Heads or tails?

Heads or tails

Editor's Introduction

Polarized notum activation at wounds inhibits Wnt function to promote planarian head regeneration

annotated by
Melissa McCartney

Humans seem to be superior to starfish, with a complex nervous system, the ability to walk upright, and opposable thumbs.  However, starfish are capable of regeneration, which is the ability to regrow organs or tissues after injury.  Understanding the molecular mechanisms underlying regeneration could lead to advances in medicine that benefit patients with amputations or nerve damage.  Can we translate what we know about regeneration to humans?

Paper Details

Original title
Polarized notum activation at wounds inhibits Wnt function to promote planarian head regeneration
Authors
Peter Reddien
Original publication date
Reference
Vol. 332 no. 6031 pp. 852-855
Issue name
Science
DOI
10.1126/science.1202143

Abstract

Regeneration requires initiation of programs tailored to the identity of missing parts. Head-versus-tail regeneration in planarians presents a paradigm for study of this phenomenon. After injury, Wnt signaling promotes tail regeneration. We report that wounding elicits expression of the Wnt inhibitor notum preferentially at anterior-facing wounds. This expression asymmetry occurs at essentially any wound, even if the anterior pole is intact. Inhibition of notum with RNA interference (RNAi) causes regeneration of an anterior-facing tail instead of a head, and double-RNAi experiments indicate that notum inhibits Wnt signaling to promote head regeneration. notum expression is itself controlled by Wnt signaling, suggesting that regulation of feedback inhibition controls the binary head-tail regeneration outcome. We conclude that local detection of wound orientation with respect to tissue axes results in distinct signaling environments that initiate appropriate regeneration responses.

Report

How an organism determines what cells or tissues are missing for regeneration is poorly understood. Planarians are freshwater flatworms that can regenerate from nearly any injury (1). The head-versus-tail regeneration decision in planarians, known as regeneration polarity, is a paradigm for studying appropriate regeneration program specification (2). Wnt signaling controls regeneration polarity, with pathway components β-catenin-1 (35) and wnt1 (formerly called wntP-1) (68) required to prevent head regeneration and promote tail regeneration at posterior-facing wounds. wnt1 expression is up-regulated near both anterior- and posterior-facing wounds (6, 8, 9). Therefore, how wnt1 and β-catenin act to promote tail formation only at appropriate wounds is unknown.

We sought factors that inhibit Wnt signaling at anterior-facing wounds to promote head regeneration, and we identified a planarian homolog of Drosophila notum (Smed-notum) (fig. S1) (10). Notum proteins are secreted α/β-hydrolase family members (11, 12), cleave glycosylphosphatidylinositol anchors of cell surface proteins (13), and can act on glypicans to modulate Drosophila Wnt signaling (11, 12, 14, 15). Glypicans are cell surface, heparan-sulfate proteoglycans that participate in several signaling pathways (16). The roles of Notum proteins in development are unknown outside of Drosophila.

notum was expressed at the planarian anterior pole (Fig. 1A). In contrast, wnt1 is expressed oppositely, at the posterior pole (3, 6). Early after head and tail amputation (6 to 24 hours), notum expression was highly up-regulated preferentially near anterior-facing wounds (Fig. 1B and fig. S2). notum expression was weaker and initiated later at posterior-facing wounds. Later after amputation (48 to 72 hours), anterior notum expression was coalesced at the pole, whereas posterior expression remained low (Fig. 1C and fig. S2). notum was expressed in subepidermal cells (Fig. 1D) that, at wounds, resemble wnt1-expressing cells (6). Indeed, notum and wnt1 were coexpressed in some cells at anterior-facing wounds (Fig. 1E).

