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Figure 1. The method of Xenopus laevis limb joint amputation. In order to observe spike regeneration from the elbow joint, the forelimb was amputated slightly distal to the elbow joint and the remaining pieces of the radio‐ulna were completely removed with forceps.
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Figure 2. The bending−stretching motion of the regenerated spike (see also Movie S1). After amputation at the elbow, the motion of the regenerated forelimb was observed. (A) The frog stretched the regenerated forelimb. (B) The frog then bent the regenerated forelimb at the elbow joint. (C) The frog then stretched the limb again. Yellow arrowheads indicate the position of the regenerated elbow between the remaining stylopod and the regenerated spike. The sides of the squares in the background are 1 cm.
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Figure 3. Joint structure was regenerated at the proximal end of the spike. Whole‐mount bone and cartilage staining image of (A) the intact forelimb and (B) the regenerated forelimb after amputation at the elbow. Black arrowheads indicate the position of the elbow joint. Bones were stained magenta, and cartilage was stained blue. 3D reconstructed images of the skeletal tissues in (C) the intact forelimb and (D) the regenerated forelimb after amputation at the elbow, which were obtained with EFIC. 3D reconstructed image showing the skeletal tissues (except for the humerus) in (E) the intact forelimb and (F) the regenerated forelimb (spike). White arrowheads indicate the concave morphology of the elbow joint.
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Figure 4. The remaining muscles were inserted into the regenerated spike cartilage. 3D reconstructed image of the skeletal tissues and muscles in (A) the intact forelimb and (B) the regenerated forelimb after amputation at the elbow. The remaining skeletal tissues were segmented in pink, and the regenerated cartilage (spike) was segmented in blue. The biceps (m. sternoradialis) and its tendons were segmented in yellow, and the triceps (m. triceps brachii) was segmented in green. Arrowheads indicate the insertion site of the biceps. The same reconstructed images are shown from different angles for (C) the intact forelimb and (D) the regenerated forelimb. Arrowheads indicate the insertion site of the triceps. Schematic images of the musculoskeletal structure of (E) the intact forelimb and (F) the regenerated forelimb.
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Figure 5. Time course and various tissue components involved in joint regeneration. Histological sections stained by Elastica van Gieson staining of (A) the intact forelimb, (B) the forelimb at 0 days, (C) 1 week, (D) 2 weeks, (E) 3 weeks, and (F) 4 weeks after amputation at the elbow. (E′) A more highly magnified view of the boxed region in (E). Elastic fibers in cartilage were stained purple, collagen fibers in bone and tendons were stained red, muscles were stained yellow, and the cell nucleus was stained black. Yellow arrowheads indicate the loss of the signal for elastic fibers in the remaining joint of the humerus. Black arrows indicate the tendon insertion into spike cartilage.
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Figure 6. Observation of the reformation of the joint cavity using serial sections. Alcian Blue/Nuclear Fast Red staining of the regenerating joint at (A)−(A″) 2 weeks, (B)−(B″) 3 weeks, and (C)−(C″) 4 weeks after amputation. Each row shows selected serial frontal sections of one particular regenerating joint. Acidic polysaccharides of cartilage were stained blue, and the cell nucleus was stained red. Arrows indicate the connecting part between the remaining articular cartilage and the regenerating tissues.
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Figure 7. Expression of joint marker genes in the regenerating joints. In situ hybridization of Gdf5 (a joint cell marker) and Sox9 (a chondrocyte marker) and Alcian Blue/Nuclear Fast Red staining to serial sections close to each other in the regenerating spike at (A)−(C) 2 weeks, (D)−(F) 3 weeks, and (G)−(I) 4 weeks after the amputation. Arrowheads in each column indicate the signals for the Gdf5 and Sox9 expression and the signals for cartilage in Alcian Blue staining, respectively. Green arrowheads indicate the connecting part between the remaining articular cartilage and the regenerating spike cartilage at 3 weeks after the amputation.
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Figure 8. Nuclear accumulation of β‐catenin in the cells of the regenerating joints. (A) Alcian Blue/Nuclear Fast Red staining in the regenerating joint at 3 weeks after amputation (magnified view of Fig. 6B). (B)−(E) Immunostaining with anti‐β‐catenin antibody of a serial section close to that shown in (A). (B′)−(E′) Hoechst staining of the same section as that in (B′). (B″)−(E″) Merged view of immunostaining with anti‐β‐catenin antibody (magenta) and Hoechst staining (green). (C), (C′) The cells in the regenerated spike cartilage in boxed area C in (B″). (D), (D″) The cells on the surface of the remaining articular cartilage in boxed area D in (B″). (E), (E″) The cells in the connecting part between the remaining articular cartilage and the regenerating cartilage in boxed area E in (B″). Bars in (A) and (B)−(B″) indicate 250 μm. Bars in (C)−(C″), (D)−(D″), and (E)−(E″) indicate 10 μm.
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Figure 9. Expression of a tendon marker gene in the regenerating spike. (A) In situ hybridization of Scx, a tendon progenitor cell/tenocyte marker. (B) In situ hybridization of Sox9, as a chondrocyte marker, in a serial section close to that shown in (A). Sox9 expression was observed in the regenerating spike cartilage. (C) Elastica van Gieson staining (EVG) of a serial section close to those shown in (A) and (B). Elastic fibers in cartilage were stained purple, collagen fibers in bone and tendons were stained red, muscles were stained yellow, and the cell nucleus was stained black. Scx and Sox9 expressions were observed in the regenerated spike cartilage (black arrowheads) and Scx expression was also observed in the extremities of the remaining muscles (green arrows).
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Figure 10. The process of joint regeneration in frogs. Left panel: Amputated frog forelimb after amputation at the elbow joint. Middle panel: At 3 weeks after amputation, the partial connection between the remaining joint cartilage and the regenerating spike cartilage is divided by a cavity between these apposing cartilages, and the interlocking structure is thereby reformed. Right panel: At 4 weeks after amputation, tenocytes are regenerated between the extremity of the remaining muscles and the regenerating cartilage.
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