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Figure 1. Medium bud stage blastemas derived from (A) the distal third of the humerus of the spotted salamander larva, Ambystoma maculatum. B: The distal tip of the radius and ulna of the axolotl, Ambystoma mexicanum. Note the thickened apical wound epidermis (AEC) covering the tip of each blastema. The epidermis consists of keratinocytes and Leydig (gland) cells that are distinguished by their large size and lighter color. M, muscle. Photos by DL Stocum.Download figure to PowerPoint
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Figure 2. Relationship between nerves (yellow), AEC (green) and blastema cells (red) in the growing blastema (based on data of Kumar et al., 2007). Nerve axons stimulate the AEC to express AGP, which diffuses into the blastema and promotes blastema cell proliferation by binding to its ligand, Prod1.Download figure to PowerPoint
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Figure 3. Intercalary regeneration in the PD axis of the axolotl limb (after Pescitelli and Stocum, 1980). A: A triploid (3N) wrist (W) blastema is heterografted to a diploid (2N) upper arm stump (S, stylopodium), discarding the intermediate structures. B: After a delay during which the host limb tissues dedifferentiate and contribute to the blastema, a normal limb is regenerated by the intercalation of the distal stylopodium (bright blue) and the zeugopodium (lower arm, green). Counts of triploid vs. diploid cells show that the 3N graft develops into carpals (C) and digits, whereas the intercalated intermediate structures are the host 2N ploidy.Download figure to PowerPoint
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Figure 4. A: Upper panel: The ulna and associated muscle (orange hatch) of the posterior half of a newt lower arm was removed while leaving the skin (green) intact (after Goss, 1957). After amputation, radial intercalation (arrows) restored the positional identities of the posterior half. Lower Panel: A half limb regenerates when all the tissues of the posterior half are removed because radial intercalation is not possible. B: Lheureux' experiment (1975). Upper panel: An unirradiated dorsal longitudinal skin strip was turned 90° and wrapped around the circumference of an irradiated limb (I, lightning bolts) at the level of the upper arm. After amputation through the strip, all blastema cells have the same positional identity and sense no discontinuity. Lower panel: Control experiment grafting rotated longitudinal strips of skin from each quadrant of the limb. Positional discontinuity is now sensed and distal transformation takes place. R, radius; S, humerus; d, dorsal; a, anterior; v, ventral; p, posterior.Download figure to PowerPoint
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Figure 5. Right: “Affinophoresis” assay in vivo for blastema cell PD adhesive gradient (after Crawford and Stocum, 1988a). Medium bud stage blastemas derived from the wrist (W), elbow (E), or mid-upper arm (UA) were autografted to a wound bed made at the junction of the stump and a medium bud blastema derived by amputation (Amp) at the mid-femur (F) level of the hindlimb. Left: As the host hindlimb blastema grows and redifferentiates, the wrist and elbow blastemas sort distally to their corresponding tarsal and knee levels, respectively, while the mid-upper arm blastema remains at the amputation level. Download figure to PowerPoint
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Figure 6. Coordinate proximalization of adhesive quality and positional identity of blastema cells (after Crawford and Stocum, 1988b). Right: RA-treated wrist and elbow blastemas (W, E) were grafted to the blastema-stump junction of a hindlimb regenerating from the mid-femur (F). Left: As the hindlimb blastema grows and redifferentiates, the proximalized wrist and elbow RA-treated blastemas develop from the upper arm level (UA) and remain at the site of grafting instead of sorting distally.Download figure to PowerPoint
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Figure 7. Right: Basic features of the polar coordinate model (after Bryant et al., 1981). Cells from the circumferential arcs of all four quadrants of the amputated limb migrate centripetally (red arrows) and interact to generate circumferential and radial intercalation (yellow arrows), forming a complete cross-section of blastema cells that assume the next distal positional identity. Successive repetitions of this process regenerate the PD axis (dark blue arrow). Left: Upper: Dorsal view of a right limb stump (light blue) to which a left blastema (green) has been grafted, reversing the AP axis. Lower: View from the lateral side of the above construct showing how this graft confronts two half circumferences (orange brackets, DPV of the stump and DAV of the graft). The graft (primary limb) develops with its handedness of origin (blue arrow). A complete right-handed cross-section of blastema cells is generated by radial intercalation within the half circumferences. Repetitive circumferential and radial intercalation/distalization, as in the diagram at the right, regenerates the PD axis. A, anterior; P, posterior; D, dorsal; V, ventral.Download figure to PowerPoint
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Figure 8. The boundary model (after Meinhardt, 1983b). The intersection of AP and DV boundaries (yellow lines) to form four compartments is necessary for distal transformation. A strip of polarizing tissue flanks the AP boundary. When the circumferences of the limb stump and axially reversed blastemas are “unrolled,” the cellular contributions of each compartment in the host and graft predict most of the known anatomical configurations of supernumerary limbs.Download figure to PowerPoint
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Figure 9. Models that link blastema self-organization and expression of Hoxa9/13, which is assumed to be a code for the prospective autopodium (after Stocum, 2006). A:Hoxa9/13 are expressed uniformly in all the cells of the accumulation blastema. Hoxa9 expression is autonomous, but that of Hoxa13 requires factors (arrow) from the AEC. As the blastema grows, its more proximal cells fall outside the range of the AEC factors and express only Hoxa9, the code for the prospective stylopodium. This separates stylopodial and autopodial domains. The code for the prospective zeugopodium is intercalated at the boundary between autopodium and styopodium. B:Hoxa9 and Hoxa9/13 are expressed by separate subsets of cells at accumulation blastema. Hoxa9 and Hoxa9/13 cells have a differential homotypic adhesive affinity and Hoxa9/13 cells have a stronger heterotypic affinity for the AEC. Prospective autopodial and stylopodial domains separated by cell sorting.Download figure to PowerPoint
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