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Figure 1.
Sodium transport is required for tail regeneration. A , Control tails amputated at st. 40 regenerate fully whereas siblings treated with 250 μM MS222 fail to regenerate. B , Effects of NaV chemical inhibition on regeneration showing dose-dependent inhibition. C , Fluorescent indicator dye (CoroNa Green) of sodium flux (red arrows). Uncut tail has scattered fluorescence. Cut tail (24 hpa) shows strong sodium influx into the regeneration bud (white circle) in contrast to MS2222-treated buds. D , Whole-mount in situ hybridization for NaV1.2 in amputated tails. Regeneration bud (6 hpa) lacks NaV1.2 expression. By 24 hpa, NaV1.2 is expressed in the regeneration bud and persists until 48 hpa. A section through an 18 hpa regeneration bud reveals NaV1.2 in mesenchymal cells but not the wound epidermis. E , Control tails amputated at st. 40 regenerate fully but animals expressing NaV RNAi construct do not. Scale bars: A , E , 1 mm; C , D , 500 μm; D , far right, 100 μm. Red arrows, expression; white arrow, lack of expression; yellow arrows, amputation plane. dpa, Days postamputation. *p < 0.001.
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Figure 2.
NaV-mediated sodium transport acts early during regeneration. A , Effect of NaV inhibition (MS222) on proliferation and innervation. Top, Immunohistochemistry of 48 hpa tails using an anti-H3P antibody (blue) in sagittal sections (yellow arrows indicate mitotic cells; melanocytes are black). Bottom, Tails (72 hpa) stained with acetylated α-tubulin antibody to identify axons. Control axon bundles run parallel to the anterior–posterior axis and concentrate at the tip (yellow arrow). MS222 treatment reduces axons (white arrow) that trace along the edge. B , Effect of NaV inhibition on genes that regulate regenerative outgrowth (as shown by RNA in situ hybridization in sagittal sections at 48 hpa). Notch RNA (top) is expressed in the neural ampulla (red arrows) and in the regeneration bud mesenchyme, whereas Msx1 (bottom) is expressed solely in the neural ampulla. Gene expression is abolished after NaV1.2 inhibition (black arrows). Scale bars, 250 μm.
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Figure 3.
Transient induction of sodium current drives regeneration. A , Regeneration rescue by human NaV1.5 (h NaV1.5). Control tail stumps (β-gal-injected) cut during the refractory period regenerate poorly, which is rescued by hNaV1.5 expression. B , Effects of regeneration rescue by hNaV1.5 during refractory period block. C , CoroNa Green analysis of sodium-current induction. C1 , Control (vehicle only), noninduced refractory stage bud has little CoroNa Green signal. C2 , Induction with 90 mM sodium and 20 μM monensin for 1 h (18–19 hpa) significantly increases intracellular sodium (green). Images are merged brightfield and fluorescence of the same exposure time. White circle, Refractory bud. C3 , Most refractory stage amputations fail to regenerate. C4 , Stimulation with sodium current restores full regeneration. D , Transient sodium current rescues nonregenerative wound epidermis. Stimulation of sodium current increased regeneration more than twofold and improved regeneration quality compared with control siblings (treated with vehicle only, 0.01% ethanol). Treatment with either monensin or 90 mM sodium alone showed no effect. Scale bars: A , 1 mm; C , 500 μm.
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Figure 4.
Salt-inducible kinase is required for tail regeneration. A , Comparison of the relative voltage patterns of tail regeneration buds at 24 hpa using the voltage dye, DiBAC4(3). Green is more depolarized than blue. Distal tail end (amputation site) is outlined in white. Scale bar, 100 μm. The regeneration bud (red circle) of controls was polarized (blue color). MS-222-treated buds show a similar pattern. B , RNA in situ hybridization for endogenous SIK in whole-mount cut tails. SIK is expressed in the regeneration bud at 24 and 48 hpa (red arrows) but not in uncut tails (black arrow). C , Effect of SIK RNAi on regeneration. SIK RNAi-expressing tadpoles fail to regenerate but develop normally.
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Figure 5.
