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 ß-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 µm 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).