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 µm. (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: 80 µm. (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 G' confirming that refractory tails fail to regenerate, even at 7 dpa. Scale bar: 250 µm. (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: 250 µm. (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).