PMC

Cartoon summarizing cellular changes during regeneration initiation. Two distinct phases of neoblast responses occur during regeneration initiation. The first phase represents a generic response to injury that spreads quickly from the wound site, is wound-size dependent, and induces increased mitotic entry of neoblasts body-wide. The second phase occurs only when a significant amount of tissue is missing and involves local signals that induce recruitment of neoblasts followed by local proliferation and differentiation at the wound site. Depicted are posterior (tail) fragments.

(A-R) Wounding triggers a widespread first mitotic peak, by 4-10 hours, and a localized, second mitotic peak by 48h. A), F), K) schematics, amputation procedure. Green line, amputation plane. Black dotted circle, region analyzed. Amputated fragments were labeled with an anti-H3P antibody to detect mitoses at indicated timepoints. (P) Change in mitotic density with time following amputation in transversely amputated fragments. Mitotic numbers were significantly higher at 48h versus 18h; **p<0.01, *p<0.05, Student’s t-test. (Q) Mitotic numbers at different distances from the wound site in posterior fragments. Numbers were significantly higher at the wound site at 48h versus 6h; **p<0.01, *p<0.05, Student’s t-test. (R) Change in mitotic density with time following parasagittal amputation. Mitotic numbers were significantly higher at 48h versus 18h; *p<0.05 by Student’s t-test. Red arrows, amputation site. White arrows, pharynx. Circles, wound site at 48h showing increased mitoses. n ≥ 3. Anterior to the top in all images, dorsal view. Scale bars 100μm. All data represent averages ± standard deviation (sd).

(A-K) Cartoons at left depict surgery types. Top and middle, amputation of the head tip (green line); bottom, black line indicates site of needle poke. White arrows on images indicate the sites of amputation or needle poke in head tips. White asterisks indicate photoreceptors. Anterior, up. Bars, 100μm. (A-D) Mitotic cells appear in front of the photoreceptors at (C) 18h and (D) 48h after amputation. Differential interference contrast (DIC) image superimposed with image of mitotic cells (anti-H3P, red). (E-K) smedwi-1⁺ cells (magenta – fluorescence at animal periphery is non-specific) and mitoses (green) accumulate in head tips (a region normally devoid of neoblasts) at 18h and 48h, respectively, when the head tip was amputated (E-H), but not following a needle-poke (I-K). (L) A head tip needle-poke induces a first mitotic peak. Data represent averages (n ≥ 3) ± sd. Mitoses were quantified from z-stacks through the entirety of the DV axis from tip of the head to the posterior base of the pharynx. Data were significantly higher at 6h than at 5min; **p<0.01 and *p<0.05 by Student’s t-test.

(A-D) Cycling cells accumulate at the wound site at 18h following wounding. Wound sites in tail fragments are shown. (A-C) smedwi-1⁺ and D) histone H2B⁺ cells accumulate at the wound site at 18h (green). Nuclei were labeled with Hoechst (blue). Anterior, to the top. (E-L) Cycling cells accumulate at the tip of gut branches at 18h (yellow arrows), but not at the nerve cords. Tail fragments were labeled with smedwi-1 (magenta) and anti-H3P (yellow), together with Smed-MAT (EG413862, Methionine adenosyltransferase, expressed in the planarian intestine) or Smed-ChAT (Choline acetyltransferase, expressed in planarian nerve cords) (green) at indicated timepoints. Anterior, left, except in I) and L). (I) 3D projection showing a gut branch opening (green) surrounded by smedwi-1⁺ cells (magenta); anterior-dorsal view. (J) Optical section from dorsal domain of a posterior (tail) fragment. Gut branch outlines can be seen by Hoechst labeling; in this dorsal domain, no ChAT expression is found. (K) Optical section from ventral domain of the same tail fragment as J). No accumulation of smedwi-1⁺ cells in areas of ChAT expression. (L) 3D projection from an anterior (head) fragment showing that the majority of smedwi-1⁺ cells accumulates around the single gut branch, but not the nerve cords - ChAT ⁺ cells (in green, white arrow heads), posterior-ventral view. Images represent superimposed optical sections, except in (J-K). Dorsal view, unless otherwise stated. White arrows, wound site. Yellow arrowheads, smedwi-1⁺ cells surrounding gut branches. Bars, 100μm.

