Phenotypes generated after Smed-βcatenin1 silencing in regenerating and in intact planarians. (A) Quantification of the different phenotypes obtained after Smed-βcatenin1 silencing in bipolar regenerating trunk fragments, after different doses of dsRNA injection. (d) Days, (inj) injections. (B–G) Stereomicroscope images of live animals originated from bipolar regenerating trunk fragments: Control (B); “tailless” planarians (C); two-headed planarians with a second ectopic pharynx (D); two-headed planarians with anterior ectopic eyes (E); two-headed planarians with anterior and posterior ectopic eyes (F); and “radial-like hypercephalized” planarians (G). (H–K) z-projections of confocal images corresponding to bipolar regenerating trunk fragments immunostained with anti-synapsin antibody: Control (H); two-headed planarians with a second ectopic pharynx (I); two-headed planarians with anterior ectopic eyes (J); and “radial-like hypercephalized” planarians (K). (L–N) Stereomicroscope images of live intact animals: Control (L); two-headed with ectopic anterior and posterior eyes (M); and “radial-like hypercephalized” planarians (N). (O–Q) z-projections of confocal images corresponding to intact animals immunostained with anti-VC1 antibody: Control (O), two-headed with ectopic anterior and posterior eyes (P); and “radial-like hypercephalized” planarians (Q). Bipolar regenerating animals correspond to 20–25 d of regeneration. Intact animals in images M and P correspond to 15 d after the last injection, and images N and Q correspond to 30 d after the last injection. Yellow asterisks indicate original pharynx, and red asterisks indicate ectopic ones. Yellow arrows indicate normal anterior regenerated eyes, and red arrows indicate ectopic eyes. Bar, 500 µm.

Disruption of the dorsoventral axis after BMP pathway silencing. (A) 21-d-old regenerating head pieces after Smed-Smad1 RNAi. White arrowheads mark the ectopic dorsoventral margin that delimits, which seems to be a second planarian differentiated on the dorsal side like a Siamese twin. Yellow arrows point to the original eyes. Red arrows point to ectopic eyes. (B) 21-d-old regenerating trunk piece after Smed-BMP RNAi. White arrowheads mark the duplicated body margin. Bars, 0.5 mm.

Regenerative capacity of freshwater planarians. (A) Schmidtea mediterranea planarian (top left). (e) Eyespots, (ph) pharynx. Bar, 1 mm. (B–E) Tail pieces at various stages of regeneration (top right). The white tissue in the most anterior tip is the regenerative blastema. Two small eyespots are evident within it after 5 d of regeneration. (F) Planarians display unique regenerative capacities, as any small fragments from almost anywhere can regenerate a new organism in 2 wk. In this diagram, we summarize the main types of planarian regeneration: (1) Terminal regeneration: After transverse sectioning, the anterior end (red line) will regenerate the missing head, whereas the posterior end (green line) will regenerate the missing tail. This indicates that the remaining tissue is polarized and knows what is missing. (2) Lateral regeneration: After longitudinal sectioning (blue line), the old tissue regenerates the missing lateral half. (3) Intercalary regeneration: After joining two distal pieces produced by transverse sections, planarians intercalate the missing region. In that case, cells from each piece participate equally in the production of an intercalary blastema (Saló and Baguñà 1985).

Model of the dynamics and the establishment of a Smed-βcatenin1 gradient activity in adult planarians. (A–H) A gradient of Smed-βcatenin1 activity, with its maximum in the posterior region, would explain the different phenotypes obtained after the different levels of Smed-βcatenin1 inhibition (C–F) and Smed-APC-1 inhibition (H). (blue line in A) Levels of Smed-βcatenin1 activity in intact wild-type planarians; (blue dot in A, G, and H) source of wnt-secreted elements; (red asterisks) levels of Smed-βcatenin1 activity in the posterior blastema of regenerating planarians (C–F) and in the anterior blastema after Smed-APC-1 silencing (H); and (orange line) levels of Smed-βcatenin1 activity established all along the planarian body during the first regeneration stages. A and G show Smed-βcatenin1 activity in wild-type planarians that regenerate posteriorly and anteriorly, respectively. Under each scheme, the corresponding phenotype generated is shown. (I) Graphic representation of eight S. mediterranea wnt expression. All show a restricted expression pattern in specific planarian structures. The putative protein gradient of the 2 posterior wnts that give an anteriorized phenotype after RNAi silencing is shown in the corresponding color. These gradients would have morphogenetic activity, and pattern the AP axis, through regulation of Smed-βcatenin1 activity.

Effects of nou-darake silencing on planarian regeneration. (A) Whole-mount in situ hybridizations with a glutamate receptor homolog (a brain-specific marker) on regenerating head (left panels), bipolar trunk (central panels), and tail (right panels) segments in control and nou-darake (ndk) RNAi animals. The schematic drawings show in red the amputation level of the regenerating pieces shown below. (Adapted from Cebrià et al. 2002.) (B) Putative combinatorial action of gradients of a brain activator (BA) and Wnt activity in regulating neural differentiation along the AP axis (see text for details). Bar, 0.5 mm (head pieces); 1 mm (trunk pieces); and 0.5 mm (tail pieces).