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We previously showed that recombinant limb buds with dissociated and reaggregated mesenchyme develop more than 30 digits in Xenopus laevis, which exhibits different capacities for limb regeneration at different developmental stages (Yokoyama et al. [1998] Dev Biol 196:1-10). Cell-cell contact among anterior- and posterior-derived mesenchymal cells is required for anteroposterior (AP) axis formation of recombinant limbs in an intercalary manner. However, whether one-way induction from posterior cells to anterior cells as proposed by the polarizing zone model or interactions between anterior and posterior cells evoke the AP axis formation in recombinant limbs remains unclear. In this study, we found, by a combination of X-ray irradiation and a recombinant limb technique, that not one-way induction but interactions between anterior and posterior cells accompanied by cell contribution are indispensable for AP axis formation in recombinant limbs. Shh was expressed in posterior-derived not anterior-derived cells. We propose that the recombinant limb is an excellent model for examining the axis formation mechanism in regenerating limbs because, as in recombinant limbs, cell-cell contact among cells derived from different positions of an amputation plane occurs in the blastema of regenerating limbs.
Figure 1. A: Preparation of Xenopus recombinant limbs. Recombinants with whole limb bud mesenchyme at stage 55–56 formed many digits. B–G: Gene expressions in recombinants. Expressions of fgf-8 (B), shh (C), and Hoxa-13 (D) were examined by in situ hybridization in serial sections at 8 days after grafting. Expressions of fgf-8 (E) and shh (F) were also examined by whole-mount in situ hybridization at 5 days and 8 days after grafting, respectively. G: Lmx-1 expression was examined by in situ hybridization on a section at 8 days after grafting. Arrowheads show the host–graft boundary. Arrows indicate expression domains. a, host anterior side; p, host posterior side; d, host dorsal side; v, host ventral side. Scale bars = 1 mm in A, 250 μm in E,F, 100 μ m in B–D,G.
Figure 2. HoxA expressions in recombinants with distal (A–D) and proximal (E,F) mesenchyme. The schematic diagram shows the limb bud at stage 55–56. Solid lines indicate incisions made for the preparation of proximal and distalmesenchyme. Hoxa-11 (A,C,E,G) and Hoxa-13 (B,D,F,H) expressions were examined at 5 days (A,B,E,F) and 8 days (C,D,G,H) after grafting. Arrowheads show the host–graft boundary. a, host anterior side; p, host posterior side. Scale bars = 100 μm in A–H.
Figure 3. Expressions of shh in recombinants with anterior (A) and posterior (B) mesenchyme. Expression of shh was found only in recombinants with posteriormesenchyme. Arrowheads show the host–graft boundary. a, host anterior side; p, host posterior side. Scale bar = 250 μm in B (applies to A,B).
Figure 4. Recombinants with anterior and posterior cell reaggregates. A: The scheme represents recombination of anterior cell and posterior cell reaggregates. B: Digit pattern in a recombinant with anterior and posterior cell reaggregates. Arrows indicate digits 3. Arrowheads show the host–graft boundary. C: Histogram showing the percentage distribution of digit types in recombinants with anterior and posterior cell reaggregates. Roman numerals indicate digit types. x, unidentifiable digit. Scale bar = 1 mm in B.
Figure 8. Digit patterns in X-ray–irradiated recombinants. A,B: Recombinants with irradiated whole limbmesenchyme (iW). Both anterior (with claws) and posterior (without claws) digits were formed. C,D: Recombinants with nonirradiated anterior cells and irradiated posterior cells (A + iP). E,F: Recombinants with irradiated anterior cells and nonirradiated posterior cells (iA + P). Mesenchyme was irradiated with 1,000 rad (A,C,E) or 2,000 rad (B,D,F). Arrows indicate claws on the digit tip. Arrowheads show the host–graft boundary. Scale bar = 1 mm in F (applies to A–F).
