Defects in gut patterning after Smed-slit RNAi. (A-D) Confocal projections of posterior regenerates visualized using anti-phospho-tyrosine immunofluorescence. (A, C) Control planarian imaged at different focal planes: (A) at level of the ventral nerve cords (vnc); (C) dorsal to those shown in (A), at the level of the gut (g). Note separation between regenerated ventral verve cords (in A) and regenerated gut branches (in C). (B, D) Different focal planes from a Smed-slit RNAi planarian: (B) at the level of ventral nerve cords; (D) dorsal to those shown in (B), at the level of gut. Note midline collapse of ventral nerve cords (in B) and improper connection between the left and right posterior gut branches (arrow in D). (E,F) Confocal projections of lateral regenerates visualized using anti-phospho-tyrosine (green) and VC-1 immunofluorescence (bright green labeling of the photoreceptors, pr). (E) Control planarian: note two posterior gut branches. (F) Smed-slit RNAi planarian: only a single main gut branch is observed posterior to the pharynx (arrow). The drawing at the bottom depicts the longitudinal amputation site for the samples shown in (E) and (F). (A, C, E, F) Anterior is to the left; (B, D) anterior is to the upper left. Scale bars: 50 μm in A-D; 200 μm in E; 200 μm in F.