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. 2018 Nov 27;25(9):2577-2590.e3.
doi: 10.1016/j.celrep.2018.11.004.

Cellular and Molecular Responses Unique to Major Injury Are Dispensable for Planarian Regeneration

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Free PMC article

Cellular and Molecular Responses Unique to Major Injury Are Dispensable for Planarian Regeneration

Aneesha G Tewari et al. Cell Rep. .
Free PMC article

Abstract

The fundamental requirements for regeneration are poorly understood. Planarians can robustly regenerate all tissues after injury, involving stem cells, positional information, and a set of cellular and molecular responses collectively called the "missing tissue" or "regenerative" response. follistatin, which encodes an extracellular Activin inhibitor, is required for the missing tissue response after head amputation and for subsequent regeneration. We found that follistatin is required for the missing tissue response regardless of the wound context, but causes regeneration failure only after head amputation. This head regeneration failure involves follistatin-mediated regulation of Wnt signaling at wounds and is not a consequence of a diminished missing tissue response. All tested contexts of regeneration, including head regeneration, could occur with a defective missing tissue response, but at a slower pace. Our findings suggest that major cellular and molecular programs induced specifically by large injuries function to accelerate regeneration but are dispensable for regeneration itself.

Keywords: TGF-β signaling; Wnt signaling; follistatin; planarians; regeneration; wound response.

