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β-Catenin Defines Head Versus Tail Identity During Planarian Regeneration and Homeostasis

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Science  18 Jan 2008:
Vol. 319, Issue 5861, pp. 323-327
DOI: 10.1126/science.1150029

Abstract

After amputation, freshwater planarians properly regenerate a head or tail from the resulting anterior or posterior wound. The mechanisms that differentiate anterior from posterior and direct the replacement of the appropriate missing body parts are unknown. We found that in the planarian Schmidtea mediterranea, RNA interference (RNAi) of β-catenin or dishevelled causes the inappropriate regeneration of a head instead of a tail at posterior amputations. Conversely, RNAi of the β-catenin antagonist adenomatous polyposis coli results in the regeneration of a tail at anterior wounds. In addition, the silencing of β-catenin is sufficient to transform the tail of uncut adult animals into a head. We suggest that β-catenin functions as a molecular switch to specify and maintain anteroposterior identity during regeneration and homeostasis in planarians.

β-Catenin is a multifunctional protein that controls transcriptional output as well as cell adhesion. During embryonic development of both vertebrates and invertebrates, β-catenin regulates a variety of cellular processes, including organizer formation, cell fate specification, proliferation, and differentiation (19). In adult animals, the Wnt/β-catenin pathway participates in regeneration and tissue homeostasis; misregulation of this pathway can lead to degenerative diseases and cancer in humans (912). In response to upstream cues, such as Wnt ligands binding to Frizzled receptors, β-catenin accumulates in nuclei (Fig. 1A) and invokes transcriptional responses that direct the specification and patterning of tissues (13, 14). Adenomatous polyposis coli (APC) is an essential member of a destruction complex that phosphorylates β-catenin, resulting in its constitutive degradation. Hence, loss of APC leads to a rise in β-catenin levels that is sufficient to drive transcriptional responses (15). The intracellular protein Dishevelled has multiple functions but plays an essential role as a positive regulator of β-catenin by inhibiting the destruction complex (16).

Fig. 1.

Signaling through β-catenin defines head versus tail during regeneration. (A) Canonical Wnt pathway. Numbers denote S. mediterranea homologs identified and silenced. (B) Experimental strategy. (C to V) Trunk fragments 14 days after amputation. Dashed lines, amputation planes; control, unc-22(RNAi). (C) to (F): Live animals. (D) and (E): Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) posterior blastemas formed heads with photoreceptors (arrowheads). Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) fragments also developed ectopic photoreceptors, often within old tissues (arrows). (F): Smed-APC-1(RNAi) anterior blastemas formed tails. (G) to (V): In situ hybridizations (n ≥ 5 per marker). Markers: CNS, prohormone convertase 2 (PC2); gut, porcupine (Smed-porcn-1); anterior, secreted frizzled-related protein (Smed-sFRP-1); posterior, frizzled (Smed-fz-4). (G) and (K): Normal brain and ventral nerve cords; single anterior and dual posterior gut branches (dotted lines). (H), (I), (L), and (M): Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) posterior blastemas developed brain tissue and head-like gut branches. (J) and (N): Smed-APC-1(RNAi) anterior blastemas developed tail-like nerve cords and gut branches. (O) and (S): Normal anterior and posterior marker expression. (P), (Q), (T), and (U): Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) induced anterior marker expression at both ends, whereas the posterior marker was virtually undetectable. (R) and (V): Smed-APC-1(RNAi) induced posterior marker expression at both ends, whereas the anterior marker was virtually undetectable. Scale bars, 200 μm.