Fig. 1. notum is expressed at anterior-facing wounds. (A to C) notum in situ hybridizations: intact animals (A); regenerating head and trunk fragments over time (hours, h) [(B) and (C)]. Brackets shown in (A) indicate regions imaged in (B) and (C). (D) notum is expressed in anterior-pole, subepidermal cells (arrow). (D and E) The area highlighted by the dashed green box is enlarged in the right panels. (E) Double-fluorescence in situ hybridization (FISH); notum (red) and wnt1 (blue) are coexpressed (arrows) at an anterior-facing wound (yellow dotted line) 18 hours after amputation. (F to I) FISH; notum expression 6 hours after incisions. (Top) The green box depicts the region that was imaged; red lines depict incisions. (Bottom) Dotted red lines show sealed wound location. In (G) and (H), triangular tissue was removed and the wound allowed to seal; tissue between incision sites (~200 μm apart) in (I) was not removed. Anterior to the left [(B) to (I)] or top (A); dorsal view, (A), 72 hours in (C) and (D); or ventral view (all other panels). Images represent ≥four of five animals per panel. Scale bars, 200 μm.
In situ hybridization

In situ hybridization:  a technique using a labeled complementary DNA or RNA strand to localize a specific DNA or RNA sequence in a portion or section of tissue (in situ).  This is distinct from immunohistochemistry, which localizes proteins. 

Click here for a video describing in situ hybridization. 

Panel A

Picture1.jpg

In panel A, the line across the planaria represents the cut, or wound, that the authors made. This results in two planaria, each regenerating a different section (remember: posterior/tail and anterior/head). The authors experiment with a variety of wound types to examine what (or what does not) influence notum upregulation at the anterior-facing side of the wound.

Panel B and C

B C.jpg

Here, the authors present a time-course study of the expression of notum at the wound site. The planaria was wounded as indicated by the line in panel A. The top panels in B and C are the head section of the planaria in panel A which has a posterior-facing wound, meaning this part of the planaria will grow a new tail. The bottom panels in B and C are the tail section of the planaria in panel A which has an anterior-facing wound, meaning this part of the planaria will grow a new head. 

Panel D and E

D E.jpg

Panel D shows the cell type where notum is expressed. Panel E shows that both notum and wnt are expressed in cells lining the anterior-facing wound.

Panel F

F.jpg

Here, tissue was not removed from the planaria as it was in panels A through E. Instead, a simple incision was made (the red line) and notum expression was still up-regulated.

Panel G and H

G H.jpg

Diagonal wounds were applied (and the tissue removed) to the planaria to show that notum up-regulation at the anterior-facing side of the wound does not depend on the wound being perpendicular to the body axis of the planaria. Nor does expression depend upon the presence or absence of poles from the planarian.

Panel I

I.jpg

Two wounds close together (with no tissue removed) still show up-regulation of notum only on the anterior-facing side of the posterior-most wound, but not on the anterior-facing side of the anterior-most wound.

To test whether wound-site notum expression is specific to head amputation, we incised animal sides without tissue removal. notum expression was detected specifically on the anterior-facing side of these sealed incisions (Fig. 1F). Therefore, asymmetric notum expression after wounding (greater at anterior-facing rather than posterior-facing wounds) does not require the loss of large tissue regions, such as the anterior pole. Asymmetric wound expression also occurred at sealed incisions diagonal to the main body axis (Fig. 1G) and was independent of anterior or posterior pole presence (Fig. 1H), indicating that local cues rather than signals from poles control notum expression asymmetry at wounds. We conclude that wounding elicits notum expression, dependent on wound-edge orientation with respect to the polarized primary body axis.

Posterior-facing wounds could be nonpermissive, and/or anterior-facing wounds could be specifically instructive, for notum expression. We therefore examined notum expression between two closely opposed wounds. Regions neighboring only an anterior-facing wound had more notum-expressing cells than did regions bordering both anterior- and posterior-facing wounds (11.0 ± 6.9 versus 1.6 ± 2.4 cells, respectively, n = 8 animals) (Fig. 1I). These data suggest that posterior-facing wounds suppress wound-induced notum expression, providing expression asymmetry.

