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

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RESEARCH REPORT| 15 March 2009

Supplementary information

Supplemental Figure S1Fig. S1. Sequence analysis of the Smed-Evi protein. (A) Alignment of Evi proteins from different species, showing four conserved cysteines (pink), and eight putative transmembrane domains (blue asterisks). Smed-Evi transmembrane domains were predicted with the help of TMpred (http://www.ch.embnet.org/software/TMPRED_form.html). (B) Phylogenetic tree of Evi proteins inferred from the accurate multiple amino-acid sequence of Evi proteins obtained using the MAFFT version 5.8 (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/). The CAT+Γ model in an MCMC framework, as implemented in PhyloBayes (http://www.lirmm.fr/mab/). The complete Evi/Wls amino acid sequences were used. Smed-Evi groups with invertebrate members. Its closer position to Ce-Evi/Wls is probably due to the phenomenon of �long branch attraction�. No Evi protein from any other Lophotrocozoan has been reported. Ag, Aedes aegypti; Ce, Caenorhabditis elegans; Ci, Ciona intestinalis; Dm, Drosophila melanogaster; Dr, Danio rerio; Hs, Homo sapiens; Mm, _Mus musculu_s; Sp, Strongylocentrotus purpuratus; Tc, Tribolium castaneum; Xl, Xenopus laevis. Accession numbers: Ag-Evi/Wls, XP_001237950; Ce-Evi/Wls, NP_001022275; Ci-Evi/Wls, XP_002123798; Dm-Evi/Wls, NP_729681; Dr-Evi/Wls, NP_998311; Hs-Evi/Wls, NP_079187; Mm-Evi/Wls, NP_080858; Sp-Evi/Wls, XP_001181848; Tc-Evi/Wls, XP_974084; Xl-Evi/Wls, Q66IZ4.

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Supplemental Figure S2Fig. S2. Phenotypic scoring of regenerating Smed-evi RNAi animals. Alive regenerating heads, trunks and tail fragments of Smed-evi RNAi animals where analyzed with a stereomicroscope. (A,A′) All regenerated head fragments showed differentiation of two or more ectopic posterior eyes and two lateral protrusions in the middle region of the animal (A′). (B-H) Bipolar regenerated trunks showed different phenotypes, depending on RNAi penetrance but also on frequent spontaneous scission during regeneration. At early stages of regeneration, all trunk fragments showed ectopic anterior eyes. However, the severity of the phenotype on the posterior part of the animal was different, giving rise to: �tailless� phenotype, without ectopic posterior eyes (C); �two-headed� phenotype with several ectopic anterior eyes, and two posterior ectopic eyes (D); and �two-headed� phenotype with several ectopic anterior and posterior eyes (E). Approximately 50% of the �two-headed� animals underwent spontaneous scission, giving rise to an anterior part, which generated another �tailless� phenotype (F), or even a secondary posterior head (G). The posterior fragment generated after scission was not able to regenerate a normal anterior head, and remained single-headed, with multiple posterior eyes (H). In all cases, animals had two lateral protrusions in the middle region of the body, which never showed ectopic eyes. (I,I′) Each regenerated tail fragment had ectopic anterior eyes, and a �heart-like� shape, due to a tissue regression of the tail region (I′). (J) Quantification of phenotypes. Planarians with an ectopic posterior head, in which two eyes were detected, are named �Two- headed�. When additional anterior or posterior eyes appeared, we refer to the phenotype as �Two-headed with ectopic anterior/posterior eyes�. Red arrows indicate ectopic eyes. Yellow arrows indicate normal anterior eyes. Orange arrowheads point at lateral protrusions. Scale bar: 300 µm in A-B′; 500 µm in C-I.

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Supplemental Figure S5Fig. S5. Expression analysis and functional characterization of Smed-wntA. (A,A′) Smed-wntA mRNA expression in intact animals. Smed-wntA is expressed in the posterior region of the cephalic ganglia (yellow arrowheads in A,A′), in the ventral nerve cords (white arrows) and in the pharynx (white asterisk). (B) Smed-wntA mRNA expression during regeneration. In head fragments, which must regenerate the rest of the body, Smed-wntA is detected in the posterior region of the cephalic ganglia (yellow arrowheads) and in the primordial of the pharynx (orange arrowhead). In bipolar regenerating fragments, Smed-wntA mRNA is detected in the new nervous tissue, both in the anterior (yellow arrows) and in the posterior blastema (white arrows), and also in the pharynx (white asterisk) and the mouth (red arrowhead). Its expression is not detected in tail fragments, which must regenerate the rest of the body, at 4 days after dissection. (C-E) Phenotype of _Smed-wntA_-silenced planarians. After 20 days of regeneration, _Smed-wntA_-silenced planarians show a defect in the shape of the new head, as it seems smaller and more round (C). Analysis with molecular markers shows that the brain of _Smed-wntA_-silenced animals is expanded posteriorly (gluR and DAPI), and that brain branches have an irregular shape (gluR; D,E). Immunodetection of Arrestin (VC-1) shows that the visual axons are also expanded posteriorly (red arrowheads; E). Even though the posterior expansion of the brain is similar to that of evi RNAi animals, the elongated visual axons after wntA knockdown are not evident in evi RNAi animals, probably because of the complexity of the phenotype. Scale bar: 500 µm in A; 300 µm in B; 250 µm in A′,C. Our results agree with the phenotype reported after DjwntA RNAi (Kobayashi et al., 2007). _DjwntA_-silenced planarians also show an expansion of the brain, with differentiation of ectopic and irregular brain branches. However, in the reported study, the analysis with different molecular markers suggests that the expanded region corresponds to the anterior part of the ganglia, resulting in the expansion of the visual center and the appearance of several ectopic eyes. We did not observe differentiation of ectopic eyes in S. mediterranea. More molecular markers should be used in order to address this issue.

