Expression patterns of Tcf and β-catenin. (A-D) Tcf. (A) Female polyp. Strong expression is visible in the developing gonads with weaker expression in the polyp head. (B) Young oocytes expressing Tcf as they enter the developing gonad. (C) Tcf expression in two oocytes located in a newly developed gonad. mRNA is concentrated uniformly around the germinal vesicle. (D) Oral (top) view of mature feeding polyp. Tcf expression is visible around the mouth. (E-I) β-catenin. (E) Ubiquitous expression in the early stages of development (16-cell embryo and late gastrula). (F) Expression in the larva is ubiquitous, but increased expression is also seen at the future oral pole in a similar domain as Wnt3 (arrowhead). (G) Female polyp. β-catenin is expressed in the head of the polyp, in early oocytes in the germinal zone and in oocytes in early gonads. (H) Male sexual polyp. β-catenin is expressed in the head of the polyp and during spermatogenesis, but not in mature sperm. i, immature male gonad expressing β-catenin during sperm development; m, mature gonad no longer expressing β-catenin. (I) β-catenin is ubiquitously expressed at low levels throughout mature feeding polyps. Increased expression is seen just under the apical tip of the mouth. h, head (A, G and H). Scale bars: 200 μm in A,D-I; 20 μm in B,C.

Altered body proportioning and gene expression after Wnt activation and Tcf knockdown during metamorphosis. (A-F) Primary polyps 1 day after metamorphosis induction. (A) Control primary polyp with both stolon (s) and tentacle buds (t) visible. (B) Azakenpaullone-treated, oralized primary polyp. Ectopic tentacles are visible but no aboral structures (body column or stolons) are present. (C) Rescued primary polyp. Treatment with azakenpaullone and Tcf RNAi results in normal development. (D-F) Tcf RNAi treatment phenotypes. (D) Axial patterning has been inhibited with no bud development of either kind. (E) Only oral development, 3 tentacle buds are visible. (F) Only aboral development, 2 stolons are visible. (G) Graph of the percentage of control and Tcf RNAi-treated primary polyps showing no bud development of either kind. (H) Oral-aboral balance in control and Tcf RNAi-treated animals. Tcf RNAi directed animals to an aboral fate with little or no head formation. However, some animals showed reduced head development and no stolons. Classes 1-8 indicate coded stolon by tentacle values calculated in Table 1. (I-P) Wnt3, Tcf and Brachyury expression 1 day post-metamorphosis, except L and P, which are 2 days after metamorphosis. Top row, control animals; bottom row, azakenpaullone-treated animals. (I-L) Polarized oral expression in normal animals. (I) Tcf. (J) Brachyury (arrowhead). (K,L) Wnt3 (arrowhead). (M-O) Depolarized, ubiquitous expression following azakenpaullone treatment. (M) Tcf. (N) Brachyury. (O) Wnt3. (P) By 24 hours after the end of treatment (48 hours after metamorphosis induction), normal polarization of Wnt3 mRNA has been re-established in oralized animals (arrow). (Q-U) Expression levels of Wnt target genes and non-target controls analyzed by qPCR. Expression levels are normalized to GAPDH. The relative quantity (RQ) of expression of Tcf (Q), Brachyury (R) and Wnt3 (S) in control animals was compared with expression levels in the three treatment groups: Tcf RNAi, 24 hours azakenpaullone and rescue (24 hours azakenpaullone + Tcf RNAi). Vasa (T) and 18s rRNA (U) expression in control and double-treated (15 hours + 24 hours) Tcf RNAi groups. Scale bars: 200 μm.

