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Figure 1. Domain organization of the predicted survivin and mortalin proteins of H. glaberrima.
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Figure 2. Diagram summarizing the anatomical features of the non-eviscerated (normal) and regenerating digestive tube and the expression patterns of survivin and mortalin in H. glaberrima. (A) Non-eviscerated animals (on the anatomical drawing, the gut mesenteria are not shown). (B) - (E) Regenerating animals at day 2, 7, 14, and 21, respectively. 1di - first descending intestine; 2di - second descending intestine; ai - ascending intestine; c - cloaca; ct - connective tissue layer; de - digestive (luminal) epithelium; dm - dorsal mesentery; e - esophagus; m - mesothelium; pb - pharyngeal bulb; vm - ventral mesentery. All anatomical drawings are positioned with the anterior to the top. The arrows indicate the position of the representative transverse sections. Colors indicate the following: blue - in situ hybridization signal; green - non-eviscerated ('old') tissues; red - regenerating ('new') tissues; black - lumen of the digestive tube. Not to scale.
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Figure 3. In situ hybridization. Expression of survivin (A and B) and mortalin (C - E) in the tissues of the digestive tube in non-eviscerated animals. (A) and (B) survivin expression in the scattered cells of the luminal epithelium in the esophagus (A) and the second descending intestine (B). (C) mortalin transcripts widely expressed in the apical region of the mesothelium in the esophagus. (D) Asymmetric distribution of mortalin transcripts in the distal region of the mesentery attached to the second descending intestine. (E) mortalin-expressing cell in the luminal epithelium of the cloaca. Arrows indicate rare cells showing in situ hybridization signal. de - digestive (luminal) epithelium; ct - connective tissue layer; m - mesothelium; vm - ventral mesentery. Scale bars = 50 μm in (A) - (C); 100 μm in (D); 200 μm in (E).
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Figure 4. In situ hybridization. Expression of survivin at early stages of gut regeneration (days 2 - 6 after evisceration). (A) Digestive (luminal) epithelium of the cloaca on day 2. (B) Free distal edge of the posterior mesentery on day 2. The inset shows a higher magnification view of the boxed area. (C) The stump of the esophagus on day 2. (D) Wall of the cloaca on day 6. (E) A low-magnification view of the posterior regenerate on day 6. (F) Higher magnification of the boxed area on (E) showing a furrow (arrowhead) of the coelomic epithelium. (G) Esophageal stump on day 6. The inset shows a lower magnification view of the cross-section of the stump. (H) Free distal margin of the anterior mesentery on day 6. de - digestive (luminal) epithelium; dm - dorsal mesentery; ct - connective tissue layer; l - lumen of the gut; m - mesothelium; vm - ventral mesentery. Scale bars = 50 μm in (A), (B inset), (F) and (G); 100 μm in (B), (G inset), and (H); 200 μm in (C) - (E).
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Figure 5. In situ hybridization. Expression of survivin at advanced stages of gut regeneration (days 12 - 21 after evisceration). (A) Section through the posterior rudiment at its attachment to the cloaca on day 12. (B) High magnification view of the boxed area on (A). (C) Section through the posterior regenerate at a more anterior level relative to (A). (D) and (E) show high magnification views of the boxed areas on (C), representing the mesenterial attachment and the digestive epithelium, respectively. (F) The wall of the esophageal stump on day 12. (G) and (H) Sequential sections trough the tip of the blindly ended anterior rudiment on day 12. (I) and (J) The wall of the posterior portion of the regenerated digestive tube on day 21, showing scatted labeled cells in the luminal (digestive) epithelium (I) and no labeling in the mesothelium (J). (K) The wall of the anterior region of the regenerated gut on day 21, showing expression of survivin in the mesothelium. de - digestive (luminal) epithelium; ct - connective tissue layer; m - mesothelium. Scale bars = 100 μm in (A), (D), (G), (H); 50 μm in (B), (E), (I), and (J); 200 μm in (C); 25 μm in (F) and (K).
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Figure 6. In situ hybridization. Expression of mortalin in gut tissues on day 2 after evisceration. (A) Patchy in situ hybridization signal in the mesothelium of the anterior region of the cloaca. The inset shows a detailed view of the mesothelium corresponding to the boxed area on the main image. (B) Low-magnification view of the posterior mesentery. (C) A higher magnification of the free distal edge of the posterior mesentery - boxed area in (B) - showing strong hybridization signal in some cells of the mesothelium. (D) Ventral (anti-mesenterial) region of the esophageal stump showing very weak and restricted in situ hybridization signal (arrow) in the mesothelium. (E) Low-magnification view of the anterior mesentery. (F) Magnified view of the boxed area in (E) showing strongly labeled cells in the mesothelium of the free distal edge of the mesentery. de - digestive (luminal) epithelium; ct - connective tissue layer; m - mesothelium. Scale bars = 100 μm in (A), (B), (D), and (E); 25 μm in (A inset) and (C); 50 μm in (F).
