Figure 1.

AP differences in bioelectric signaling exist before the earliest asymmetrically expressed gene notum appears. (A and B) V_mem reporter assay using DiBAC₄(3) is shown. Images are pseudocolored blue-green-red. Brighter pixels (red) are most positively charged on the inside of cells relative to the outside, i.e., relatively depolarized. Pixels of lower intensity (blue) are relatively hyperpolarized or more negatively charged on the inside of cells relative to the outside. Green arrows indicate anterior blastema, and blue arrows indicate posterior blastema. (A) Untreated wild-type (WT) D. japonica fragments cut from the same worm 2 h before DiBAC₄(3) imaging are shown. Boxes indicate paired wound sites that were once in the same location in the animal. Anterior-facing blastemas on posterior fragments are significantly depolarized compared with posterior-facing blastemas on anterior fragments, which were located in the same position in the whole worm before amputation (p < 0.05, N = 14, paired t-test). (B) WT fragments in DiBAC₄(3) at (a) 1 h, (b) 7 h, (c) 14 h, and (d) 21 h after amputation with anterior blastema oriented toward the top and posterior blastema oriented to the bottom are shown. (C) Quantification of DiBAC₄(3) fluorescence intensity at the anterior and posterior blastema of the same individual fragments during regeneration at 3 h (N = 19), 6 h (N = 23), 18 h (N = 17), 24 h (N = 11), and 48 h after cutting (N = 24) is shown. Blastemas from the same fragment are connected by a line. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.005, paired t-test. (D) Timeline indicating notum expression in WT regenerating D. japonica at (a) 0 h, (b) 3 h and (c) 6 h postamputation, as determined by in situ hybridization, is shown. See Fig. S1 for notum expression at later time points. Amputation plane is indicated in red on the sketch. Each panel representative of a time point includes the posterior wound site of the anterior portion of an amputated worm (top) and the anterior wound site of the posterior portion of an amputated worm (bottom). Purple arrows indicate punctate expression pattern. White arrows mark the edge of the blastema with no signal. Yellow boxes in (A) demarcate regions of interest. Scale bars, 1 mm throughout. To see this figure in color, go online.