Nerve-blastema interactions induce fibroblast growth factor-1 release during limb regeneration in Pleurodeles waltl.

Previous studies have shown that both fibroblast growth factor (FGF)-1 and nerves play an important function during limb regeneration, but no correlation between these two regeneration factors has yet been demonstrated. In the present study we first establish that exogenous FGF-2, a member of the FGF family that binds to the same high-affinity receptors as FGF-1, is able to stimulate both [3H]-thymidine incorporation and the mitotic index in the mesenchyme and the epidermal cells of denervated blastemas. We then use cocultures of spinal cord and blastema on heparin-coated dishes, an in vitro system mimicking the in vivo interactions during limb regeneration, to show that interactions between nerve fibers from the spinal cord and the blastema enhance the release of bioactive FGF-1. Release of this growth factor seemed to correlate with nerve fiber regeneration, as it decreased in the presence of the dipeptide Leu-Ala, known to inhibit neurite outgrowth, while the inverse dipeptide Ala-Leu was inactive. Therefore, these results support our hypothesis that the interaction between nervous tissue and blastema is permissive for the release of FGF-1, which in turn stimulates blastema cell proliferation.

Experimental evidence for FGF-1 control of blastema cell proliferation during limb regeneration of the amphibian Pleurodeles waltl.

During regeneration, blastema cell proliferation depends on several different factors which are, as yet, not fully understood. Previous studies showing the presence of FGF-1 and FGF receptors in the limb blastema make FGF-1 a potentially important molecule for limb regeneration but they do not demonstrate that this factor is active during the process. In the present study, we have first of all confirmed the presence of FGF-1 in limb blastemas of the amphibian Pleurodeles waltl using immunochemistry. Second, we provide evidence in vivo that FGF-1 controls blastema cell proliferation by using different reagents which interfere with FGF activity. Sulfated polysaccharides which bind FGFs, such as heparin, iota-carrageenan and pentosan polysulfate, are able to decrease both 3H-thymidine incorporation and the mitotic index in regeneration blastemas. In addition, suramin which inhibits the binding of growth factors to their receptors, induces the same effect. The presence of receptors in blastema cells is also demonstrated by using the FGF-saporin complex which is known to bind to FGF receptors and to kill cells bearing these receptors. This complex decreases the mitotic index in mesenchyme, while saporin alone did not influence cell proliferation. Finally, results obtained using a neutralizing monoclonal antibody against FGF-1 which was able to specifically reduce blastema cell proliferation, suggests that FGF-1 plays an important function in limb regeneration.