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Supplemental Figure S4Fig. S4. Phylogenetic analysis of the S. mediterranea Wnt family. Phylogenetic tree inferred from the accurate multiple amino-acid sequence alignment of Wnt proteins using MAFFT version 5.8 (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/). The CAT+Γ model in an MCMC framework, as implemented in PhyloBayes (http://www.lirmm.fr/mab/) was used. Only the Wnt domain, as determined in SMART (http://smart.embl-heidelberg.de) and the Protein families database (http://www.sanger.ac.uk/Software/Pfam/index.shtml), was used. Confidence values are indicated in the main nodes. Smed-Wnt5 is the only S. mediterranea Wnt that clusters with one of the already described Wnt subfamily (Wnt5) with a high confidence value. Smed-Wnt2-1, together with Dj-WntB (the homolog from the planarian species Dugesia japonica) and Sj-Wnt10 (from Schistosoma japonicum, also a Platyhelminth) cluster into the Wnt2 subfamily; Smed-Wnt11-1 and Smed-Wnt11-2 into the Wnt11 subfamily. Although with a low confidence value, both results are reproducible when inferring different phylogenetic trees. Smed-WntA, Smed-WntP-2 and Smed-WntP-3 cluster with Dj-WntA and Sj-Wnt4 (from Dugesia japonica and Schistosoma japonicum, Platyhelminthes), but do not group with any of the already described Wnt subfamilies. Smed-WntP-1 and Smed-WntP-4 cluster, but also do not fall into any of the already described Wnt subfamilies. The clustering of Nv-WntA with Smed-WntP-1 and Smed-WntP-4 is probably due to the phenomenon of �long branch attraction�. S. mediterranea Wnts are labeled with a red circle. Ag, Anopheles Gambia; Bf, Branchiostoma floridae; Ce, Caenorhabditis elegans; Dj, Dugesia japonica; Gt, Girardia tigrina; Hs, Homo sapiens; Nv, Nematostella vectensis; Pd, Platynereis dumerilii; Pv, Patella vulgata; Sj, Schistosoma japonicum; Smed, Schmidtea mediterranea; Sp, Strongylocentrotus purpuratus; Tc, Tribolium castaneum. Accession numbers: Ag-Wnt1, XP_553580; Ag-Wnt10, XP_318815; Ag-Wnt5, XP_319487; Ag-Wnt6, XP_318816; Ag-Wnt7, XP_001238162; Ag-Wnt9, XP_318818; Ag-WntA, XP_557821; Bf-Wnt1, AAC80432; Bf-Wnt11, AAF80555; Bf-Wnt3, AAL37555; Bf-Wnt4, AAC80431; Bf-Wnt5, AAL37556; Bf-Wnt7b, AAC80433; Bf-Wnt8, AAF80559; Ce-cWn-1, NP_493668; Ce-cWn-2, NP_501822; Ce-egl-20, NP_501754; Ce-lin-44, NP_001021081; Ce-mom-2, NP_505154; Dj-WntA, BAD93239; Dj-WNTB, BAD93245; Gt-Wnt5, AAO17782; Hs-Wnt1, NP_005421; Hs-Wnt10a, NP_079492; Hs-Wnt11, CAA73223; Hs-Wnt16, NP_476509; Hs-Wnt2b, NP_004176; Hs-Wnt3a, AAI03922; Hs-Wnt4, NP_110388; Hs-Wnt5A, NP_003383; Hs-Wnt6, NP_006513; Hs-Wnt7b, NP_478679; Hs-Wnt8a, Q9H1J5; Hs-Wnt9A, NP_003386; Hs-Wnt9B, O14905; Nv-Wnt1, AAT00640; Nv-Wnt10, AAT00641; Nv-Wnt11, AAV87175; Nv-Wnt16, ABF48091; Nv-Wnt2, AAW28132;Nv-Wnt3, ABF48092; Nv-Wnt4, AAV87174; Nv-Wnt6, AAW28134; Nv-Wnt7, AAV87176; Nv-Wnt8, AAW28136; Nv-WntA, AAT02182; Pd-Wnt1, CAD37164; Pd-Wnt4, CAD37166; Pd-WntA, CAD37169; Pv-Wnt1, CAD37170; Pv-Wnt10, CAD37172; Pv-Wnt2, CAD37171; Pv-WntA, CAD37173; Sj-Wnt10, ABG73039; Sj-Wnt4, ABG73038; Smed-Wnt2-1, FJ463753; Smed-Wnt11-1, ABY85210; Smed-WntP-1, ABY85207; Smed-WntP-2, ABY85208; Smed-WntP-3, ABY85209; Sp-Wnt1, NP_001116972; Sp-Wnt10, XP_781564; Sp-Wnt3, XP_790595; Sp-Wnt4, XP_797603; Sp-Wnt4b, XP_786346; Sp-Wnt5, XP_779946; Sp-Wnt6, XP_790077; Sp-Wnt7, XP_796616; Sp-Wnt7b, XP_787051; Sp-Wnt8, NP_999832; Tc-Wnt1, NP_001107822; Tc-Wnt10, XP_968210; Tc-Wnt11, XP_969261; Tc-Wnt5, XP_974684; Tc-Wnt6, XP_968055; Tc-Wnt7, XP_973159; Tc-Wnt9, XP_967898; Tc-WntA, XP_972893; Tc-WntD/8, XP_971439.

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