FGFs Treatment on Amputated Lizard Limbs Stimulate the Regeneration of Long Bones, Opening New Avenues for Limb Regeneration in Amniotes: A Morphological Study

Abstract

Previous studies indicated that Fibroblast Growth Factors (FGFs) are present during tail and early limb regeneration in lizards, but FGFs disappear in the limb that turns into a scar and does not regenerate at 25-40 days post-amputation. Based on these indications, the aim of the present study was to evaluate the influence of administered FGFs on limb regeneration in lizards by injections of FGF1-2 into amputated hind-limbs that were studied between 40 and 70 days post-amputation. Outgrowths of 2.0 to 3.5 mm were produced but they did not develop an autopodium during this period. The skin remained most un-scaled, resembling that of a tail blastema. Four hours before sacrifice, the animals were injected with 5BrdU to study cell proliferation using microscopic and immunofluorescent methods. Histological examination of the outgrowths at 40-70 days of regeneration showed the presence a rod of cartilage (femur), or partially or completely sub-divided into two parts likely corresponding to a tibia and fibula. The regenerated cartilage was in continuity with the transected long bones and was surrounded by a perichondrium and a dense connective tissue, sparse nerves while muscles were reduced or absent. Qualitative observations on 5BrdU-immunolabeling indicated that most proliferating cells were present in the apical wound epidermis, the apical-most perichondrium and in the regenerating scales at 40-60 days post-amputation, but decreased at 70 days. Few 5BrdU-labeled cells were seen in other tissues, including in the regenerated cartilages. The present study indicates that FGF1-2 treatment in lizards mainly stimulate cartilage regeneration and the formation of a thick epidermis with an Apical Epidermal Peg, the epidermal micro-region that favors regeneration. In summary, these results suggest that FGFs treatments on amputated limbs could also be attempted in others amniotes, including mammals. However FGFs are not capable to induce an autopodium, which requires further signaling factors for its formation.

Proliferating Cells in Knee Epiphyses of Lizards Allow for Somatic Growth and Regeneration after Damage

“…It is likely that the regenerated cartilaginous epiphyses formed after knee injury, later mature into epiphyses with secondary centers, the initial condition present before injury in adult epiphyses [31,32,34,36]. This is also supported from the experiments using FGF1-2 that indicated at 50-70 days post-amputation of the limb, the beginning of formation of ossification centers in the induced cartilaginous anlagen of the tibia-fibula or of the femur ( [26], this volume). When large and multiple incisions are produced in the epiphyses of lizard knees and extensive damage is produced in the epiphyses, the strong inflammatory reaction determines a more limited production of cartilage that is mixed to a dense fibrotic connective.…”

“…Lizard perichondrium in both vertebrae and knees also appears sensitive to diffusible signals derived from the lesion or amputation of nearby tissues, and it seems to react by chondroblasts proliferation. The latter can be stimulated by the administration of growth factors that favor the regeneration of a cartilaginous anlage of the femur, tibia and fibula, as it has been shown by treating regenerating lizard limbs with FGF1-2 ( [26], this volume). The potential of cartilage regeneration in lizards, and perhaps in other ectothermic reptiles, appears higher than in mammals and birds where instead a fibrous connective tissue is formed after injury.…”

“…This proliferative response suggests that lizard epiphyses and the periosteum have a broad regenerative potential, likely due to the permanence of a reserve of stem cells in these regions. These premises can explain the broad induction of cartilaginous tissue in the femur, tibia and fibula, after limb amputation followed by stimulation of the healing process using FGF1-2 ( [26], this volume). In other experiments, after the damage of the epiphyses in the lizard P. muralis, by producing small cuts in the articular cartilage or in the diaphyses, the formation of irregular holes and cartilage degeneration occurs [34,36].…”

“…It is indicated that these putative progenitor cells give rise to the new, regenerated cartilage after un-direct or direct damage such as amputation of the limb or direct knee injury, providing the damage is not excessive. The Review introduces to the following study on the massive regeneration of cartilaginous tissue that forms the cartilaginous anlagen of a new femur, tibia and fibula in the amputated hindlimb of lizards after stimulation with Fibroblast Growth Factor FGF1-2 ( [26], this volume).…”

Appendage regeneration in anamniotes utilizes genes active during larval‐metamorphic stages that have been lost or altered in amniotes: The case for studying lizard tail regeneration

“…The wound epidermis does not form an AEP, like in the tail, and FGFs and Wnt are absent. Attempts to stimulate the growth of the blastema by growth factors such as FGF1-2, EGF or hyaluronate coating the stump have managed in numerous cases to obtained blastema-like outgrowths (Alibardi, 2017d(Alibardi, , 2019c. These softer outgrowths initially contain numerous proliferating cells and can elongate for few mm, but proliferation stops after 50-70 days F I G U R E 9 Schematic drawings comparing hypothetic gene pathways activated during tail development (a) and inactive/partially activated during tail regeneration (b) in lizard.…”