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Fig. S1. Expression pattern of Smed-βcatenin1 and Smed-βcatenin2 by whole-mount in situ hybridization. (A) Smed-βcatenin1 mRNA shows a ubiquitous expression with high levels in the central nervous system (cephalic ganglia and ventral nerve cords) and in the pharynx. (B) Smed-βcatenin2 mRNA is detected in the digestive system and in the periphery of the cephalic ganglia. Anterior is to the left. Asterisks indicate the pharynx. agb, anterior gut branch; cg, cephalic ganglia; pgb, posterior gut branch; vnc, ventral nerve cords. Scale bar: 300 µm.
Fig. S2. Expression pattern of Smed-βcatenin1 and Smed-βcatenin2 during regeneration. Whole-mount in situ hybridization of Smed-βcatenin1 (A-D) and Smed-βcatenin2 (E-H) at different stages of regeneration. Both Schmidtea mediterranea β-catenins are expressed in both blastemas during regeneration (A-C,E-G). Smed-βcatenin1 is also detected in sensory cells in the head periphery (arrowheads in C). Smed-βcatenin2 expression in the digestive system appears first as two spots in the posterior blastema (arrowheads in F). Smed-βcatenin2 is also detected in some cells around the cephalic ganglia (arrowheads in G). The dotted lines indicate the blastema edges. Anterior is to the left. dR, days of regeneration; b, blastema; pb, postblastema. Scale bar: 300 µm.
Fig. S3. Efficiency of Smed-βcatenin1 and Smed-βcatenin2 silencing after dsRNA injection. Smed-βcatenin1 (A) and Smed-βcatenin2 (C) are expressed in both the anterior and posterior blastemas in trunk pieces after 3 days of regeneration. After Smed-βcatenin1 dsRNA (B) and Smed-βcatenin2 dsRNA (D) injections, Smed-βcatenin1 and Smed-βcatenin2 expression is not detected in regenerating trunk pieces. Anterior is to the left. Scale bar: 300 µm.
Fig. S4. Protein alignment showing the conservation of functional domains between Smed-βcatenin1, Smed-βcatenin2 and β-catenins from other species. The domains involved in Wnt-signaling and in adhesion are marked in red and blue, respectively. The GSK3-binding domain contains the residues phosphorylated by CKI (Ser45) and GSK3 (Ser33, Ser37, Thr41), which are indicated by arrows. Sequential phosphorylation of β-catenin by CKI and GSK3 target it for ubiquitination and proteolytic degradation. The domain is conserved in Smed-βcatenin1 but not in Smed-βcatenin2. Two domains implicated in α-catenin binding are located N-terminal to the Armadillo repeats. The distance between them (21 aa) is also a conserved feature. These domains are conserved in Smed-βcatenin2 but not Smed-βcatenin1. A PDZ-like target sequence at the most C-terminal end is also conserved in β-catenins from all species. This domain has been linked to E-cadherin binding and is conserved in Smed-βcatenin2 but not Smed-βcatenin1. The C-terminal region of β-catenin functions in transactivation and contains strong activation domains that bind to chromatin-remodeling subunits. This region is marked with a dashed red line. Y142 (marked with a black diamond) is a conserved residue located just before the first Armadillo repeat. The phosphorylation state of this residue modulates the switch between adhesion and transcription. Its phosphorylation induces BCL9/legless binding and inhibits the α-catenin interaction. It is conserved in Smed-βcatenin1 but not Smed-βcatenin2. The 12 Armadillo repeats are underlined (R1-R12). Note that the domains conserved in Smed-βcatenin1 and Smed-βcatenin2 correspond to the same conserved regions in Ce-BAR1 (with a signaling function) and Ce-HMP2 (with an adhesion function), respectively. Accurate multiple amino-acid sequence alignments of β-catenin proteins were obtained using MAFFT version 5.8 (http://timpani.genome.ad.jp/%7Emafft/server/) and edited using the BioEdit Sequence Alignment Editor. Hs, Homo sapiens; Bf, Branchiostoma floridae; Lv, Litechinus variegatus; Hv, Hydra vulgaris; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans.
Fig. S5. Phylogenetic analysis of β-catenin homologs from different species. Phylogenetic analysis demonstrates that the Schmidtea mediterranea β-catenin duplication is independent of C. elegans β-catenin duplication. The two S. mediterranea β-catenins are shown in bold and red. The three homologs of C. elegans are in blue. Plakoglobin (shown in green), which is a vertebrate-specific β-catenin duplication, has also been included in the phylogenetic study. Accurate multiple amino-acid sequence alignments of β-catenin and plakoglobin proteins were obtained using MAFFT version 5.8 (http://timpani.genome.ad.jp/%7Emafft/server/), and, to infer the phylogenetic tree from the amino-acid alignment, we used the neighbor-joining method with a JTT matrix using the MEGA software version 3.1. For the neighbor joining, a bootstrap analysis (500 replicates) was also done. Full-length amino-acid sequences were used for all proteins. Accession numbers are: Bf-bcat, AAY34439; Ce-BAR-1, AAC17424; Ce-HMP-2, AAB94552; Ce-wrm-1, AAC47748; Ci-bcat, BAA92185; Cv-bcat, AAL49497; Dd-Aardvark, AAG17931; Dj-bcat, BAD93243; Dm-armadillo, P18824; Dr-plakoglobin, AAH58305; Hs-bcat, P35222; Hs-plakoglobin, CAA92522; Hv-bcat, AAQ02885; Lv-bcat, AAC06340; Mm-plakoglobin, AAH40757; Pp-bcat, AAX47336; Sp-bcat, NP_001027543; Tg-bcatg, P35223; Tt-bcat, BAD90106; Uc-bcat, P35224. Bf, Branchiostoma floridae; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Cv, Chaetopterus variopedatus; Dd, Dictyostelium discoideum; Dj, Dugesia japonica; Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; Hv, Hydra vulgaris; Lv, Lytechinus variegatus; Mm, Mus musculus; Pp, Pristionchus pacificus; Sp, Strongylocentrotus purpuratus; Tg, Tripneustes gratilla; Tt, Tubifex tubifex; Uc, Urechis caupo.
Movie 1. Radial-like hypercephalized planarian in vivo. The animal, which has undergone a striking transformation from bilateral to radial symmetry, has lost its sense of direction following abolition of the anteroposterior axis as a result of Smed-βcatenin1 RNAi. The movie shows the regenerating trunk of a Smed-βcatenin1 RNAi-treated animal after 24 days of regeneration.
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