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Abstract
During outgrowth of the developing limb, signals from the apical ectodermal ridge, such as fibroblast growth factors, are paramount for limb patterning. Similarly, fibroblast growth factor molecules and their receptors are synthesized in the wound epithelium of the regenerating limbblastema, implicating an analogous function to limb development. To address this issue further and to understand the role of fibroblast growth factor receptor signaling in limb regeneration, we have examined the expression patterns of x-fibroblast growth factor receptors-1, -2, -3, -4a, and -4b in Xenopus laevis. This amphibian model provides a system in which both regenerating (premetamorphic; tadpole or larva stage) and nonregenerating (postmetamorphic; froglet stage) hindlimbs can be studied. In premetamorphic hindlimbs (stage 53), all of the receptors were expressed in the wound epithelium and the underlying mesenchyme. In postmetamorphic limbs (stage 61), however, transcripts for x-fibroblast growth factor receptors-1 and -2 were absent from the wound epithelium. The expression results for x-fibroblast growth factor receptors-1 and -2 were corroborated at the protein level by employing specific antibodies. Thus, it appears that expression of both fibroblast growth factor receptors-1 and -2 is associated with the ability for limb regeneration. The role of these receptors in regeneration was further investigated by using specific inhibitors to fibroblast growth factor receptors during premetamorphic hindlimb regeneration. These compounds inhibited the normal limb outgrowth and resulted, in the majority of the cases, in outgrowths of cones or spikes reminiscent of growth that is seen in amputated postmetamorphic limbs. Thus, fibroblast growth factor receptors-1 and -2 expression and function should be regarded as paramount for the ability of limb regeneration in Xenopus.
Figure 1 Expression patterns of FGFFs in Xenopus hindlimb regeneration pression of xFGFR-1 in a 15-day blastema after amputation of a stage 53 hindlimb . Note the high expression (depicted as blue color) in the wound epithelium (we), the blastema (b), perichondrium (p), and cartilage (c). B, Expression of xFGFR-1 in a 15-day regenerate after amputation of a stage 61 hindlimb, There is some ex- pression in the mesenchyme (m), but the wound epithelium is negative. C. Expresssion of xFGFR-4b in a 15-day blastema
after amputation of a stage 53 hindlimb Similar to xFGFR-1, note the expression in the wound epithelium and the under- neath mesenchymal cells of the blastema. D, Expression of xFGFR-4b in a 15-day regenerate after amputation of a
stage 61 hindlimb.Note the expression in the blastema and in the basal layer of the wound epithelium. E and F, Hybridization with a sense probe (xFGFR-1) to show the background in a 15-day regenerate after amputation of a stage 53 and stage 61 hindlimb, respectively.
Reproduced with permission of the publisher, John Wiley & Sons.
Figure 2 Immunohistological detection of FGFR-1 and FGFR-2 protein in premetamorphic and postmetamorphic hindlimb blastema. All panels are sections obtained from paraffin-embedded X. laevis tissue. A, Regenerating hindlimb blastema (stage 53, 15 days postamputation); FGFR-1 (flg) shows high expression in the wound epithelium (we), the mesenchyme (m), and the cartilage condensations (c), B, Nonregenerating hindlimb (stage 61, 15-days postamputation)
FGFR-1 (flg) is absent from the wound epithelium (we), as well as its underlying mesenchyme. C, Regenerating hindlimb blastema (stage 53, 15-days postamputation)
FGFR-2 (bek) shows expression in the wound epithelium (we) only. D, Nonregenerating hindlimb (stage 61, 15-days postamputation);
FGFR-2 (bek) is absent from these tissues. E. anf F. Negative controls of 15-day hindlimb blastemas (stage 53 and stage 61, respectively). Pictures were taken with a fluorescent microscope at a magnification of x200.
Reproduced with permission of the publisher, John Wiley & Sons.
Figure 3 Effects of FGFR inhibitors on X. laevis hindlimb regeneration . Limbs from stage 53 animals were amputated and treated. All of these figures represent regenerates at 23-days postamputation. A, Untreated control with normal regeneration. B, Treatment with SU4984 has resulted in only a cone formation spike formation. C aregenerate at late cone stage resulted after treatment with SU5402.D. A spike formation after treatment with Tyrophostin A-23. Arrows indicate the level of amputation.
Reproduced with permission of the publisher, John Wiley & Sons.
Figure 4 Histological examination of the effects of inhibitors on X. laevis hindlimb regeneration . Limbs from stage 53 animals were amputated and treated. . After 23 days, representative samples of affected regenerates were embedded in paraffin, sectioned, and stained with hematoxylin and eosin to reveal their tissue organization. A, Untreated control regenerating early cone 7 days after amputation, note the well formed blastema (b) covered by the wound epithelium (we). . B, Section through a regenerate treated with SU4984. Considerable tissue disorganization can be seen in the cone containing cartilaginous elements (c). C, A section through a regenerate (spike) after treatment with Tyrophostin A-23 with cartilage (c). D, A section through a spike formed after treatment with SU5402.
Reproduced with permission of the publisher, John Wiley & Sons.
