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In Xenopus laevis, amputation of the adult limb results in the formation of a simple (hypomorphic) spike-like structure without joints, although tadpolelimb bud regenerates complete limb pattern. The expression of some joint marker genes was examined in limb development and regeneration. Bmp-4 and gdf-5 were expressed and sox-9 expression was decreased in the joint region. Although developing cartilages were well-organized and had bmp-4 expressing perichondrocytes, the spikecartilage did not have such a structure, but only showed sparse bmp-4 expression. Application of BMP4-soaked beads to the spike led to the induction of a joint-like structure. These results suggest that the lack of joints in the spike is due to the deficiency of the accumulation of the cells that express bmp-4. Improvement of regeneration in the Xenopus adult limb that we report here for the first time will give us important insights into epimorphic regeneration.
Figure 3. Expression patterns of bmp-4, gdf-5, and sox-9 during hindlimb development. A,B: Bmp-4 expression in Xenopus st52 hindlimb bud. Section revealed that bmp-4 was expressed in both proximal and distal regions (A). Bmp-4 was also expressed in the distal region (B). C: In st54 limb bud, bmp-4 was expressed in the interdigital regions. D: The section of the st54 hindlimb bud revealed the expression in perichondrial cells surrounding the skeletal element. E: Higher magnification of the boxed region in D. F,G: In st59 hindlimb, bmp-4 was expressed in the joint region (F,G, arrows) and the perichondrium (F, arrowheads). Bmp-4 expression was detectable in the digit-joint region, that is, before the differentiation into three layers (G, arrowheads). H-L: Gdf-5 expression in the developing hindlimb bud. In st52 hindlimb bud, gdf-5 was expressed at low levels in the posterior region, which was similar to the expression of bmp-4 (H). In the st53 hindlimb bud, gdf-5 expression was detectable in the autopodium (I). Gdf-5 expression was restricted to the joint region (J). In the st58 hindlimb bud, clear signals of gdf-5 expression were detectable in the joint region (K). The section revealed that the expressions were restricted to the joint region and also slightly detectable in chondrocytes (L). M: Sox-9 was strongly expressed in chondrocytes. The sox-9 expression was detectable broadly from the distal to proximal regions. N: In the st55 hindlimb bud, sox-9 expression in the presumptive joint region was decreased (arrows). Scale bar = 200 mu m.
Figure 1. A: A normal forelimb of the Xenopus froglet. B: The autopodium of a normal hand. Well-developed diaphysis stained with alizarin red and epiphysis stained with alcian blue were observable. Two to three joints are formed. C: A cone-shaped hypomorphic structure formed on the forelimb at 5 weeks after amputation. This was the spike formation. D: The spike was stained with alcian blue. This shows that the spike consists of a single continuous cartilage. (E) A section of the spike that was formed on the forelimb at 5 weeks after amputation. Lines indicate the amputation level.
Figure 2. Comparison between st58 tibia and spike cartilage. Hematoxylin-eosin and alcian blue staining of the section of st58 tibia. A,B: The cartilage was well differentiated and the obvious boundary between the cartilage and other tissue was formed in the diaphysis. Some flat cells forming the perichondrium were observed (B, arrows). C–F: Double-immunolabelling of st58 tibia with antibodies specific for type I and type II collagen. C,D: In the diaphysis, immunolocalization of type I collagen in the outer layer of the perichondrium (C, arrows) and type II collagen in the inner cellular layer of the perichondrium and cartilage matrix was visualized, using rhodamine- and fluorescein isothiocyanate-conjugated secondary antibodies, respectively. E,F: In the epiphysis, many chondrogenic cells exhibited immunoreactivities for both type I and type II collagen. G,H: Morphology of the spike cartilage. Marginal region of the spike cartilage was slightly stained with alcian blue (H, arrows). I,J: Immunoreactivities for type I and type II collagen. Obviously separated signals of type I and type II collagen were not observed in the marginal region of cartilage (solid line). Nuclei were stained with DAPI (C–F, I, J). Scale bar = 50 μm.
Figure 4. Expression of bmp-4, gdf-5, and sox-9 in regenerating froglet blastema. A-C: Middle stage of a blastema. Bmp-4 expression was detectable in the distal region of the blastema (A, arrowheads). A′: Higher magnification of A. Bmp-4 was expressed in the mesenchyme beneath the epidermis. Gdf-5 expression was also observed in the region corresponding to bmp-4 expression (B, arrowheads). B′: Some signals of gdf-5 transcription were present not only beneath the epidermis but also in the interior. Sox-9 was expressed in chondrocytes (C,C′). D–F: Late stage of a blastema. Arrowheads point to several bmp-4 expressing cells (D,D′). Note the “salt and pepper” pattern characteristics of the bmp-4 signal. The accumulation of bmp-4 expressing cells was not detectable in the peripheral region of the cartilage. In the peripheral region of the cartilage, bmp-4 was expressed sparsely (D′). E: Gdf-5 expression in the late stage of blastema. E′,E′: Higher magnifications of E. In many cases, blastema consisted of some cartilage elements. The blastema expressed Gdf-5 in the boundaries of each element (E,E′, arrowheads), which expressed sox-9 (F). Gdf-5 was also expressed in the proximal cartilage (E, E′, arrows). Scale bar = 500 μm (A′,B′,C′,D′,E′,E′) and 100 μm (A–F).
Figure 5. A: BMP4-soaked beads were implanted into the spike. B: The spike in which BMP4/Chordin-soaked beads were implanted was not segmented. C: Segmented skeletal elements that were formed by implantation of BMP4 beads at 1 mg/ml concentration. Whole mount alcian blue staining. Arrows indicate the segmentation, joint-like region. D: Enlarged view of C. E: Normal joint of st57 hindlimb bud. F: Enlarged view of E. Arrowheads indicate beads.
Figure 6. Immunoreactivity for phosphorylated Smad-1/5/8 (pSmad-1/5/8). The joint-like formation was stained for pSmad-1/5/8 (A,D) and for nuclei by DAPI (B,D). Bright field (C). Arrowheads show identical cells. D: Double labeling for pSmad-1/5/8 (green) and nuclei (blue) in the joint-like region showing nuclear-localization of pSmad-1/5/8. E: The intact spike was stained for pSmad-1/5/8 (E) and for nuclei by DAPI (F). Comparing with A, weaker signals of pSmad-1/5/8 proteins were observed in the intact spike cartilage.
Figure 7. A: Histological analysis of a segmented spike. The section was stained with alcian blue, eosin, and hematoxylin. B,C: Analysis of molecular markers in the BMP4-implanted spike by section in situ hybridization. B: Gdf-5 expression. Although gdf-5 expression was still observed in the peripheral cartilage, remarkable expression was not observed in the joint-like region. C: sox-9 expression. Sox-9 transcriptions were detectable in the joint-like region. D,E: Immunoreactivity for type I and type II collagen. E: Decrease in the signals of type II collagen was observed in the joint-like region. Scale bar = 200 μm.