The specificity of strong notum expression for anterior-facing wounds suggests that notum might control regeneration polarity. After head and tail amputation, notum(RNAi) animals [RNA interference (RNAi)] failed to regenerate a head with photoreceptors (47%, n = 113) and regenerated posterior-facing tails apparently normally(Fig. 2A). notum(RNAi) animals that did regenerate at least one photoreceptor did so aberrantly, possibly reflecting a weakly expressive notum(RNAi) phenotype (fig. S3). To characterize notum(RNAi) anterior blastemas lacking photoreceptors, we assessed axial marker expression (Fig. 2, B to F). notum(RNAi) anterior blastemas lacked cephalic ganglia and anterior-pole marker expression (sFRP-1) (Fig. 2, B and C). In contrast, notum(RNAi) anterior blastemas expressed the posterior markers wnt1 and frizzled-4 (Fig. 2, D and E). Furthermore, notum(RNAi) animals regenerated an anterior gut with posterior-specific morphology (two main branches) (Fig. 2F).

We conclude that notum inhibition caused regeneration of an anterior-facing second tail after head and tail amputation. notum double-stranded RNA (dsRNA) delivery only after amputation also resulted in a regeneration polarity reversal (fig. S4), indicating a requirement for new notum expression after wounding.

planaria 2.jpg

Fig. 2. notum is required for head-tail regeneration polarity.(A) notum(RNAi) fragments failed to regenerate a head by 14 days after amputation (47%, n = 133; controls were normal, 100%, n = 101). (B to F) Control or notum(RNAi)-regenerating animals lacking photoreceptors were probed for expression of (B) PC2 (prohormone convertase 2, central nervous system marker), (C) sFRP-1 (anterior-pole marker), (D) wnt1, (E) fzd-4 (posterior markers), or (F) madt (gut marker, green). Blue, Hoechst stain. Arrows indicate lack of anterior marker [(B) and (C)], posterior marker presence [(D) and (E)], or posterior gut morphology (F) in notum(RNAi) animals. cg, cephalic ganglia; pr, photoreceptors. Images are representatives: (B) 9/11, (C) 8/25, (D) 7/24, and (E) 11/38 notum(RNAi) animals; other panels, 100%, n ≥ 7. (G) Anterior- or posterior-facing wounds, probed for wntP-2 expression. wntP-2 has been proposed to be Wnt11-related with the name wnt11-5 (8). Arrows indicate ectopic wntP-2 expression. Images represent ≥five of six animals per panel. Anterior to the left. Scale bars, 500 mm (A); 200 mm (all other panels).
RNAi

RNAi refers to the introduction of homologous double-stranded RNA to specifically target a gene's product (mRNA, which in turn encodes a protein).  Basically, it allows scientists to turn off specific genes.

Panel A

Notum RNAi planarians, meaning notum was not expressed, were unable to regenerate a head.

Panel B and C

The anterior blastemas of notum RNAi planarians lack a brain and do not express a gene normally expressed specifically in the head (sFRP-1). This demonstrates that anterior-facing blastemas are not heads.

Panel D and E

The anterior blastemas of notum RNAi planarians expressed genes that are normally only expressed in the tail. This demonstrates that anterior-facing blastemas have the characteristics of tails instead of heads.

Panel F

The intestine of planarians is visualized. In the tail there are two primary branches of intestine, and in the head there is one primary branch. In notum RNAi anterior blastemas, there are two primary intestine branches indicating that the anterior blastemas are tails.

Panel G

wntP-2 is normally up-regulated at posterior-facing, but not anterior-facing, wounds. wntP-2 has been demonstrated to be activated by wnt1 and β-catenin at posterior-facing wounds in prior published research. notum RNAi planarians (notum expression turned off) expressed wntP-2 abnormally at anterior-facing wounds, indicating that notum normally prevents activation of β-catenin targets at anterior-facing wounds.

wntP-2 [proposed to be Wnt11-related (8)] expression is up-regulated at posterior- and not anterior-facing wounds, and this requires wnt1 and β-catenin-1 (6). Therefore, wntP-2 expression reflects an early readout (i.e., well before the majority of tissue forms) of wnt1/β-catenin–mediated polarity specification. notum(RNAi) fragments expressed wntP-2 ectopically at anterior-facing wounds by 48 hours after injury (Fig. 2G), indicating that notum normally prevents activation of β-catenin targets at anterior-facing wounds.

