Fig. 1. Characterization of Smed-roboA and defects in visual-system regeneration after Smed-roboA RNAi. (A) Organization of the predicted SMED-ROBOA extracellular domain containing five immunoglobulin-like (Ig) and three fibronectin (FNIII) domains, compared to ROBO proteins from C. elegans (AF041053), Drosophila (AF040989) and human (AF040990). Numbers indicate percentage of identical amino acids in each domain. (B-E) Smed-roboA in situ hybridization in an intact planarian (B) and during anterior regeneration (C-E); days postamputation are indicated. (C) Arrowheads show cephalic ganglia primordia. (F-K) Efficacy and specificity of SmedroboA RNAi revealed by whole-mount in situ hybridization. (F-H) Control samples; (I-K) Smed-roboA samples. (F,G) Controls reveal normal expression of Smed-roboA during anterior and posterior regeneration, respectively. (I,J) After Smed-roboA RNAi, roboA transcript is not detected in newly regenerated or pre-existing tissues. (H,K) Smed-roboA RNAi does not affect roboB expression. (F,I) Trunk pieces at 6 days of anterior regeneration; (G,H,J,K) head pieces at 6 days of posterior regeneration. (L-O) Double immunofluorescence to label the cephalic ganglia (anti-phospho-tyrosine; pale green) and photosensitive cells (VC-1; bright green). (L) Control: photoreceptor axons project to the visual center in the brain, forming a normal optic chiasm (oc); arrow indicates normal anterior commissure. (M-O) Smed-roboA RNAi: ectopic projections appear (magenta arrowheads) and the shape of the chiasm is altered (magenta arrow in N). The anterior commissure is reduced (arrow in M) or absent (arrow in O). (L,M,O) After 16 days of regeneration. (N) After 35 days of regeneration. cg, cephalic ganglia; pr, photoreceptors; vnc, ventral nerve cords; asterisk, pharynx. Scale bars: 500 µm in B; 200 µm in C-E; 200 µm in F,I; 500 µm in G,J; 500 µm in H,K; 100 µm in L-O.