A model integrating NaV in regeneration. By 6 hpa, the H+ pump V-ATPase is expressed in the regeneration bud where it regulates the membrane voltage of the bud. V-ATPase activation results in the upregulation of NaV1.2 by 18 hpa. Ablation of NaV1.2 expression (RNAi) or NaV function (pharmacological treatment) inhibits regeneration. NaV activity enables sodium ions to enter regeneration bud cells and, potentially through SIK, to activate downstream pathways (such as BMP and Notch) by 24 hpa, driving regenerative outgrowth and patterning. By 7 d after injury, the rebuilding of the tail is largely complete. Importantly, monensin-mediated induction of a transient sodium flux into nonregenerative buds is sufficient to restore full tail regeneration, demonstrating that intracellular sodium signaling is a key regulator of regeneration able to initiate repair even after a nonregenerative wound epithelium has formed.
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Figure S1. Tail Regeneration Assay.
Individual animals for each specific treatment were scored as follows: Full: complete regeneration
(indistinguishable from uncut controls). Good: robust regeneration with minor
defects (missing fin, curved axis). Weak: poor regeneration (hypomorphic/defective regenerates).
None: no regeneration. Shown are representative examples of each regenerate
class. To quantify and compare regeneration efficiency of tadpoles treated with different
reagents, we also determined the Regeneration Index (RI) - a single number expressing
a composite metric of regenerative response in a group of individuals. The Rl
evaluates the efficiency of regeneration for each treatment and allows for comparison of
the effect of chemical inhibitors to controls. For each treatment, the percentage of regenerates
belonging to each category were calculated, and then multiplied by 3, 2, 1 or 0, respectively
for: Full, Good, Weak and None. The resulting Rl for each condition tested
ranges from 0 to 300, with 0 corresponding to no regeneration, and 300 for complete regeneration.
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Figure S2. NaV Activity is a Determinant of Regenerative Ability.
(A) Immunohistochemistry using a NaV1.2 antibody in amputated tails shows the expression
of NaV1.2 in the regeneration bud (red arrows) whereas NaV1.5 is not expressed.
(B) MS222 treatment reduced expression of genes involved in driving regenerative
outgrowth in the tail bud region (black arrows) at 48 hpa. (C) In contrast to
NaV1.2 expression at 24 hpa (red arrow), NaV expression is missing (white arrows) in:
Concanamycin-treated (V-ATPase-inhibited), non-regenerative Refractory stage, and
non-regenerative depolarized buds (Palytoxin-treated). This shows that NaV expression
is regulated by the membrane potential state of the regenerate, downstream of VATPase
activity.
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Figure S3. Sodium Current Induction Is Sufficient to Rescue a Regeneration
Block by an Apoptosis Inhibitor.
Apoptosis is required for tail regeneration. Treatment of amputated tails with the apoptosis
inhibitor, M50054, blocks regeneration (Tseng et al., 2007). Induction of Na+ current
using monensin rescued the M50054-mediated block of tail regeneration
(RI=59.1, n=11 0) as compared to controls (RI=22. 7, n=88, p<0.01 ).
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Figure S4. A Model for Sodium Transport Control of Regeneration.
After tail amputation, V-ATPase-mediated re-polarization of the bud cells up-regulates expression
of NaV1.2 by 18 hpa. NaV1.2 enables sodium ions to enter the regeneration
bud cells, leading (perhaps through the salt-inducible kinase SIK) to the activation of
downstream regenerative signaling pathways (induction of proliferation and axonal guidance).
By 7 dpa, the rebuilding of the tail is complete. In contrast, tails amputated during
the refractory period remain depolarized and instead form a non-functional wound epidermis
by 18 hpa, which fails to regenerate. Remarkably, induction of a transient sodium flux
into non-regenerative refractory buds at 18 hpa provides the necessary activating signal
to proceed with regeneration, demonstrating that intracellular Na+ signaling is a key regulator
of regeneration that can initiate repair even after a non-regenerative wound epithelium
has formed.
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Figure S5. Differences in Regenerative and Non-Regenerative Wound Epithelium.
Tails amputated during the refractory period show a notably thickened, non-regenerative
wound epidermis (WE) by 24 hpa. Consistent with the observation that tails amputated at
non-regenerative stages exhibit altered healing, 18 hpa refractory caudal stumps already
have a thickened WE (B-B'--width of epidermis outlined by dashed red line) compared to a
regenerative bud WE at the same timepoint (A-A').
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