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First published online 11 February 2009
doi: 10.1242/dev.033761

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Smed-Evi/Wntless is required for β-catenin-dependent and -independent processes during planarian regeneration

¹ Department of Genetics and Institute of Biomedicine of the University of Barcelona (IBUB), 08028 Barcelona, Spain.
² German Cancer Research Center, Division of Signaling and Functional Genomics, and University of Heidelberg/Faculty of Medicine Mannheim, Department of Cell and Molecular Biology, 69120 Heidelberg, Germany.

View larger version (62K): [in this window] [in a new window]   Fig. 1. An Evi homolog is expressed in planarians. (A) Human, fly and planarian Evi proteins. Protein sizes and sequence identities are indicated. Putative transmembrane domains are blue. (B,C) Whole-mount in situ expression analysis of evi mRNA in Schmidtea mediterranea. In intact animals, Smed-evi mRNA is expressed in the cephalic ganglia (CG), the ventral nerve cords (VNCs), the mouth, the pharynx, and cells of the posterior parenchyma (B). During regeneration (C), Smed-evi is expressed in the regenerating nervous system, and is upregulated in the pharynx and in posterior blastemas (arrows). Shown are head, trunk and tail fragments six days after dissection. Anterior is left, posterior is right. Scale bar: 500 µm.

View larger version (61K): [in this window] [in a new window]   Fig. 2. Smed-evi RNAi phenotypes. (A-E) Smed-evi RNAi results in anteriorized planarians. Images of live animals (A,D,D'), and animals stained for visual Arrestin (A',D''). Note the formation of a posterior head, indicated by white arrows pointing towards ectopic eyes. Orange arrows indicate ectopic lateral protrusions (see also I'). Loss of the optical chiasm is indicated by the white arrowhead in D''. (B,E) Whole-mount in situ hybridization against Smed-glutamate receptor (gluR) mRNA shows the formation of an ectopic brain instead of a tail in Smed-evi RNAi animals (arrow). (C) Whole-mount in situ hybridization against Smed-hoxD mRNA indicates the loss of posterior and medial identities after Smed-evi RNAi. (F) RNAi against Smed-β-catenin1 leads to fully anteriorized planarians. (G,G',I,I') Smed-evi RNAi results in growth and patterning defects of the regenerating nervous system. The nervous system was labeled with an anti-Synapsin antibody (green). The position of Arrestin-positive eyes is indicated in red (pseudocolor). In control head fragments (G) CG (red arrows) are located dorsally above the VNCs (blue arrows). After Smed-evi RNAi (G'), the posterior CG are detected in dorsal and ventral sections, laterally to the VNCs (deflected-brain phenotype). (I,I') Regenerating trunk fragments. After Smed-evi RNAi, both anterior and posterior nervous tissue show the deflected-brain phenotype. CG,red arrows; VNCs, blue arrows, putative VNCs projected from posterior CG, purple arrows; disconnections between the old and the new nervous tissue, white arrows. (H,J) Synapsin expression in regenerating head and trunk fragments after Smed-β-catenin1 RNAi. Regenerating head and trunk animals correspond to 20 and 25-30 days of regeneration. Images are z-projections of several confocal sections. Dorsal and ventral images are single sections. Anterior is left, posterior is right.

View larger version (45K): [in this window] [in a new window]   Fig. 3. Smed-wnt11-2 and Smed-wntP-1 are required for posterior identity during planarian regeneration. (A-D) Whole-mount in situ hybridization analysis of Smed-wnt11-2 and Smed-wntP-1 mRNAs (arrows) in intact (A,B) and regenerating animals at day 4 of regeneration (C,D). (F,G) RNAi against Smed-wnt11-2 and Smed-wntP-1 leads to a `tailless' phenotype (red arrows, compare with control in E). In some animals (see quantification in G''), Smed-wntP-1 RNAi causes the generation of ectopic posterior heads (white arrows in G). Shown are images of live regenerating trunk fragments (E-G), and confocal z-projections of trunk fragments stained with anti-Synapsin (E'-G'), at day 20 of regeneration.

View larger version (92K): [in this window] [in a new window]   Fig. 4. Smed-wnt5 regulates growth and patterning of the planarian regenerating nervous system. (A,B) In situ hybridization analysis of Smed-wnt5 mRNA in intact (A) and regenerating (B) animals. Smed-wnt5 is expressed in discrete cells along the VNCs and the CG (red arrows), as well as in cells in the periphery and along the DV boundary (blue arrows). It is upregulated in the regenerating CG of the anterior blastema (white arrows in B). (C,D) Control and Smed-wnt5 RNAi trunk fragments at day 20 of regeneration, stained with anti-Synapsin. Red arrows point to CG, blue arrows to VNCs. Note the deflection and expansion of regenerating nervous tissue in Smed-wnt5 RNAi animals. White arrows indicate the disconnection between old and new nervous tissue. Images are z-projections, dorsal and ventral views are single sections. (E) In situ hybridization analyis of gluR mRNA. No brain tissue is detected in the tail. (F) Summary of RNAi phenotypes of regenerating trunk fragments. Shown are dorsal and lateral views. Yellow indicates CG; green, VNCs. A, anterior; P, posterior; D, dorsal; V, ventral.

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