Regeneration. (A) Schematic illustration of the cutting procedure showing the source of the body column tissue used for the experiments. (B-E) Polyp regeneration 2.5 days following decapitation. (B) Seawater control polyp has regenerated a single head. (C) pGEM-T RNAi-treated control polyp, at the same concentration and time of incubation as Wnt3 and Tcf RNAi-treated polyps, also shows normal head regeneration. (D) Tcf RNAi treatment inhibits head regeneration, despite wound healing occurring normally. (E) Similarly, Wnt3 RNAi-treated polyps have also failed to regenerate heads. (F) Control body column, 3 days post-cutting. The head has regenerated at the oral pole and no development past wound healing occurs at the aboral pole. (G-J) Tcf RNAi-treated body columns. G is 3 days post-decapitation and H-J are over 1 week post-decapitation. (G) Still no head regeneration. (H) Double head regeneration after RNAi knockdown has faded. Heads are separated by branching of the body column. (I) Two smaller offshoot polyps deviating from the oral-aboral axis of the original body column. (J) Phenotype showing the development of four heads and small polyps from the original body column. (K-O) Wnt3 RNAi-treated body columns. K is 4 days post-decapitation; L-O over 1 week post-decapitation. (K) Development of stolon bud (bottom left corner) in the absence of head regeneration. (L) Further development and branching of stolons over time. Head regeneration failed to occur. (M) Multiple heads forming side-by-side (arrowheads) and a smaller polyp budding from the opposite end of the body column. (N) Two small offshoot polyps formed at either end of the body column (arrowheads) with the remainder of the tissue developing into contorted stolon-like tissue. (O) Offshoot polyp formed at a right angle to the original oral-aboral axis. The rest of the body column has lost the tissue identity of a polyp body. (P-S) Azakenpaullone-treated body columns. (P) A body column treated for 24 hours post-cutting develops heads at both ends. (Q) A body column treated for 48 hours lost polyp body shape, forming a ball-like structure. (R,S) Following the ball stage, the columns that had been treated for 48 hours have been repatterned to an oralized fate. Ectopic tentacles develop while body tissue is absent. Scale bars: 200 μm. t, tentacles; s, stolons.

Wnt3 expression in regeneration. (A-E) Control regeneration. (A) 1 day post-decapitation. A spot of Wnt3 expression appears at the future site of head formation. (B) 36 hours post-decapitation. Regenerated hypostome, expressing Wnt3 at the oral tip, and early tentacle buds are visible. (C) 36 hours post-decapitation. Broader Wnt3 expression domain at the oral pole. (D) 2 days post-decapitation. Wnt3 expression remains restricted to around the mouth. (E) 7 days post-decapitation. Fully regenerated polyp. Wnt3 expression remains restricted to the oral pole. (F-I) Wnt3 RNAi-treated body columns. (F) 1 day post-decapitation. Treated polyps fail to re-establish Wnt3 expression. (G) 36 hours post-decapitation. An additional Wnt3-expressing spot appears at the top and bottom of the polyp after RNAi inhibition had dissipated. (H) 2 days post-decapitation. A stolon bud developed in the absence of a Wnt3 signal (bottom right of polyp) and a single expression spot has later been established (top of polyp). (I) 2 days post-decapitation. Multiple Wnt3 expression domains remain established (top and bottom left of polyp). (J,K) Tcf RNAi-treated body columns. (J) 36 hours post-decapitation. Two broad expression domains have been established (putative head regeneration sites). (K) 2 days post-decapitation. Multiple expression sites are still present although heads have yet to regenerate. (L-O) Late-stage regenerated Wnt3 RNAi polyps. (L) Ectopic expression in two polyps after head regeneration. The asterisk denotes ectopic Wnt3 expression along the extended hypostome of the first polyp. The second polyp (left) has ectopically regenerated two heads, each showing Wnt3 expression localized around the mouth, visible in insets 1 and 3 at higher magnification. The left polyp also has a stolon-like outgrowth (inset 2) that does not express Wnt3. (M) Polyp in which, after RNAi treatment, stolonal tissues developed. Stolons do not express Wnt3. After the RNAi inhibitory phase ended, a single head regenerated, showing normal oralized expression (asterisk). (N) Ectopic Wnt3-expressing oral outgrowth (asterisk). The outgrowth is located on the side of the polyp body column, not at a pole. (O) Ectopic stolon bud formation. Stolon budding occurred in the absence of Wnt3 expression. Scale bars: 200 μm in A-K; 500 μm in L,M; 50 μm in N,O.