|
Display additional annotations [+]
|
| |
Fig. 1. krt62.L and fgf8 expression in froglet blastema. (A–C) Blastema in the early bud stage. (D–F) Blastema in the mid bud stage. (G–I) Blastema in the late bud stage. (J–L) Early spike stage. (M–O) Spike 2 months after amputation. (A, D, G, J, M) Alcian blue, eosin and Alizarin red staining. (B, E, H, K, N) fgf8 expression was visualized by in situ hybridization. (C, F, I, L, O) krt62.L expression was visualized by in situ hybridization. Scale bar in A and B1 = 1000 µm. Scale bar in B2 = 100 µm. Scale bar in E2 = 200 µm. Scale bar in H1 = 400 µm. A, D, G, J and M are the same magnification. B1, C1, E1, F1 and L1 are the same magnification. B2, C2, H2, I2, L2 and O2 are the same magnification. E2, F2 and N are the same magnification. H1, I1, K and O1 are the same magnification. |
|
Display additional annotations [+]
|
| |
Fig. 2. Fgf8, krt62.L and Krt12.6.S expression in the froglet blastema. The middle bud blastema was sectioned. In situ hybridization (for Fgf8 or krt62.L) and immunofluorescence (for Krt12.6.S) were performed on an identical section. (A) Histological observation. B–G are the higher magnification views of the arrowed region in A. (B–D) krt62.L and Krt12.6.S expression. D is the merged image with the high magnification of B and C. Krt12.6.S color was changed using Photoshop for easier recognition. (E–G) fgf8 and Krt12.6.S expression. G is the merged image with the high magnification of E and F. Krt12.6.S color was changed by Photoshop for easier recognition. The dotted lines in D and G indicate the border of the blastema epithelium. Scale bars in A, B and D are 500, 200 and 100 µm, respectively. |
|
|
| |
Fig. 4. Denervation effect on krt62.L expression. (A, C, E, G) Histology. (B, D, F, H) krt62.L expression. (A, B, E, F) Sham operated limbs (control). (C, D, G, H) Denervation procedure was performed 11 days after limb amputation. (C, D) The blastema was grown for 3 days after denervation. krt62.L expression domain expanded from the basal to the surface layer. (G, H) The blastema was grown for 7 days after denervation. krt62.L expression had almost vanished from the blastema epithelium. Scale bar in A= 500 µm. Scale bar in B= 300 µm. Scale bar in B′= 100 µm. |
|
|
| |
Fig. 6. Functionality of froglet blastema epithelium for supporting limb development. (A) Schematic diagram of the experimental design. A developing limb bud was isolated from a st. 52 tadpole. Next, the epidermis was removed. The epidermis-stripped mesenchyme was grafted into a froglet blastema in the middle bud stage. To distinguish between host and graft cells, a transgenic Xenopus (H2B-GFP) was used as a graft. (B) Development of the grafted tissues in the regenerating froglet limb bud. The GFP positive graft was gradually patterned. (C) Phenotype of the grafted limb 1 month after surgery. (D) Skeletal pattern. (E) The section of the blastema containing the graft. The blastema was fixed and sectioned 5 days after the surgery. F and G are the higher magnifications of the adjacent section of the boxed region in E. (F) krt62.L expression was visualized by in situ hybridization. (G) The section shown in F was processed for immunofluorescence using anti-GFP antibody. GFP positive cells were derived from a graft. Scale bar in C and D = 1 mm. Scale bar in E = 500 µm. Scale bar in F= 100 µm. |