(A-B) A needle-poke and a hole-punch both induced a strong first peak, but only a hole-punch induced a second, localized peak. Animals were labeled with anti-H3P at indicated timepoints. At left, cartoon schematic depicts the surgical strategy with representative images shown in (A) and quantification shown in (B). Green circles, wound site. A needle was used to inflict piercing of the epidermis (poke, diameter 10-20μm), whereas a broken needle was used to inflict hole-punches (hole, diameter >50μm) that removed a cylindrical region of tissue. Black circles, analyzed areas. (A) Insets, wound-site magnification. (B) At left, cartoon depicts the regions quantified following infliction of poke or hole wounds. Mitotic numbers were determined for two different 267 μm diameter, cylindrical tissue regions (dotted circles): I) centered at the wound site and II) at a region distal from the wound. n ≥ 5. **p<0.01 by Student’s t-test.(C-D) Amputations, I), were accomplished by surgical removal of the anterior two-thirds of the body (green line). Incisions, II), were made through the entirety of the body at indicated locations (green lines) and allowed to re-seal. Amputation, I), triggered a localized second mitotic peak, but incisions, II), which caused a larger wound size than in I) but no significant loss of tissue, did not. Blue and red circles, area analyzed. (C) Mitotic density with time following amputation. n ≥ 4. **p<0.01 by Student’s t-test. (D) Representative images of animals analyzed in (C). Red arrows, wound site. Bars, 100μm. Data represent averages ± sd.

(A-D) The first mitotic peak magnitude depends on wound size. A) Green lines, wound. Light blue, blue, and red circles, area analyzed. (B) Change in mitotic density with time following wounding. (C-D) A small injury (needle-poke) induces a widespread first mitotic peak by 6h; n ≥ 5. (E-H) The signal that causes the first peak spreads from the wound site. (E) Tail tips were amputated; blue and red circles, area analyzed at right. (F) Change of mitoses in areas far from the wound (anterior) and close to the wound (posterior) following wounding. The number of mitoses for each data point was divided by the average number of mitoses determined for each body region present immediately after amputation (5 min). (G-H) Animals from E) labeled with anti-H3P at 4.5h. Images, stages (G) with mitotic numbers elevated near but not far from the wound (2/8 animals), and (H) with mitotic numbers elevation having spread along the animal periphery (5/8 animals); n ≥ 6. (I-K) The signal that causes the first mitotic peak likely acts on G2/M transition. Graphs, quantification of in situ hybridizations on fixed cells from animals macerated at indicated timepoints, probing for (I) histone H2B (DN290330), (J) rnr1 (DN309701), and (K) smedwi-1 (DQ186985) mRNA. The number of S phase-marker-positive cells did not robustly change before the first mitotic peak; n=5, triplicate. Red arrows, wound sites. Anterior to the top, dorsal view. Asterisks, photoreceptors. Scale bars, 100μm in all images. Data represent averages ± sd.

(A) Lineage relationship schematic. smedwi-1⁺(mRNA, magenta)/SMEDWI-1⁺(protein, green) neoblasts will become SMEDWI-1⁺ cells as they differentiate. (B-C) Tail fragments probed with anti-SMEDWI-1 antibody (green) and smedwi-1 riboprobe (magenta). White arrow, wound site. Anterior, up. (B) smedwi-1⁺/SMEDWI-1⁺ cells accumulate at the wound site of tail fragments at 18h. White arrow, wound site. Anterior, left. (C) A layer of smedwi-1⁻/SMEDWI-1⁺ cells formed in front of actively proliferating smedwi-1⁺/SMEDWI-1⁺ cells at 48h, indicating increased differentiation. (D) smedwi-1⁺ neoblasts (magenta) will turn on the NB.21.11E and Smed-AGAT-1 genes (green) as they differentiate. (E-L) Timecourse, following amputation, of tail fragments (E-H) labeled with an NB.21.11E riboprobe (green); (I-L) merge of (E-H) labeling with anti-H3P antibody for mitoses (blue epidermal fluorescence is non-specific), and with a smedwi-1 riboprobe (magenta). Insets, magnified view of wound site. White arrow, wound site. Anterior, left. (M-N) Timecourse, following amputation, of tail fragments. Smed-AGAT-1⁺ (EC616230) cells (green) accumulate at the wound site in front of smedwi-1⁺ cells (magenta) at 48h (mitoses in blue, anti-H3P), indicating increased differentiation. Anterior, left. (O-P) Neoblast descendants at the wound site show an increase in nucleolar size at 48h. Tail fragments labeled with anti-SMEDWI-1 (green) and anti-NST (red) antibodies. NST signal was also found in SMEDWI-1⁺ cells, white arrowheads. (O) At 6h, NST was localized to a small nuclear region, inset (Hoechst, blue). (P) At 48h, NST signal occupies up to 1/3 of the nucleus. SMEDWI-1 and NST are in the cytoplasm and nucleus, respectively. However, both antibodies are from rabbits, leading to some artificial double labeling where there was high signal intensity (e.g., at 48h, nucleolar SMEDWI-1 signal is an artifact). (Q-S) Increased neoblast descendant formation at wounds is specific to tissue loss. NB.21.11E⁺cells (green) increased at the wound site (white arrow) between 18h and 48h following head tip amputation (Q-R), but not following a poke in the head tip (S-T). (U) Number of NB.21.11E⁺ cells in the wound area shown in images (Q-T). Data represent averages; n ≥ 3 ± sd. Green line, amputation plane. Red and blue regions, analyzed areas. (B, C, E-T) Images represent superimposed optical sections, dorsal view, unless otherwise stated. Scale bars, 100μm.