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We prepared recombinant limb buds of Xenopus tadpoles by grafting a mesenchyme mass of the hindlimb bud. The Xenopus recombinant limb buds with dissociated and reaggregated mesenchyme developed more than 30 digits with cartilage segmentation, while those with undissociated mesenchyme developed a limb with normal cartilage pattern. Before the formation of multiple digits, a patchy expression pattern of fgf-8, an AER marker, was observed in the distal region of recombinant limb buds. shh, a ZPA (zone of polarizing activity) marker, was expressed broadly in the distal region of recombinants. Recombinant limb buds with the reaggregated mesenchyme of anterior halves formed anterior digits with claws, and those with the mesenchyme of posterior halves formed posterior digits without claws. The temporal and spatial changes in the potency of multiple digit formation are discussed with reference to the regenerative capacity of Xenopus limb buds.
FIG. 1. Multiple digit formation with reaggregated whole mesenchyme. (A) Cartilage pattern of a recombinant with undissociated mesenchyme. Ectoderm-free limb bud mesenchyme at stage 51 was grafted onto the stage 56 limb stump amputated at knee level and allowed to develop for 30 days. An ectopic hindlimb with normal cartilage pattern was formed which included femur (eF), tibiofibula (eTF), tarsus (eT), anterior three digits with claw (eD1), and posterior two digits without claw (eD4). Host limb bud stump regenerated an incomplete pattern which included a femur (hF), a set of tibiofibula (hTF), a tarsus (hT), and two digits (hD). (B) Recombinant limbs with reaggregated stage 512 whole limb bud mesenchyme. (B) 15 days after operation. (C) 28 days after operation, before staining. Note the formation of many digits. (D) Alcian blue-stained multiple digits, 70 days after operation. Small arrows indicate cartilage segments in the digits. (E) Multiple digit formation in the recombinant with reaggregated stage 56 whole mesenchyme. Alcian blue staining. (F) Digit formation in the recombinant with reaggregated stage 58 whole mesenchyme. Alcian blue staining. Arrowheads show hostraft boundary. Bars, 1 mm.
FIG. 2. PKH-26-labeled recombinant limb made from stage 51 mesenchyme. (A and B) Whole-mount preparation of a recombinant limb. (C and D) Cryosection of the same recombinant limb. (A and C) In ordinary light. (B and D) In fluorescent light. 18 days after operation. Small arrows indicate protrusions which later form digits. Note that the protrusion-forming regions are of graft origin. Bars, 250 mm.
FIG. 3. Whole-mount in situ hybridization of fgf-8 (A) and shh (C) in recombinant limbs. (A) Dorsal view of the recombinant made from stage 56 mesenchyme showing that fgf-8 expression is restricted in the distal region of the recombinant. (B) Frontal view of the same recombinant as in (A), showing that there are multiple separate expression domains in the recombinant. (C) Dorsal view of the recombinant made from stage 52 mesenchyme showing widespread distribution of shh. (D) Dorsal view of the recombinant made from stage 56 mesenchyme showing shh expression in whole recombinants. Arrowheads show the hostraft boundary. Bars, 250 um.
FIG. 4. Digit formation in the recombinants with proximal and distalmesenchyme. (A) Schematic diagram showing the limb bud at stage 556. Solid lines indicate incisions made for the preparation of proximal and distalmesenchyme. (B) Ventral view of the recombinant with multiple digits produced from proximalmesenchyme. (C) Dorsal view of the recombinant produced from the distalmesenchyme. Alcian blue staining. Note that the recombinant from the proximalmesenchyme developed many radially elongated digits, but that from the distal one developed a few digits. Arrowheads show the hostraft boundary. Bars, 1 mm.
FIG. 5. Multiple digit formation in the recombinants with anterior, posterior, and whole mesenchyme. (A) Schematic diagram showing stage 556 limb buds used for the preparation of mesenchyme. The solid line indicates the incision dividing the limb bud into the anterior and posterior halves. (B) Dorsal views of the recombinants made from the anterior (B), posterior (C), and both anterior and posterior (D) mesenchyme. Note that all digits have a claw in (B) but no digits have a claw in (C), and some digits have a claw in (D). Arrowheads show the hostraft boundary. Bars, 1 mm.