As part of a systematic effort to define the roles of signaling pathways in planaria, we analyzed the canonical Wnt signaling system in Schmidtea mediterranea. We cloned and determined the expression patterns of all identifiable homologs of core pathway components (Fig. 1A) and silenced them, individually or in combinations, on the basis of likelihood of redundancy as gleaned from the expression data (figs. S1 to S6 and table S1). RNAi-treated animals were then amputated to assess the role of the silenced genes during regeneration (Fig. 1B). After head and tail amputations of control worms, the remaining trunk formed one anterior and one posterior blastema, which then differentiated to replace the missing structures (Fig. 1C; n = 28). However, RNAi of a single β-catenin (Smed-βcatenin-1), both dishevelled homologs (Smed-dvl-1;Smed-dvl-2), or APC (Smed-APC-1) caused striking alterations in the anteroposterior (A/P) identity of regenerating tissues (Fig. 1, D to F). Both blastemas of Smed-βcatenin-1(RNAi) worms adopted an anterior fate, resulting in animals with two heads of opposite orientation (penetrance = 100%, n = 39). Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) worms also regenerated heads from both blastemas but displayed additional phenotypes, including ectopic and supernumerary photoreceptors in the anterior region. This is consistent with the multiple roles of Dishevelled in different pathways (Fig. 1E; penetrance = 75%, n = 20). On the other hand, Smed-APC-1(RNAi) animals regenerated tails from both amputation planes (Fig. 1F; penetrance = 60%, n = 43).

To address whether these changes were superficial or reflected a fate transformation of internal cell types and organ systems, we used anatomical and molecular markers of A/P identity. Anatomically, two organ systems with characteristic asymmetries along the A/P axis were examined: (i) the central nervous system (CNS), composed of two anterior cephalic ganglia (brain) and two ventral cords projecting posteriorly (Fig. 1G), and (ii) the digestive system, consisting of a single anterior and two posterior gut branches (Fig. 1K). The “posterior” head of Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) animals contained a characteristically anterior nervous system and gut, as did the “anterior” head (Fig. 1, H, I, L, and M). In contrast, the “anterior” tail of Smed-APC-1(RNAi) animals was devoid of discernible brain tissue and exhibited posterior structures, as did the “posterior” tail (Fig. 1, J and N).

To define the molecular extent of A/P misspecification, we used two markers identified during our in situ analyses that are specifically expressed at the anterior and posterior ends of intact and regenerating animals (Fig. 1, A, O, and S). Smed-sFRP-1, a homolog of secreted Frizzled-related proteins (sFRP), was expressed in an arch of cells capping the anterior edge of the animal. In contrast, Smed-fz-4, a homolog of Frizzled receptors, was expressed at the posterior edge in a posterior-to-anterior gradient. We refer to Smed-sFRP-1 and Smed-fz-4 as the “anterior marker” and the “posterior marker,” respectively, in all subsequent analyses.

In Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) worms, the “posterior” head expressed the anterior marker, whereas the posterior marker was severely reduced or absent (Fig. 1, P, Q, T, and U). Conversely, the “anterior” tail of Smed-APC-1(RNAi) animals expressed the posterior marker, whereas the anterior marker was severely reduced or absent (Fig. 1, R and V). We noted that misspecified heads and tails in RNAi-treated worms moved independently from the rest of the animal, hence this tissue was functioning autonomously (movies S1 to S3). We conclude that silencing Smed-βcatenin-1, Smed-dvl-1(RNAi);Smed-dvl-2(RNAi), or Smed-APC-1 is sufficient to misspecify blastema identity.

The inappropriate regeneration of complete heads and tails in RNAi-treated animals suggested that regulators of β-catenin act early during regeneration. We therefore used the anterior and posterior markers to investigate the onset of blastema differentiation in control and RNAi-treated trunk fragments (Fig. 2, A and B). In control animals at 12 hours after amputation, the anterior marker was virtually undetectable, whereas the posterior marker was clearly evident in the posterior blastema; 24 hours after amputation, the two markers recapitulated the A/P specificity seen later during regeneration and in adult animals (Fig. 1, O and S; Fig. 2, A and B). However, in Smed-βcatenin-1(RNAi) trunks, the anterior marker was expressed at both ends by 12 hours and maintained throughout the experiment, whereas the posterior marker remained markedly reduced (Fig. 2, A and B). The reverse was observed in Smed-APC-1(RNAi) trunks (Fig. 2, A and B). Consistent with the inferred time window for β-catenin signaling, Smed-βcatenin-1 and Smed-APC-1 were expressed at both ends in wild-type animals by 12 hours (fig. S7). These results indicate that β-catenin and APC act very early to determine blastema identity.

