(A) Experimental design to test for the sufficiency of injury to induce new organizer formation in tissue with or without pre-existing organizers. (B) Prolonged injuries induce ectopic head regeneration when pre-existing organizers are removed. Scale bars indicate 1 mm. (C) Quantification of the number of ectopic tentacles induced by various impalement injury conditions. Both the presence of pre-existing organizers and the duration of the injury signal significantly contributed to the ectopic head regeneration phenotype. –Org indicates that organizers were removed just prior to impalement, and +Org indicates that organizers were left intact. (D) RT-qPCR results for head-specific transcripts in tissue that had been impaled for 12 hr. The Wnt signaling genes wnt3, wntless, and wnt9/10c and the head-specific transcription factor brachyury1 (bra1) are significantly upregulated after 12 hr of impalement. Three biological replicates, each consisting of tissue originating from 15 polyps, were generated for each treatment. Error bars indicate standard deviation. All statistical analyses were performed on 2-∆Cq values using Tukey’s HSD in R. Numbers in parenthesis indicate p-values. (E) Experimental design to test both the sufficiency of injury to induce ectopic heads during foot regeneration and the inhibitory capacity of oral-facing amputations. (F) Prolonged injuries induce ectopic heads at aboral-facing amputations, but only if prolonged oral-facing amputations are removed. Scale bars indicate 0.5 mm. The white arrowhead indicates a regenerated basal disk. (G) Quantification of the number of ectopic tentacles induced by various impalement injury conditions. +reamp indicates that the oral half of the regenerating tissue fragment was removed after 12 hr of impalement. HR: head regeneration; FR: foot regeneration, * indicates p-value≤0.05, ** indicates p-value≤0.01, *** indicates p-value≤0.001. All animals were imaged 4 days post injury, giving sufficient time for any removed structures to regenerate.