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Figure 7. In situ hybridization. Expression of mortalin in gut tissues on day 6 after evisceration. (A) In situ hybridization signal in the mesothelium of the posterior rudiment. Note that the signal is weak or completely absent from the anti-mesenterial region of the rudiment (arrow) and from the bottom of mesothelial furrows (arrowhead). The inset shows a low-magnification view of the posterior rudiment with the boxed area corresponding to the main image. (B) The posterior tip of the esophageal stump. Note strong in situ hybridization signal in the mesothelium and moderate signal in the luminal (digestive) epithelium. (C) Strong expression of mortalin in the mesothelium of the anterior mesentery. de - digestive (luminal) epithelium; ct - connective tissue layer; m - mesothelium. Scale bars = 50 μm in (A); 200 μm in (A inset); 100 μm in (B) and (C).
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Figure 8. In situ hybridization. Expression of mortalin at advanced stages (days 12 - 21) of gut regeneration. (A) Posterior rudiment on day 12 after autotomy. (B) Growing posterior tip of the anterior rudiment on day 12 after evisceration. (C) The wall of the esophageal stump on day 12 after evisceration. (D) The wall of the newly regenerated posterior regions of the esophagus on day 21 after evisceration. (E) The second descending part of the newly regenerated intestine on day 21 after evisceration. The inserts show higher magnification view of the asymmetrical expression of mortalin in the mesothelium of the mesentery attachment and also strongly labeled singly scattered cells in other regions of the mesothelium. de - digestive (luminal) epithelium; ct - connective tissue layer; m - mesothelium. Scale bars = 200 μm in (A) and (E); 500 μm in (B); 25 μm in (C), (D) and (E insets).
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Figure 9. Real-time RT-qPCR. Overall abundance of survivin and mortalin transcripts in the regenerating digestive tube. (A) and (B) Survivin expression in the anterior and posterior regenerates, respectively. (C) and (D) Mortalin expression in the anterior and posterior regenerates, respectively. Transcript abundance is expressed as x-fold relative to the normal gut. Results are represented as mean ± standard error. *P < 0.05, **P < 0.01
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Figure 10. TUNEL assay. Percentage of apoptotic cells in tissue layers of the normal and regenerating digestive tube. (A) Cell death in the anterior regenerate. (B) Apoptosis in the posterior regenerate. Results are represented as mean ± standard error. *P < 0.05, **P < 0.01
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Figure 11. Representative micrographs of the distribution of TUNEL-positive cells (green) in the normal gut and in the early regenerates (days 3 - 7). (A) Wall of the esophagus in a non-eviscerated animal. (B) Wall of the second descending intestine in a non-eviscerated animal. (C) and (D) The anterior and posterior regenerates, respectively, on day 3. (E) and (F) General view of the anterior and posterior regenerates, respectively, on day 7. (E') and (F') Higher magnification of the boxed areas on (E) and (F), respectively. de - digestive (luminal) epithelium; ct - connective tissue layer; m - mesothelium. TUNEL-positive cells are green; nuclei were stained with DAPI and are shown in blue. Scale bars = 50 μm in (A), (E'), and (F'); 100 μm in (B) - (D); 200 μm in (E) and (F).
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Figure 12. Representative micrographs of the distribution of TUNEL-positive cells during the late phase (days 14 and 21) of visceral regeneration. (A) and (B) The anterior and posterior regenerates, respectively on day 14. (C) and (D) The newly regenerated posterior region of the esophagus and the second descending intestine on day 21 after evisceration. Insets show higher magnification views of the gut wall. de - digestive (luminal) epithelium; ct - connective tissue layer; l - gut lumen; m - mesothelium; vm - visceral mesentery. TUNEL-positive cells are green; nuclei were stained with DAPI and are shown in blue. Scale bars = 200 μm in (A) - (D); 50 μm in all insets.
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Figure 13. Double labeling with riboprobes for survivin and mortalin (blue) and TUNEL assay (green) on the posterior regenerate on day 7. (A) - (C) Survivin riboprobe and TUNEL assay. (D) - (F) Mortalin riboprobe and TUNEL assay. vm - ventral mesentery. Arrowhead on (D) - (F) marks the anti-mesenterial region of the rudiment, where mortalin transcript are absent. Note a negative correlation between the localization of survivin in situ hybridization signal and the density of the TUNEL-positive cells (A) - (C). Scale bars = 100 μm.
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Figure 14. Double labeling with riboprobes for survivin and mortalin (blue) and BrdU immunocytochemistry (green) on the posterior regenerate on day 7. (A) - (C) Survivin riboprobe and BrdU immunohistochemstry. (D) - (F) Mortalin riboprobe and BrdU immunohistochemistry. Note that BrdU-incorporating cells are mostly distributed within the expression domains of survivin (A) - (C) and mortalin (D) - (F). Scale bars = 100 μm.
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