The notum(RNAi) phenotype is similar to that caused by inactivation of APC, an intracellular β-catenin inhibitor (4). Additionally, Drosophila notum inhibits Wnt signaling in imaginal discs (11, 12). Therefore, we performed double-RNAi experiments to assess the candidate pathway of action involving notum, β-catenin-1, and wnt1. β-catenin-1 and notum double RNAi resulted in a polarity phenotype identical to that of β-catenin-1 RNAi alone: anterior- and posterior-facing head regeneration (Fig. 3A). Similarly, wnt1 inhibition suppressed the polarity phenotype caused by notum RNAi (Fig. 3A). notum RNAi efficiency was not reduced in double-RNAi animals (fig. S5), indicating that suppression of the notum phenotype by wnt1 and β-catenin-1 dsRNA is unlikely to be explained simply by competition with notum dsRNA for RNAi. These data suggest that the notum(RNAi) phenotype requires wnt1 and β-catenin genes, supporting a model in which notum normally inhibits wnt1 and β-catenin-1 function to allow head regeneration.

Fig. 3. notum is a Wnt signaling–dependent Wnt inhibitor that controls regeneration polarity. (A) Double RNAi between notum and Wnt signaling components. The chart shows phenotypes (PR, photoreceptor) as percentages of animals. wnt1 RNAi can cause tail-regeneration failure and/or head regeneration at posterior-facing wounds (6–8). Competition between wnt1 and notum dsRNA probably accounts for tail-regeneration failure rather than ectopic head regeneration in wnt1(RNAi); notum(RNAi) animals. (B) β-catenin-1(RNAi) reduced, and APC(RNAi) enhanced, notum expression 18 hours after amputation. notum expressing cell numbers at anterior-facing wounds: controls, 102 ± 17 cells; β-catenin-1(RNAi), 17 ± 23 cells (P = 6.5 × 10−8 ); APC(RNAi), 186 ± 37 cells (P = 8.1 × 10−6 ). Number of notum-expressing cells at posterior-facing wounds: controls, 9 ± 5 cells; β-catenin-1(RNAi), 1 ± 3 cells (P = 0.003); APC(RNAi), 30 ± 24 cells (P = 0.014). Errors, standard deviations; P values, two-tailed t tests. Anterior to the top (A) or left (B). Scale bars, 200 mm. (C) Proposed pathway: selective feedback inhibition of wound-induced Wnt signaling by notum at anterior-facing wounds controls switchlike behavior of regeneration polarity.

Panel A

The chart summarizes the phenotypes of the double RNAi experiments shown in panel A. PR stands for photoreceptor, or eye. Regenerated planarians can have 2 eyes, 1 eye, or no eyes in their anterior section.  

In the phenotype images, β-catenin-1 and notum double RNAi resulted in a double headed planaria!  

wnt1 inhibition reversed the polarity phenotype caused by notum RNAi, suggesting that the notum(RNAi) phenotype requires the expression of both wnt1 and β-catenin genes. Therefore, a model for this pathway would be notum inhibiting wnt1 and β-catenin-1 function to allow head regeneration.

Panel B

The authors inhibit β-catenin-1  before amputation and find reduced notum expression levels near wounds, showing that Wnt signaling is necessary at wounds for notum expression. The adenomatous polyposis coli gene (APC) is a negative regulator of β-catenin. RNAi of APC caused increased levels of notum expression at wounds, further demonstrating notum is a target of wnt/β-catenin signaling in regeneration.

Panel C

The authors update their model to include the data found from experiments in Figure 3B. notum is a feedback inhibitor of wnt signaling at anterior-facing wounds to promote head regeneration.

notum is a Wnt signaling–dependent Wnt inhibitor that controls regeneration polarity. (A) Double RNAi between notum and Wnt signaling components. The chart shows phenotypes (PR, photoreceptor) as percentages of animals. wnt1 RNAi can cause tail-regeneration failure and/or head regeneration at posterior-facing wounds (68). Competition between wnt1 and notum dsRNA probably accounts for tail-regeneration failure rather than ectopic head regeneration in wnt1(RNAi); notum(RNAi) animals. (B) β-catenin-1(RNAi) reduced, and APC(RNAi) enhanced, notum expression 18 hours after amputation. notum-expressing cell numbers at anterior-facing wounds: controls, 102 ± 17 cells; β-catenin-1(RNAi), 17 ± 23 cells (P = 6.5 × 10−8); APC(RNAi), 186 ± 37 cells (P = 8.1 × 10−6). Number of notum-expressing cells at posterior-facing wounds: controls, 9 ± 5 cells; β-catenin-1(RNAi), 1 ± 3 cells (P = 0.003); APC(RNAi), 30 ± 24 cells (P = 0.014). Errors, standard deviations; P values, two-tailed t tests. Anterior to the top (A) or left (B). Scale bars, 200 μm. (C) Proposed pathway: selective feedback inhibition of wound-induced Wnt signaling by notum at anterior-facing wounds controls switchlike behavior of regeneration polarity.

