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First published online 24 January 2007
doi: 10.1242/dev.02794

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Morphogenesis defects are associated with abnormal nervous system regeneration following roboA RNAi in planarians

Francesc Cebrià^* and Phillip A. Newmark

Department of Cell and Developmental Biology, Neuroscience Program, University of Illinois at Urbana-Champaign, B107 Chemical and Life Sciences Laboratory, 601 South Goodwin Avenue, Urbana, IL 61801, USA.

View larger version (91K): [in this window] [in a new window]   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.

View larger version (102K): [in this window] [in a new window]   Fig. 2. Supernumerary pharynx formation after Smed-roboA RNAi. (A,B) Confocal projections showing ectopic pharynges after 16 days of regeneration and two rounds of RNAi treatment. Anti-phosphotyrosine and anti-VC-1 labeling is shown. In A, arrows indicate pharyngeal opening; in B, arrows indicate connections between ectopic and pre-existing gut. (C,D) laminin (DN293829) in situ hybridization in 16-day regenerates. (C) Control; (D) Smed-roboA RNAi. In all panels, white asterisks indicate original pharynges; magenta asterisks show ectopic pharynges. Anterior is to the left. cg, cephalic ganglia; g, ectopic gut branches. Scale bars: 200 µm in A; 100 µm in B; 300 µm in C,D.

View larger version (92K): [in this window] [in a new window]   Fig. 3. Development of ectopic cephalic outgrowths after SmedroboA RNAi. Living planarians imaged at 16 days (A) and 33 days (B) after amputation. (C-E) Cephalic neural markers in outgrowths. (C) Visual-cell marker, VC-1; confocal projection at 35 days after amputation. (D) Sensory-cell marker, cintillo (Oviedo et al., 2003); arrow indicates photoreceptor pigment cells. (E) Clone H.10.2f, marker of cephalic ganglia. (D,E) At 16 days after amputation. In A-E, arrowheads indicate ectopic outgrowths. (F) Ectopic Smed-netrin1 expression (arrowheads) near an ectopic pharynx (red asterisk); 18-day regenerate. (G-L) Expression of the ventral marker anosmin-1 (AY066061) in an 18-day regenerate. (G) Control: anosmin-1-positive cells are not observed in dorsal views. (H) Smed-roboA RNAi: anosmin-1-positive cells are detected dorsally. (I-L) Control (I,J) and Smed-roboA RNAi (K,L) planarians viewed ventrally (I,K) and dorsally (J,L). Anterior to the left. Scale bars: 1.5 mm in A (bar shown in B); 1 mm in B; 100 µm in C-E; 100 µm in F; 500 µm in I-L.

View larger version (71K): [in this window] [in a new window]   Fig. 4. Defects in CNS regeneration after Smed-roboA RNAi. (A-F) Anti-tubulin immunofluorescence in control (A,C,E) and SmedroboA RNAi (B,D,F) 16-day regenerates. Arrowheads in A and B delimit the anterior commissure. (C,D) Regenerated cephalic ganglia in control (C) and Smed-roboA RNAi (D) planarians; magenta lines mark the position of the VNCs in more-ventral sections (shown in E and F). (E) Control: normally regenerated VNC; arrowheads show regularly spaced lateral processes. (F) Smed-roboA RNAi: improperly regenerated VNC; lateral processes are disorganized; magenta arrows indicate curvature. (G-J) Anti-phosphotyrosine immunofluorescence (green) in 5-day (G,H) and 16-day (I,J) regenerates. Hoechst staining is shown in blue. (G,I) Controls: regenerated cephalic ganglia overlap the regenerated nerve cord (arrows). (H,J) Smed-roboA RNAi: regenerated cephalic ganglia are not connected properly to the truncated nerve cord (arrows). (A-F) Anterior to the top left. (G-J) Anterior to the left. Images are confocal projections. Scale bars: 100 µm. cg, cephalic ganglia; vnc, ventral nerve cords; ph, pharynx.