Fig. 2.

Smed-βcatenin-1(RNAi) and Smed-APC-1(RNAi) phenotypes manifest early, and the Smed-APC-1(RNAi) phenotype depends on Smed-βcatenin-1. (A) Anterior (Smed-sFRP-1) and(B) posterior(Smed-fz-4) 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 Smed-sFRP-1 expression in anterior trunk blastemas 4 days after amputation; animals were scored as high, low, or absent (see examples, top row). Proportions of scored animals are listed for each category (n ≥ 24 per condition). Control, unc-22(RNAi).

We next used animals silenced for both Smed-βcatenin-1 and Smed-APC-1 to test whether the Smed-APC-1(RNAi) phenotype results from increased β-catenin activity. Under these conditions, anterior blastemas were properly fated, indicating that the misspecification phenotype of Smed-APC-1(RNAi) depends on Smed-βcatenin-1 (Fig. 2C). Additionally, posterior blastemas adopted an anterior fate, indicating that the Smed-βcatenin-1(RNAi) phenotype does not depend on APC activity. The combined data show that signaling through β-catenin occurs at posterior amputations and is necessary and sufficient to specify tail fate. In contrast, signaling through β-catenin is blocked or never occurs at anterior amputations, and this is necessary and sufficient to specify head fate. The premature expression of the anterior marker in Smed-βcatenin-1(RNAi) worms may indicate that in wild-type planarians, β-catenin inhibition does not immediately follow amputation (Fig. 2A). We suggest that β-catenin activity acts as a molecular switch to specify head versus tail fate in planarians.

We then explored whether the β-catenin switch plays a role in blastema identity regardless of the A/P location or angle of amputation. Indeed, the head fragments of Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) worms regenerated a head from the posterior wound (penetrance = 79%, n = 24; penetrance = 82%, n = 33, respectively), and the tail fragments of Smed-APC-1(RNAi) worms regenerated a tail from the anterior wound (penetrance = 67%, n = 27; Fig. 3, A to E, and movie S4). After longitudinal amputation along the midline, control animals formed a blastema along the A/P axis and regenerated mediolaterally (Fig. 3, F, J, N, and R). In contrast, Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) worms regenerated anterior tissue and developed multiple ectopic heads along the lateral edge (Fig. 3, G, H, K, L, O, P, S, and T). The most severely affected Smed-APC-1(RNAi) worms regenerated posterior tissue and failed to replace the lost head structures (Fig. 3, I, M, Q, and U). Thus, the laterally regenerating tissue in RNAi-treated animals was misspecified. Our data indicate that β-catenin activity is regulated during lateral regeneration and that the β-catenin switch can dominantly misspecify regenerating tissues regardless of A/P position or amputation angle.

Fig. 3.

Smed-βcatenin-1, Smed-dvl-1; Smed-dvl-2, and Smed-APC(RNAi) phenotypes do not depend on position or orientation of amputation. Fragments 14 days after amputation are shown; control, unc-22(RNAi). (A to E) PC2 in situ hybridization (CNS) (n ≥ 5). Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) fragments regenerated posterior brain tissue; Smed-APC-1(RNAi) fragments failed to regenerate anterior brain tissue. (F to M) During lateral regeneration, Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) animals developed supernumerary heads and photoreceptors; Smed-APC-1(RNAi) prevented proper regeneration of these structures (n ≥ 15). Photoreceptors were visualized with VC-1 antibody (25). (N to U) Insituhybridization (n ≥ 5 per marker). Anterior marker (Smed-sFRP-1) expression expanded posteriorly along the blastema in Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) animals, whereas Smed-APC-1(RNAi) restricted expression to preexisting tissues. Posterior marker (Smed-fz-4) expression was severely reduced in Smed-βcatenin-1(RNAi) and Smed-dvl-1(RNAi);Smed-dvl-2(RNAi) animals but expanded throughout regenerated tissues in Smed-APC-1(RNAi) animals. (F) to (I): Live animals. White arrowheads, photoreceptors; black arrowheads, extent of anterior marker expression. Scale bars, 200 μm.

