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

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.

Figure 1. notum is expressed at anterior-facing wounds

(A-C) notum in situ hybridizations: intact animals (A); regenerating head and trunk fragments over time (hours, h) (B, C). (A) Brackets: regions imaged in (B, C). (D) notum is expressed in anterior-pole, subepidermal cells (arrow). (D, E) Dotted green line is enlarged in right panels. (E) Double-fluorescence in situ hybridization (FISH); notum (red) and wnt1 (blue) are co-expressed (arrows) at an anterior-facing wound (dotted line) 18h after amputation. (F-I) FISH; notum expression 6h after incisions. Above, green box depicts region imaged. Red lines depict incisions. Below, red line shows sealed wound location. In (G, H) triangular tissue was removed and the wound allowed to seal; tissue between incision sites (~200 microns apart) in (I) was not removed. Anterior, left (B-I) or top (A). Dorsal view (A, 72h in C, D), or ventral view (all others). Images represent ≥4 of 5 animals per panel. Bars, 200 microns.

Figure 2. notum is required for head-tail regeneration polarity

(A) notum(RNAi) fragments failed to regenerate a head by 14 days following amputation (47%, n=133; controls were normal, 100%, n=101). (B-F) Control or notum(RNAi) regenerating animals lacking photoreceptors were probed for expression of (B) PC2 (prohormone convertase 2, CNS marker), (C) sFRP-1 (anterior-pole marker), (D) wnt1 and (E) fzd-4 (posterior markers), and (F) madt (gut marker, green). Blue, Hoechst. Arrows, lack of anterior marker (B-C), posterior marker presence (D-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 name wnt11-5 (8). Arrows, ectopic wntP-2 expression. Images represent ≥5/6 animals per panel. Anterior, left. Bars, 500 microns (A) and 200 microns.

Figure 3. notum is a Wnt signaling-dependent Wnt inhibitor that controls regeneration polarity

(A) Double RNAi between notum and Wnt signaling components. Chart shows phenotypes (PR, photoreceptors) as animal percentages. wnt1 RNAi can cause tail regeneration failure and/or head regeneration at posterior-facing wounds (–8). Competition between wnt1 and notum dsRNA likely 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 18h after amputation. notum-expressing cell numbers at anterior-facing wounds: controls, 102+/−17 cells; β-catenin-1(RNAi), 17+/−23 cells (p=6.5x10⁻⁸); APC(RNAi), 186+/−37 cells (p=8.1x10⁻⁶). 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, 2-tailed T-tests. Anterior, top (A), or left (B). Bars, 200 microns. (C) Proposed pathway: selective feedback inhibition of wound-induced Wnt signaling by notum at anterior-facing wounds controls switch-like behavior of regeneration polarity.