(A) Microinjection schedule of double-stranded RNA (dsRNA) in intact planarians. A total of five injections (arrows between green squares) were performed over 19 days. Injecting a mix (1:1) of Dj-Inx-5 + 13 dsRNA for 3 consecutive days and 2 injections of Dj-Inx-12 ds-RNA 2 weeks after first injections led to strong and reproducible phenotypes ~14 days after last injection. Control animals were always run in parallel and injected with water under same schedule. (B) Representative phenotypes following 30-35 days since first injection. Conditions: control (water injected) and Dj-Inx-5 + 13, −12(RNAi). Control animals (left column) did not display any behavioral or anatomical abnormalities as assayed by markers of CNS (anti-synapsin antibody), mitotic cells (anti-H3P antibody), proliferative neoblasts (Dj-Inx-11 antisense probe), and digestive system (Dj-Inx-7 antisense probe). In contrast, animals subjected to Dj-Inx-5 + 13, −12(RNAi) displayed obvious behavioral and morphological changes (right column). Random contractions, ectopic photoreceptor pigmentation (yellow arrow), and extra pharynxes (blue arrows) were evident, especially in post-pharyngeal areas. Ectopic pharynxes, brain tissue (white arrows) and striking alterations in the distribution of neoblasts and the digestive system were observed. Remarkably, the orientation of ectopic brain and pharynx were sometimes reversed with respect to the original A/P axis, and the distribution of neoblasts and digestive system adopted anatomical changes consistent with the new structures developed (e.g.: no mitotic activity surrounding brain and pharynx and formation of primary intestinal branch in post pharyngeal area followed by posterior bifurcation around ectopic pharynx). Bar is 500 μm. (C) Microinjection dsRNA schedule for worms amputated (red arrow) after ~25-28 days of first injection. (D) To analyze the roles of Dj-Inx-5 + 13, −12(RNAi) during regeneration, injected animals were amputated at different levels along the A/P axis producing 5 fragments. Pharyngeal and post-pharyngeal fragments regenerated animals with polarity problems (inset representative picture). Data are represented as mean +/− confidence intervals (95%) and red dotted line indicates plane of amputation. (E) Summary illustration of phenotypes obtained in intact and regenerating of Dj-Inx-5 + 13, −12(RNAi) animals. In both cases, polarity problems and ectopic organs appeared (as after pharmacological inhibition of GJs). The original anterior ends are shown to the left.