Figures

Figure 1.
Figure 1.. follistatin Is Required for Regeneration Only after Complete Head Amputation in the Posterior
(A) follistatin is only required for head regeneration. For each surgery: top: live images 20 days after surgery; bottom: fluorescence in situ hybridization (FISH) for differentiated tissue markers 20 days after surgery. opsin, photoreceptor neurons (green, yellow); grh-1, pharynx neurons (magenta); NB.22.1e, dorsal-ventral (DV) boundary, mouth and esophagus (yellow); cintillo, sensory neurons (magenta); gad, GABAergic neurons (magenta); notum, anterior pole (magenta); wnt1, posterior pole (magenta). Arrowheads indicate lack of regeneration. Two independent experiments. dpa, days post amputation. (B) The requirement for follistatin in head regeneration diminishes at amputations made at serially anterior locations along the anterior-posterier (AP) axis. “n” describes the number of animals that look like the representative image. % head regeneration is provided in yellow. Bottom: FISH for differentiated tissue markers notum, cintillo, gad pool (anterior pole, sensory neurons, GABAergic neurons; magenta), and opsin (photoreceptor neurons; green) at 20 dpa (dorsal view). Two independent experiments. Scale bars, 200 μm. See also Figure S1.
Figure 2.
Figure 2.. follistatin Is Required for the Missing Tissue Response at Multiple Injury Types
(A) follistatin is required for the second mitotic peak after tail amputation and removal of tissue wedges from the head. Phospho-histone H3 (H3P) antibody labeling to mark mitotic cells (green) after the indicated injury at the specified time point (ventral view). Two independent experiments. (B) Graph displays the number of H3P+ cells counted in the region marked by the blue box at the indicated times after head wedge removal. ****p < 0.0001. n > 8 per time point; n > 30, 2 dpa; n > 20, 0 hours post amputation (hpa). (C) Graph displays the number of H3P+ cells counted in the region marked by the blue box at 0 hpa and 36 hpa. ****p < 0.001, ***p < 0.001, *p < 0.05. (D) follistatin is required for perduring wound-induced gene expression at posterior-facing wounds. FISH for the wound-induced gene inhibin-1 (yellow) at 24 hpa in regenerating head fragments (dorsal view). Black box indicates the region shown. Two independent experiments. (E) follistatin is required for the 72-hr wave of apoptosis after tail amputation. TUNEL marked cells undergoing apoptosis (orange) at 72 hpa in regenerating head fragments (ventral view). Four independent experiments. (F) Graph displays the number of TUNEL+ cells counted in regenerating head fragments at the indicated time points. ****p < 0.0001. (G) follistatin is required for the missing tissue response after AP1 amputation. FISH for the wound-induced gene inhibin-1 (yellow) at 24 hpa (dorsal view). H3P antibody labeling to mark mitotic cells (green) at 48 hpa (ventral view). TUNEL marked apoptotic cells (orange) in pharynges at 72 hpa. Black box indicates the region shown. Two independent experiments. (H) Graph displays the number of H3P+ cells counted near the wound, indicated by the blue box. Anterior regeneration at AP1 displayed a higher second mitotic peak than posterior regeneration in control RNAi animals. n ≥ 8 for each time point. n > 20, 0 hpa; n > 25, 2 dpa. ****p < 0.0001. (I) Graph displays the number of TUNEL+ cells counted in the pharynges of regenerating trunks indicated by the blue box. n ≥ 6 for each time point. ***p < 0.001, *p < 0.05. (J) Live images of head regeneration outcome 10 dpa at AP1 after follistatin RNAi. Scale bars, 200 μm. Error bars represent mean ± SD. NS indicates no significant difference. See also Figure S2.
Figure 3.
Figure 3.. Posterior Regeneration Occurs with a Defective Missing Tissue Response, but at a Slower Rate
(A) follistatin RNAi head fragments regenerate slowly with small blastemas. Live images at the indicated time points post amputation. Asterisks mark a newly formed pharynx. Arrowheads indicate a blastema. (B) Multiple differentiated tissue structures appear small or absent at 7 days post tail amputation after follistatin RNAi. FISH to mark marginal adhesive gland cells (mag-1+, green), the mouth (NB.22.1e+, yellow), and pharyngeal neurons (grh-1, magenta) (ventral view). Black box indicates the region shown. Two independent experiments. (C) follistatin RNAi animals regenerate pharynges slowly and are indistinguishable from controls by 14 days post tail amputation. Graph indicates the number of grh-1+ cells counted per animal at the specified time points post tail amputation. Data are plotted as mean ± SD. n > 8 for each time point. ****p < 0.0001, *p < 0.05. (D) The posterior pole forms slowly in regenerating head fragments after follistatin RNAi. FISH for wnt1 to mark the posterior pole (magenta). Dorsal view. Black box indicates the region shown. Two independent experiments. (E) Posterior patterning occurs slowly in regenerating head fragments after follistatin RNAi. FISH for posterior patterning genes wntP-2 (magenta) and fz-4, wnt11-1, and wnt11-2 pool (green) at the indicated time points after tail amputation (ventral view). Black box indicates the region shown. Two independent experiments. Scale bars, 200 μm. See also Figure S3.
