muscle (c), blood vessel (d), ventral fin muscle (e). (B) Immediately after amputation (day 0), large outward currents (positive values) were detected at all positions. (C) At all positions, 3–4 days after cutting, stump currents in tadpoles of stage 45, which do not regenerate, remained outward . However, currents in stage 40 tadpoles reversed direction, becoming inward (negative values) at all positions of measurement. Data from 3 different batches of tadpoles; total numbers: 14 (stage 40), 16 (stage 45).
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Fig. 4. Electric currents at stumps correlate with regeneration. (A) Chamber constructed to hold tadpoles for measurement and imaging (scale bar 1 cm). (B) Anatomy of tadpole tail. Measurements were made adjacent to the spinal cord. Scale bar 1 mm. (C, D) Tail stump current during regeneration, 0–24 h, and up to 12 days. Tadpole tail electric current correlates with tail regeneration. Immediately following amputation, large currents flow out of the stump in tadpoles of both stages 40 and 45. The currents then decreased. In regenerating tails, the direction of current reversed. Current at stage 40 and 45 tails was significantly different at 24 h after amputation (âŽP < 0.03) and at the time that stage 40 tails begin to re-grow (day 5; âŽâŽP < 0.01). Data from 4 different batches of tadpoles; total numbers: 25 (stage 40), 18 (stage 45). (E) The inward currents at day 5 were confirmed in regenerating stage 40 and 48 tadpoles. Only non-regenerating stage 45 had outward currents. αP < 0.01; #P < 0.02. Stage 48 n = 18 from 3 different batches.
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Fig. 5. Sodium in the bathing solution is important for stump currents and tail regeneration. Tadpoles (stage 40) were incubated in solution lacking sodium. (A) In sodium-free solution, the stage 40, day 5 tail current was significantly reduced (âŽP < 0.01) to 33% of control ((− 0.299/− 0.893)âŽ100). Tail current in chloride-free solution was unchanged. “Normal†data taken from Fig. 4D (day 5). (B) Regeneration rate (“normal†n = 25 from 4 different batches) was also significantly reduced in sodium-free solution (âŽâŽP < 0.03) but unchanged in chloride-free solution (AU = arbitrary units). (C) The percentage of tails that regenerated (stage 40) was significantly less in sodium-free solution (αP < 0.01), but unchanged in chloride-free solution. “Normal†data taken from Fig. 1C. (D) Drugs that alter ion transport in mammalian epithelia did not affect stump currents or regeneration. Tadpoles (stage 40) were incubated in drugs which increase (aminophylline) or decrease (ouabain) ion pumping, and are known to alter wound current and wound healing in rat cornea. Neither drug had any effect on tail current, regeneration rate or % regeneration (data not shown).
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Fig. 6. Sodium and wound healing. (A, B) Fin punch wounds (stage 48) healed normally in Na-free solution (both Na-free and Normal n = 8 from 2 different batches). (C) Punch-wounds in fin also healed normally at stage 45 (n = 12 from 2 different batches). Scale bar 500 μm.
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Fig. 7. Possible roles of depolarized cells and epithelial morphology. (A) Using the membrane potential-sensitive fluorescent dye DiBAC4(3), aggregates of highly-depolarized cells were observed in regenerating (stage 40) tails 5 days after cutting. Non-regenerating tails had no such aggregates, but had a thick skin-like epithelial membrane covering the cut stump (arrows in C) which was not present in stage 40 tails (B). Scale bar 500 μm.
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Fig. 8. Schematic model depicting the possible role(s) of electrical events during tail regeneration. Amputation breaks the epithelial barrier, collapses the transepithelial potential difference, and induces large outward currents. The outward electric currents last only a few hours in the tails that regenerate. A sequence of cellular events that may regulate electric current flow occur 4–24 h after amputation and persist for up to a few days. These events may include expression of the V-ATPase that drives induction of NaV1.2 and KCNK1 (Adams et al., 2007). The epithelium covering regenerating stumps was rough and had no visible basement layer under phase optics. In contrast, non-regenerating stumps quickly (1–2 days) grew a thick, smooth, skin-like epithelium over the cut stump. In regenerating tails, a group of highly depolarized cells appeared under the site of tail re-growth. These cells might cause the electric current in the regenerating stump to reverse direction and became inward. Altering the inward currents decreased regeneration. In contrast, tails during refractory stages did not reverse the current direction, which appeared to contribute to the inability to regenerate.
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