Hedgehog signaling affects planarian regeneration polarity (9, 17), so we tested whether notum requires or influences Hedgehog signaling. patched RNAi overactivates Hedgehog signaling and increases wnt1 wound expression; hedgehog inhibition reduces wnt1 wound expression (9, 17). In contrast, notum(RNAi) animals displayed normal wnt1 expression after amputation (fig. S6A), suggesting that notum does not act in polarity by influencing Hedgehog activity. Second, patched(RNAi) animals regenerated anterior tails (n = 3 of 10 animals) but had normal asymmetric notum expression at wounds (n = 8/8) (fig. S6B), suggesting that Hedgehog signaling does not act in regeneration polarity to drive asymmetric notum expression at wounds.

notum can function as a wingless (Wnt) feedback inhibitor in Drosophila (11, 12), so we tested whether Wnt signaling is required for wound-induced notum expression. β-catenin-1 inhibition before amputation robustly reduced notum expression levels near wounds (Fig. 3B and fig. S7). Conversely, APC RNAi caused notum up-regulation near wounds (Fig. 3B and fig. S7, A to C). Therefore, Wnt signaling is necessary and can be sufficient at wounds for notum expression. Whether Smed-notum is a direct or indirect β-catenin transcriptional target is unknown, but notum is a direct target of Wnt signaling in cultured Drosophila and mammalian cells (18, 19). Wnt signaling perturbation affected notum expression regardless of wound orientation (fig. S7, A and B), so we propose that some other process ensures asymmetric notum expression at wounds. Specifically, in APC(RNAi) animals, notum was up-regulated at wounds, but expression asymmetry remained. Because Smed-notum inhibits β-catenin-1 activity and requires β-catenin-1 for its effects, these results suggest that regulation of feedback inhibition controls the regeneration polarity decision (Fig. 3C).

Wnt signaling is used broadly in regeneration (2024), and our results suggest that Notum proteins can be an important determinant of the outcome of Wnt expression in regeneration. Additionally, primary body-axis development involves anterior Wnt inhibition in many animals (25); notum is an ancient gene present in many metazoans (fig. S1), making it a candidate for controlling anterior identity broadly. Feedback inhibitors operate in many signaling pathways (26) and frequently simply attenuate pathway output. Here, we present evidence that a target and inhibitor of Wnt signaling, the secreted hydrolase NOTUM, controls the switchlike behavior of the head-versus-tail regeneration decision in planarians. These results raise the possibility that control of whether feedback inhibition occurs could, in general, be used to mediate binary developmental decisions.

In principle, decisions of which tissues to regenerate could be accomplished only by sensing the absence of particular structures. In contrast, our results indicate that regeneration programs elicited at wounds can involve local responses to tissue orientation regardless of the identity of missing tissue. We conclude that initiation of correct regeneration programs involves responses to wounding that depend on local tissue polarization, such as along a body axis.

References and Notes

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Acknowledgments

We thank the Reddien Lab for discussions and S. Lapan for hedgehog constructs. P.W.R. is a Howard Hughes Medical Institute early career scientist. We acknowledge support by NIH R01GM080639, ACS RSG-07-180-01-DDC, the Keck Foundation, and an American Cancer Society postdoctoral fellowship to C.P.P.  C.P.P. and P.W.R. are inventors on a pending patent on the use of Notum inhibitors to enhance regeneration.

Supplementary Materials

www.sciencemag.org/cgi/content/full/332/6031/852/DC1
Materials and Methods
Figs. S1 to S7
References