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Fig. 2. Characterization of V-ATPase expression and physiology. Whereas NaV1.5 (A,A′) and most transporter genes tested, including Kir6.2 and subunits of the H+/K+-ATPase, are absent, both V-ATPase mRNA and protein are detected in the regeneration bud (B,B′,C,C′). High magnification of section reveals the predicted cell-membrane localization for the protein (C′; scale similar to Fig. 1F′). (D) As in regenerating tails, amputation during the refractory period (st. 46) results in V-ATPase protein expression at 24 hpa. The same is true in irradiated larvae examined at 48 hpa (E), which exhibit no cell proliferation, as detected by the phosphorylated Histone H3B marker (F, compare with normal pattern of proliferation in G). Sections were generated as described by Levin (Levin, 2004). Larvae shown in A-E are oriented with anterior towards the bottom; sections A′,B′,C′,F′,G′ are oriented with anterior to the right and dorsal upwards. Larvae in F,G are shown at 48 hpa. White arrowhead points to regeneration bud with lack of expression. Red arrows and arrowheads indicate positive signal (expression of RNA or protein marker).
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Fig. 4. V-ATPase function is required for the upregulation of cell proliferation in the tail regeneration bud. Proliferating cells were labeled with immunohistochemical staining for phosphorylated Histone H3B (H3-P). (A) At 24 hpa, proliferating cells in the G2-M transition are randomly distributed. (B) By 48 hpa, the proliferating cells cluster in the regeneration bud and have largely disappeared from the caudal flank. Red arrows indicate H3P-positive cells. (C) Incubation in concanamycin immediately after amputation reduces proliferation in the bud; sample proliferation data are from D (control) and D′ (concanamycin-treated) larva. A,B are oriented anterior to the left; all other panels are oriented anterior upwards. n=8 for each condition. (E-G′) KCNK1 expression requires V-ATPase activity. The regeneration bud normally expresses the KCNK1 channel by 24 hpa (E,E′). By contrast, larvae in which the V-ATPase was inhibited by concanamycin (F,F′) or anti-c subunit antibody (G,G′) exhibit no KCNK1 expression at 24 hpa. n=10 for each condition. White arrows indicate lack of expression; red arrows indicate positive signal (expression of KCNK1).
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Fig. 1. The V-ATPase is required for tail regeneration in Xenopus. When amputated at st. 41 (A), the Xenopus laevis larva (n>1000) rapidly rebuilds a tail (A). Regeneration, but not anterior development, primary tail growth or wound healing, is abolished by specific inhibition of the V-ATPase H+ pump (n=226) by concanamycin treatment immediately after cutting (B; close-up shown in B). Blue lines in A-B' indicate approximate amputation plane. This inhibition of regeneration is not due to an upregulation of apoptosis, as concanamycin-treated tails (C) do not show a greater degree of staining for the apoptosis marker activated Caspase-3 than controls (D) when processed at 48 hpa. When the dominant-negative V-ATPase E subunit is expressed in tails, regeneration is not observed (E, compare with B', n=66), while otherwise normal development continues. For statistical analyses, see Table S2 in the supplementary material. (F-H) We attempted to rescue the V-ATPase-inhibited phenotype with misexpression of a single-subunit concanamycin-insensitive H+ pump, PMA. (F-F") Immunohistochemistry with antibody to PMA. Larvae were microinjected with PMA construct+GFP at the 1-cell stage, and sorted at tailbud or later stages for GFP expression in the tail. (F) Negative control larva injected with gal and probed with PMA antibody, showing no signal.
(F') Positive staining in regeneration bud confirms that injected constructs lead to robust levels of expression in the tail during regeneration when mRNA is injected at 1-cell stage. (F") Close- up of PMA-expressing cells demonstrating the predicted cell- membrane localization of H+ pump (red arrows). Scale bar: 50 um in panel F" (G,G') Such expression of the PMA pump is sufficient to rescue regeneration after V-ATPase inhibition by concanamycin. (G) Tails of larvae injected with PMA at the 1-cell stage, amputated at st. 41, and then incubated in concanamycin regenerate (compare with phenotype in B'). Newly regenerated tissue is indicated by the blue bracket. (G') Normal neuronal pattern revealed by immunohistochemistry. (H) Quantification of rescue effect. Abrogation of regeneration (red bars) can be largely prevented by misexpression of PMA (dark yellow bars, n=127), demonstrating the necessity for the H+ flux during regeneration (off-white bars, no treatment; green bars, PMA injection only).
