Research Article

Self-organization and progenitor targeting generate stable patterns in planarian regeneration

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Science  27 Apr 2018:
Vol. 360, Issue 6387, pp. 404-409
DOI: 10.1126/science.aap8179
  • Fig. 1 Discordance between positional coordinates and anatomy during regeneration.

    (A) Eye progenitors emerge from neoblasts in a broad specification zone and migrate anteriorly to form eyes. (B) Eye nucleation after unilateral eye resection. (C) An amputated head fragment morphallaxed slowly, with eyes moving anteriorly, matching final animal proportions over time (30 days shown). (D) Eye positioning occurs relative to existing body size. Head fragments slowly morphallax to resolve proportions. (E) Positional control gene expression in head fragments [ndl-2 and wntP-2 fluorescence in situ hybridization (FISH)] rescales before anatomy changes occur. White arrowheads indicate brain posterior. (F) Positional information pattern regenerates faster than do anatomical changes. Experimental strategy to reveal a predicted target zone (nucleation target) shift in a decapitated and day 3 eye-resected head fragment. Scale bars, 200 μm.

  • Fig. 2 Planarian eyes act as attractors and renucleate at predictable positions.

    (A) Resected eyes on day 3 after decapitation nucleate more anteriorly. (B) Eye progenitors [SMEDWI-1+ (19)] target both nonresected eyes and regenerating eyes at different positions in the same animal (white arrowheads). (C) Resected eyes on day 3 after a head-tip cut nucleate more posteriorly. (D) Partially resected eyes prevent anterior eye nucleation. ER, eye resection. In (A), (C), and (D), red arrowheads indicate regenerating eyes. Student’s t test, **P ≤ 0.01, ****P ≤ 0.0001. Scale bars, 200 μm.

  • Fig. 3 Generation of an alternative stable neuroanatomical state.

    (A and B) Decapitation and parasagittal amputation in large sexual animals plus day 3 unilateral eye resection results in three-eyed animals. Red arrowhead indicates regenerating eye. (C) Map of ovo+ cells from 20 three-eyed animals (day 16 after surgery). (D and E) SMEDWI-1+/opsin+ newly differentiated cells (white arrowheads) were detected in all three eyes (n = 144 eyes examined). (F) Arrestin immunohistochemistry and opsin FISH. Ectopic eyes integrate into visual circuitry. White arrowheads indicate axonal projections. (G) Decapitation and left parasagittal amputation on three-eyed animals resulted in five-eyed animals. (H) Behavior analyses: Misplaced eyes drive negative phototaxis. CA, control arena; TA, test arena. Statistical significance: one-sample t test comparing each column mean with a hypothetical value of 6 corresponding to chance (n = 8 animals per cohort); Bonferroni correction was applied. Scale bars, (A), (D), (E), and (G), 200 μm; (B), (C), and (G), 100 μm. ER, eye resection. Statistical significance: one-way analysis of variance (ANOVA): *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, *****P ≤ 0.0001. NS, not significant.

  • Fig. 4 Eyes nucleate at predictable positions during dynamic positional information shifting.

    (A) Unilateral eye resection time course (days 1 to 6 after decapitation) reveal a continuously shifting TZ. (B and C) Unilateral eye resections after decapitation and parasagittal amputation. In (A) and (B), red arrows and arrowheads indicate regenerating eyes. Statistical significance: one-way ANOVA: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, *****P ≤ 0.0001. NS, not significant. Scale bars, 200 μm.

  • Fig. 5 slit, wnt5, and notum are involved in establishing a TZ.

    (A) wnt5, slit, and control RNAi in uninjured animals (more than eight double-stranded RNA feedings, more than 4 weeks). FISH: slit expression expansion and reduction after wnt5 and slit RNAi, respectively. (B) Unilateral eye resections after wnt5, slit, and control RNAi reveal the local attractive nature of eyes, preventing de novo eye nucleation and a mediolateral TZ shift. (C) Unilateral eye resection after notum RNAi led to anteriorly shifted eye nucleation without decapitation, implicating notum in AP TZ regulation. Red arrowheads indicate regenerating eyes. Asterisks indicate ratio of animals with outcome as shown. Scale bars, 200 μm.

  • Fig. 6 Ectopic eyes are maintained in a broad TAZ.

    (A) Long-term feeding of wild-type three-eyed animals. All eyes are stably maintained. (B) Selective eye resections reveal a TAZ where ectopic eyes can be maintained but not regenerated (white arrowheads). (C) Eye transplantation strategy into or outside of the TAZ. Donor animals were irradiated so as to lack progenitors. (D) Transplanted eyes sent projections into the visual circuit. (E) Transplanted eyes (red arrowheads) were maintained in the TAZ, but not outside of it (tail). (F) Only TAZ-transplanted eyes incorporated progenitors (SMEDWI-1+/opsin+ cells, analyzed 12 to 16 days after transplantation) and SMEDWI-1+/opsin+ cell quantification (**P ≤ 0.01). White arrowhead indicates SMEDWI-1+/opsin+ cell. (G) ndk(RNAi) animals, off RNAi, with unilateral eye resection. Only one eye regenerated (in TZ); nonresected eyes remained. Red arrowheads in (B) and (G) indicate resected and regenerating eyes. (H) Eye progenitor specification zone by mapping ovo+ cells in 13 animals and generalized model for progenitors integrating self-organization, a TZ, and a TAZ. Red arrowheads indicate newly specified migratory progenitors and an example for system-level behavior predicted by the model in response to a regenerative challenge. Scale bars, 200 μm.

Supplementary Materials

  • Self-organization and progenitor targeting generate stable patterns in planarian regeneration

    Kutay Deniz Atabay, Samuel A. LoCascio, Thom de Hoog, Peter W. Reddien

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S13
    • References
    Table S1
    GenBank accession numbers and sequences used for all dsRNA preparations are provided in Table S1.

    Images, Video, and Other Media

    Movie S1
    Movie S2
    Movie S3
    Movie S4
    Negative phototaxis assay. Animals with intact eyes (S1), intact eyes in a control arena (S2) bilaterally resected eyes (S3), and misplaced eyes (S4) in light graded and control arenas.

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