FIGURE 1. Neural cues mediate polarity during regeneration.

(A) Schematic of transverse amputations in the presence of octanol. The incidence of bipolar head phenotype gradually increases toward posterior areas. Inset is representative of bipolar-head regenerate (white arrows indicate heads). Red dotted lines indicate level of amputation. Data are presented as mean +/− confidence intervals (95%). (B) Schematic representation of transverse amputations along the A/P axis to obtain fragments with a pre-existing head (left). Fragments generated after amputations exhibited very few bipolar-head regenerate. (C) Planarians were dissected in different ways (illustrative representation is shown for each case within columns) and the regeneration pattern was recorded after 2 weeks of regeneration. Animals reestablishing the original pattern were considered normal, while worms developing abnormalities were classified into seven categories by macroscopic observations, A/P polarity, and CNS morphological abnormalities. Seven abnormalities were: middle head; side head; bipolar + middle head; side protrusion; bipolar head and double side head (color-coded at the top of the graph). Percentage of animals regenerating each pattern is displayed along with the number of animals (within parentheses) assayed in each condition (Roman numbers). Untreated animals always regenerated normally (not shown for simplicity). Most cases (3/4) where dissections did not disrupt VNC integrity at pre-pharyngeal level regenerated 100% normal animals (conditions I, II, VII and X). Conversely, almost all cases (7/8) where VNC integrity was disrupted in pre or post-pharyngeal area led to regeneration of abnormalities characterized by alterations in CNS patterning and polarity (conditions III-IX). These results suggest that VNC integrity and GJC play important role in regenerative patterning and polarity. Since all animals regenerated tissues and developed blastemas, treatment with GJ inhibitor does not alter normal response to wound damage but rather instructively influences the identity of the newly formed tissue. Importantly only animals with disrupted VNC integrity and octanol treated led to novel phenotypes (D) characterized by multiple heads (white arrows) and pharynxes (gray arrows). Representative images of immunostaining with anti-synapsin antibody (green signal) over a pseudocolored red background are shown. In all cases original anterior end is to the top. Bars represent 200μm.