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Fig. 3. Imaging of membrane voltage using DiBAC. Red indicates more depolarized than green, which is more depolarized than blue (see key under A'). For further description of the collection and analysis of the DiBAC data see Figs S1, S2 and S3 in the supplementary material. (A,A') An uncut tail from a st. 41-43 larva shows generally even voltage levels (blue areas), with scattered cells (green to red) that are depolarized relative to the rest of the tail. Scale bar: 250 ﰀm. (B) Transmitted light image of a regenerating tail bud with the different regions labeled. The shoulder is defined as the region underlying the abnormally shaped melanocytes; this region appears darker in lower magnification images (e.g. F). Scale bar: 80um. (C) Regenerating tail bud imaged at 6 hpa. At this early time point, the bud is depolarized relative to the surrounding tissue. Yellow circles indicate bud and shoulder regions of interest (ROIs) that were used in quantifying this image (trunk ROI not shown on these cropped images). (D) Tail imaged at 24 hpa. The bud has repolarized relative to the 6 hpa state. The appearance of DiBAC fluorescence is similar to that in uncut tails, with the exception of the shoulder region that has an island of depolarization. Although not visible in every tail, this was a very common pattern. Yellow circles indicate ROIs; arrow indicates the depolarized region of shoulder cells. (E) Comparison of the relative voltage patterns found in regenerating 24 hpa (green), regenerating 6 hpa (orange), refractory (red) and PMA-rescued refractory (yellow) tails. Both regenerating 24 hpa and PMA-rescued refractory tails are depolarized in the shoulder region relative to the bud and trunk. By contrast, refractory and 6 hpa tails are highly depolarized in the bud, becoming more polarized in anterior regions. (F,F') Tail cut at st. 41 and treated with concanamycin. The large region of red and green indicates relatively strong depolarization, as predicted (V-ATPase polarizes, therefore the inhibitor should cause depolarization). (G,G') At 24 hpa, the buds of refractory tails are depolarized relative to a regenerating tail. Yellow circles indicate ROIs. (G") The same tail shown in G and Gconfirming that refractory tails fail to regenerate, even at 7 dpa. Scale bar: 250um. (H,H') Uncut st. 46-47 (refractory) tail. DiBAC images of refractory tails vary; in this image, dorsal and ventral blood vessels are obvious. The most consistent difference between refractory and regeneration-competent tails is the relatively even staining across the refractory tail (compare with the 'spottiness' of staining in A' and D). (H") The same tail as H and H' shown at 7 dpa, confirming that this tail was refractory when it was amputated. Scale bar: 250um.
(I) Refractory tail shown 4 dpa. (I P-type pump- expressing tail, 4 dpa at the refractory stage. (I') P-type pump-expressing tail, 7 dpa at the refractory stage, showing full regeneration. Red bracket indicates newly regenerated tissue absent in control tails. (J) DiBAC reveals that, unlike control refractory tails (G'), the bud of PMA-expressing refractory tails is relatively polarized, as with tails cut at st. 41 (D). Yellow circles indicate ROIs. (J') The same tail as J, showing significant regeneration at 7 dpa. (K) Analysis of the rescue of regeneration in refractory tails by misexpression of a yeast P-type H+ pump. Off-white bars, refractory tails; red bars, PMA-injected tails. Injection of PMA caused a significant augmentation of the ability of refractory tails to regenerate. *, indicates significantly different from controls (see Table S2 in the supplementary material).
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Fig. 5. Axonal growth pattern is dependent on H+ fluxes. Green arrows indicate normal axon patterning; black arrows indicate abnormal axon number and/or location. (A) The normal pattern of regenerating axons is parallel to the main body axis, extending to the tip of the bud at 72 hpa. This is altered in V-ATPase-inhibited tails, where axons are absent from the central core of the new tissue of partial regenerates at 72 hpa (B), or are present in a tangled mass and not aligned parallel to the tail primary axis (B'). The expression of the concanamycin-insensitive yeast PMA H+ pump in the tail can rescue the normal axonal patterning (C). (D) In refractory-stage larvae, axons terminate in a loop at a considerable distance from the edge, and perpendicular to the main tail axis. Expression of PMA rescues the neuronal phenotype, resulting in axon growth to the very edge of the wound in non-regenerating tails (E), and parallel outgrowth of axons into the new tissue of regenerates (E'). The normal pattern of axons, reaching to the end of the tissue at 7 dpa (F), is not affected by irradiation (G), which abolishes cell proliferation and regeneration; axons reach to the distal edge of the irradiated tissue. Tails shown in A-E are 72 hpa; those in F-G are 7 dpa.
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