Smedβ-catenin-a(RNAi) and SmedAPC(RNAi) phenotypes manifest early and the SmedAPC(RNAi) phenotype depends on Smedβ-catenin-a. (A) Anterior (SmedSfrp-a) and (B) posterior (SmedFz-d) marker expression time-course in regenerating trunk fragments. Anterior (ant.) and posterior (post.) blastemas from the same representative fragments are shown, n≥15 per condition. Scale bars: 200 μm. (C) Double-silencing experiments assaying SmedSfrp-a expression in anterior trunk blastemas 4 days post-amputation; scored as High, Low, or Absent (see examples, top row). Percent of scored animals are listed for each category. n≥24 per condition. Control: unc-22(RNAi).

Smedβ-catenin-a(RNAi) transforms non-regenerating tissues. (A through F) Tail fragments. (A,B,D,E) Anterior marker (SmedSfrp-a) analyses 24 and 48 hours post-amputation demonstrated early ectopic expression in Smedβ-catenin-a(RNAi), n≥5 per condition. (C,F) Live tail fragments 14 days post-amputation. Smedβ-catenin-a(RNAi) caused lateral ectopic protrusions (arrowheads, ectopic photoreceptors; arrows, abnormal protrusions). (G through R) Transformation of tail into head tissue in uncut Smedβ-catenin-a(RNAi) animals 14 days after final RNAi-feeding. (G, M) Living animals. (I, O) PC2 in situ hybridization (CNS), n≥4. (K, Q) Anterior marker expression, n≥4. (H,J,L,N,P,R) Magnification of tail tips (boxes in G, M). (H, N) VC-1 antibody staining (photoreceptors). Control: unc-22(RNAi). Scale bars: 200 μm.

Signaling through β-catenin defines head vs. tail during regeneration. (A) Canonical Wnt pathway. Numbers: S. mediterranea homologs identified and silenced. (B) Experimental strategy. (C through V) Trunk fragments 14 days post-amputation. Dashed lines: amputation planes. Control: unc-22(RNAi). (C through F) Live animals. (D, E) Smedβ-catenin-a(RNAi) and SmedDvl-a/b(RNAi) posterior blastemas formed heads with photoreceptors (arrowheads). SmedDvl-a/b(RNAi) fragments also developed ectopic photoreceptors, often within old tissues (arrows). (F) SmedAPC(RNAi) anterior blastemas formed tails. (G through V) In situ hybridizations, n≥5 per marker. Markers: CNS, pro-hormone convertase 2 (PC2); Gut, Porcupine (SmedPorcn-a); Anterior, secreted Frizzled-related protein (SmedSfrp-a); Posterior, Frizzled (SmedFz-d). (G,K) Normal brain and ventral nerve cords; single anterior and dual posterior gut branches (dotted lines). (H,I,L,M) Smedβ-catenin-a(RNAi) and SmedDvl-a/b(RNAi) posterior blastemas developed brain tissue and head-like gut branches. (J,N) SmedAPC(RNAi) anterior blastemas developed tail-like nerve cords and gut branches. (O,S) Normal anterior and posterior marker expression. (P,Q,T,U) Smedβ-catenin-a(RNAi) and SmedDvl-a/b(RNAi) induced anterior marker expression at both ends while the posterior marker was virtually undetectable. (R,V) SmedAPC(RNAi) induced posterior marker expression at both ends while the anterior marker was virtually undetectable. Scale bars: 200 μm.

Smedβ-catenin-a, Dvl-a/b, APC (RNAi) phenotypes do not depend on position or orientation of amputation. Fragments shown: 14 days post-amputation. Control: unc-22(RNAi). (A through E) PC2 in situ hybridization (CNS), n≥5. Smedβ-catenin-a(RNAi) and SmedDvl-a/b(RNAi) animals regenerated posterior brain tissue; SmedAPC(RNAi) animals failed to regenerate anterior brain tissue. (F through M) During lateral regeneration, Smedβ-catenin- a(RNAi) and SmedDvl-a/b(RNAi) animals developed supernumerary heads and photoreceptors; SmedAPC(RNAi) prevented proper regeneration of these structures, n≥15. Photoreceptors visualized with VC-1 antibody (Kind gift from Dr. K. Agata) (25). (N through U) In situ hybridization, n≥5 per marker. Anterior marker (SmedSfrp-a) expression expanded posteriorly along the blastema in Smedβ-catenin-a(RNAi) and SmedDvl-a/b(RNAi) animals, while SmedAPC(RNAi) restricted expression to pre-existing tissues. Posterior marker (SmedFz-d) expression was severely reduced in Smedβ-catenin-a(RNAi) and SmedDvl-a/b(RNAi) animals, but expanded throughout regenerated tissues in SmedAPC(RNAi) animals. (F through I) Live animals. White arrowheads: photoreceptors. Black arrowheads: extent of anterior marker expression. Scale bars: 200 μm.