Smed-βcatenin-1(RNAi) animals also displayed striking changes in the nonregenerating portions of regenerating fragments. Smed-βcatenin-1(RNAi) tail fragments at 24 and 48 hours after amputation expressed the anterior marker in cell clusters around the circumference of the fragment (Fig. 4, A, B, D, and E). By day 14, in addition to a new head, moving head-like protrusions developed from the periphery (Fig. 4, C and F, and movie S5). Similar protrusions also developed in trunk fragments (movie S2).

Fig. 4.

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

Such fate changes may have been initiated by amputation. We therefore observed unamputated (intact) worms 14 days after the final RNAi feeding. Ectopic photoreceptors were visible in the tail of all (n = 20) Smed-βcatenin-1(RNAi) animals and none of the control animals (Fig. 4, G, H, M, and N). Intact RNAi-treated animals also exhibited ectopic lateral protrusions, formed a brain in the tail region, and expressed the anterior marker posteriorly (Fig. 4, I to L and O to R, and movie S6). The molecular basis for such a change of A/P polarity in an adult organism was previously unknown.

Together, our data demonstrate the fundamental importance of β-catenin in the maintenance of polarity and cell fate during tissue regeneration and homeostasis in planarians (fig. S8). Our findings reveal a dynamic control of β-catenin in adult animals that is not readily apparent during the progression of embryogenesis: The precise quantity and location of regenerating tissue is different for each individual and for each regeneration event, newly regenerated tissues must integrate with the old, and ongoing homeostatic cell turnover may require sustained instructive cues. It is interesting that we did not observe any head or tail misspecification phenotypes for any of the upstream components of canonical Wnt signaling (Wnts, Frizzleds, or Porcupines; see table S1). Although we cannot rule out protein perdurance, incomplete gene silencing, or redundancy with known or unidentified components, the intracellular components of β-catenin signaling may be regulated by an unconventional upstream mechanism to specify polarity during regeneration and/or homeostasis. Indeed, β-catenin regulation can be Wnt-independent in vertebrate cells, and Dishevelled remains the most upstream known β-catenin regulator during early sea urchin development (14, 17, 18). With respect to putative downstream effectors, planarians can regenerate double heads after pharmacological gap junction inhibition, and β-catenin is implicated in gap junction formation and function (1921). Finally, whether specification and maintenance of the planarian A/P axis via β-catenin is or is not independent of Hox proteins remains to be determined.

More than 100 years ago, T. H. Morgan reported that fragments with closely spaced anterior and posterior amputation planes occasionally regenerate two-headed animals (22, 23). He termed these animals “Janus heads” and suggested that “something in the piece itself determines that a head shall develop at the anterior cut surface and a tail at the posterior cut surface” (24). Our results indicate that β-catenin activity is a key target of polarity specification in planarians, providing mechanistic insight into the old, unanswered question of how blastema fate is controlled. We propose that the evolutionarily ancient β-catenin protein, in a manner reminiscent of its role during metazoan embryogenesis (6, 8), acts as a molecular switch in adult planarians and that it may play a similar role in the adult tissues of other animals.

Supporting Online Material

www.sciencemag.org/cgi/content/full/1150029/DC1

Materials and Methods

Figs. S1 to S8

Tables S1

Movies S1 to S6

References and Notes

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