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While Xenopus is a well-known model system for early vertebrate development, in recent years, it has also emerged as a leading model for regeneration research. As an anuran amphibian, Xenopus laevis can regenerate the larval tail and limb by means of the formation of a proliferating blastema, the lens of the eye by transdifferentiation of nearby tissues, and also exhibits a partial regeneration of the postmetamorphic froglet forelimb. With the availability of inducible transgenic techniques for Xenopus, recent experiments are beginning to address the functional role of genes in the process of regeneration. The use of soluble inhibitors has also been very successful in this model. Using the more traditional advantages of Xenopus, others are providing important lineage data on the origin of the cells that make up the tissues of the regenerate. Finally, transcriptome analyses of regenerating tissues seek to identify the genes and cellular processes that enable successful regeneration. Developmental Dynamics 238:1226-1248, 2009. (c) 2009 Wiley-Liss, Inc.
Figure 4. Regeneration of the froglet forelimb. A: A normal froglet forelimb has 4 digits (far left). Amputation of the Xenopus froglet forelimb midway along the forearm (zeugopod, white arrowheads) results in epimorphic regeneration of a hypomorphic spike. Regenerative progress is shown at 0 (time of cutting) to 42 days afterward. From days 0-3, soft tissue contracts away from the wound surface and the wound becomes covered. A blastema is present by day 7 and extends distally forming a spike. B: In transgenic animals in which the Bmp signalling pathway is inhibited by induction of ectopic noggin expression with daily heat shocks over 14 days, regeneration of the spike is completely inhibited. C: Skeletal preparation of a normal froglet forelimb showing the ossified bone in red and the cartilage in blue (alizarin red S/alcian blue). The white arrowheads show the position of amputation. D: Skeletal preparation of normal wild-type regenerating forelimb after 42 days. The extent of the regenerated spike is outlined by white dots. Only cartilage is present in the spike, and there are no joints. E: Bmp inhibited animals show no regeneration of cartilage or other soft tissues. c, cartilage; h, humerus; r/u, radius and ulna; p, phalanges.
Figure 1. Regeneration of the tail. A: Time course of tail regeneration. Unoperated tail is shown at the top (WT), labeled to show the arrangement of the axial tissues. Note that the spinal cord lies on top of the notochord and is hard to see. After removal of the posterior 20% of the tail length, the extent of regeneration is shown after 1, 4, 10, and 14 days. Note the continuity of the axial structures between the original and regenerated tissues. B: Schematic of a transverse section through a Xenopus tadpoletail to show the arrangement of the tissues that must be regenerated. C: Sagittal section through a 3 day regenerating tadpoletail to show the spinal cord terminating in the blind-ended neural ampulla. The neural ampulla typically contains an enlarged lumen, continuous with the lumen of the stump spinal cord. The vacuolated notochord cells of the stump are continuous with a bullet-shaped structure composed of cells resembling the sheath cells that surround the notochord; later on, these cells will vacuolate and enlarge, becoming indistinguishable from the stump notochord cells. There are also undifferentiated, mesenchymal-like cells found around these two structures. White arrows indicate amputation plane. Scale bar = 200 μm.
Figure 2. Origins of the axial tissues in tail regeneration. A–D: Schematic representation of lineage labeling by tissue grafts expressing constitutive green fluorescent protein (GFP). A: GFP-positive neural plate grafted at st. 13, will constitutively label a section of the ventral spinal cord in the tadpole (see A′ and inset). After amputation, the entire ventral spinal cord regenerate will be GFP-positive (A′). B: GFP-positive notochord grafted at st. 13 will give rise to a GFP-positive section in the tadpole (B′). After amputation, the complete notochord in the regenerate is GFP positive (B′). C: GFP positive presomitic mesoderm grafted at st. 13 does not contain any muscle satellite cells, but labels muscle fibers in a section in the tadpoletail (C′). After amputation, no GFP-positive cells are found in the regenerate (C′). D: GFP-positive presomitic mesoderm grafted at st. 17 contains muscle satellite cells and labels similar to the early muscle grafts muscle fibers and satellite cells in a section of the tadpoletail (D′). After amputation, GFP-positive cells are seen in the regeneration bud and later in the newly formed muscle fibers of the regenerate (D′).
Figure 3. Regeneration of Xenopus hindlimbs declines with age. A–C: Developing limbs at stages indicated. D–F: Whole-mounts of 5 day blastemas amputated at indicated stages through the ankle. G–I: Longitudinal sections of 5 day blastemas stained with hematoxylin and eosin, amputated at indicated stages through the ankle. The 5 days of regeneration adds at least one developmental stage; therefore, proximallimb histology corresponds to st. 53 (G), st. 55 (H), and st. 58 (I). Note that 5 day blastemas in younger stages are larger than in older stages (compare G–I). G′–I′: Higher magnification of distalblastema from pictures above. Note the prominent distalepidermis forming the apical epithelial cap (AEC; aec), while columnar basal epithelial cells (be) are lining the blastema. In st. 57 regenerates, no AEC forms and the wound is just covered by a simple wound epithelium (e). Black arrowheads indicate the amputation plane. fe, cartilage condensation for femur; at st. 57 the femur is partially ossified, t/f, cartilage condensation for tibia and fibula, at st. 57 tibia and fibula also contain bone; m, muscle; b, blastema. Scale bars = 250 μm.
Figure 5. Regeneration of the tadpolelens. A: Schematic showing the process of lentectomy in Xenopus tadpoles and regeneration of the lens from the outer cornea. The outer cornea is cut with iridectomy scissors and flipped back, and the lens is extracted through the inner cornea using watchmaker's forceps. The outer cornea is then replaced. B–E: Histological sections of eye after lentectomy at st. 50, stained with hematoxylin and eosin. Freeman's 1965 regeneration stages are used, but B = 24 hr after lentectomy, C 2 days, D 3 days, and E 8 days. c, cornea; r, retina; i, iris; v, vitreous; l, lentoid. Arrows denote the outermost layer of the cornea. Arrowheads in C mark aggregation of transdifferentiating cells. F: transgenic tadpoleeye showing green fluorescent protein (GFP) in the lens, driven by the γ-crystallin promoter. F′: same eye after lentectomy. G–I: Fluorescent views, and brightfield views (G′–I′). G,G′: Transgenic eye 5 days after lentectomy, green cells in G are newly differentiated lens cells. H,H′: Transgenic eye 14 days after lentectomy, a new lens and a rare ectopic lentoid can be seen (white arrow). I, I′: Eye on the left was subjected to lentectomy and has regenerated a new lens. Scale bar = 50 μM in E (applies to panels B–E).