Figure 4.
Figure 4.. Head Regeneration at AP1 Occurs in the Absence of a Detectable Missing Tissue Response, but at a Slower Rate
(A) follistatin RNAi animals regenerate heads slowly after amputation at AP1. Live images at the indicated time points post amputation. Arrowheads indicate the first appearance of eyes. (B) Differentiated anterior tissues are less developed than in controls 7 days post AP1 amputation in follistatin RNAi animals. FISH for differentiated tissue markers opsin (photoreceptor neurons, green; dorsal view) and cintillo (sensory neurons, magenta; ventral view). Black box indicates the region shown. Two independent experiments. (C) Number of cintillo+ sensory neurons at 7 dpa. Blue box indicates the region quantified. ****p < 0.0001. (D) Number of opsin+ photoreceptor neurons at 7 dpa. Blue box indicates the region quantified. ****p < 0.0001. (E) Anterior patterning and pole formation do not occur after amputation below the pharynx in follistatin RNAi animals. Top: FISH for notum (magenta) and sFRP-1 (green) at 5 dpa (ventral view). Bottom: FISH for ndl-2 (magenta) and ndl-5 (green) at 5 dpa (ventral view). Black box indicates the region shown. Two independent experiments. (F) Anterior patterning and pole formation occur slowly after amputation at AP1 in follistatin RNAi animals. Top: FISH for notum (magenta) and sFRP-1 (green) at 72 hpa (ventral view). Bottom: FISH for ndl-2 (magenta) and ndl-5 (green) at 5 dpa (ventral view). Black box indicates the region shown. Two independent experiments. Scale bars, 200 μm (A, E, and F) and 100 μm (B). Error bars represent mean ± SD. See also Figure S4.
Figure 5.
Figure 5.. follistatin Inhibits Wound-Induced Expression Levels of wnt1 at Diverse Injuries
(A) Expression levels of wound-induced notum and inhibin-1 are normal after follistatin RNAi. FISH for notum (green) and inhibin-1 (yellow) at 6 hpa in regenerating tail fragments (ventral view). Black box indicates the region shown. Two independent experiments. (B) follistatin is required to inhibit wnt1 expression early after wounding at many injuries. FISH for wnt1 (magenta) at 6 hr after the indicated surgeries (ventral view). Black box indicates the region shown. Two independent experiments. (C) follistatin is not required to inhibit expression of other wound-induced genes at 6 hpa. Heatmap depicts RNA sequencing (RNA-seq) data of anterior-facing wounds collected from follistatin RNAi and control RNAi tail fragments (Scimone et al., 2017). Data are presented as log2 fold change in gene expression between follistatin RNAi and control RNAi at the indicated time points post amputation. *padj < 0.05, **padj < 0.01, ***padj < 0.001, ****padj < 0.0001. Best human BLAST hits. (D) follistatin-mediated inhibition of wnt1 is dependent on activin-1. Top: feeding regimen for RNAi. Bottom: FISH for wnt1 (magenta) at 6 hpa in each RNAi condition. Black box indicates the region shown. Two independent experiments. Scale bars, 200 μm. See also Figure S5.
Figure 6.
Figure 6.. wnt1 Inhibition Restores Head Regeneration after follistatin RNAi Despite a Defective Missing Tissue Response
(A) wnt1 RNAi suppresses the head regeneration defect after follistatin RNAi. Top: feeding regimen for RNAi. Bottom: live images of tail fragments at 14 dpa. Two independent experiments. (B) Simultaneous inhibition of wnt1 and follistatin does not restore the secondary mitotic peak in regenerating tail fragments. Graph displays the number of H3P+ cells counted in the region indicated by the blue box at 48 hpa for each RNAi condition. ***p < 0.001. Three independent experiments. (C) Inhibition of wnt1 does not suppress the defect in the 72-hpa apoptotic wave caused by follistatin RNAi. Graph: TUNEL+ cell numbers per pharynx (indicated by the blue box) at 72 hpa, in regenerating trunks, for each RNAi condition. ****p < 0.0001. Three independent experiments. (D) wnt1; follistatin double-RNAi animals regenerate eyes slowly compared to controls. FISH for opsin (photoreceptor neurons, green) at 7 dpa (dorsal view). Black box indicates the region shown. (E) Graph displays the number of opsin+ photoreceptor neurons counted per animal for each RNAi condition at the specified time points. Scale bars, 200 μm (A) and 100 μm (D). Error bars represent mean ± SD. NS indicates no significant difference. See also Figures S6 and S7.
Figure 7.
Figure 7.. Model for the Roles of the Missing Tissue Response and follistatin in Regeneration
(A) Model for the role of follistatin in planarian regeneration. (B) Schematic depicting head regeneration after follistatin RNAi in high- (posterior) and low- (anterior) Wnt signaling environments. Head regeneration can occur in a low-Wnt signaling environment even in the absence of a detectable missing tissue response, but at reduced speed. Amputation in a high-Wnt signaling environment requires appropriate regulation of wound-induced Wnt signaling. Elevated wound-induced wnt1 expression after follistatin RNAi causes regeneration of anterior patterning information and anterior tissue formation to fail.

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