Immunodetection of FGF1-2 and FGFR1-2 Indicates that these Proteins Disappear in the Wound Epidermis and Blastema of the Scarring Limb in Lizard

Abstract

In lizards while the tail broadly regenerates after amputation, the limb generally forms short scarring outgrowths. FGFs are important growth factors that are present in regenerating tissues and appear to stimulate tail regeneration in lizards. In the limb, immunohistochemistry at 15-16days post-amputation indicates that FGFs and FGFR1-2 are present with different intensity in the wound (regenerating) epidermis and sparsely in connective, muscle and cartilaginous cells of the healing tissues of the stump. However the immunofluorescence study indicate that FGF and FGF1-2 receptors are little or no immunodetectable at 25-26days post-amputation, especially in the wound epidermis, when the limb blastema is turning into a scarring connective tissue of the short limb outgrowth. Western blot analysis shows no low MW bands for FGF1-2 and altered or degraded bands of FGFR1-2 in the limb at 25-26days postamputation when scarring is taking place. The present observations indicate that the disappearance of these proteins is related to loss of cell proliferation that leads to the failure of limb regeneration in lizards. The study further stress the essential role of FGFs and their receptors in the stimulation of organ regeneration in lizards and likely in other amniotes. after injection of FGF1 obtained from Sigma. 18 The rabbit FGF2 antibody was purchased from Sigma (F3393). The antibody against FGFR1 was a rabbit polyclonal antibody directed against an epitope of the C-terminus of the receptor. 19 The FGFR2 antibody from rabbit was obtained from Santa Cruz (c-17, the beck isoform), and is directed against an epitope located in the C-terminal cytoplasmic side of the receptor. All the antibodies were generously provided from Dr. Frank Lovicu, University of Sydney, Australia, and the rabbit FGFR1 antibody 803 derived from Dr. A Baird laboratory Wittier Institute, La Jolla, CA, USA,. 19 Sections were de-waxed with xylene, rehydrated and pre-incubated for 30minutes in Buffer containing 5% of Normal Goat Serum and 2% Bovine Serum Albumin (BSA). For the detection of FGFs and their receptors, the sections were incubated for 4-5 hours at room temperature with the rabbit antibodies at a dilution 1:100 in 0.1M Phosphate buffer containing 2% BSA, while in control sections the primary antibody was omitted. After rinsing for 10minutes with 3 changes in the Buffer, the sections were incubated for 1h at room temperature with a Tetramethyl Rhodamine Isotiocyanate (TRITC, Sigma, USA) labeled anti-Rb secondary antibodies (dilution 1:150). After the final rinsing, the sections were mounted in Fluoromount (Sigma, USA), and later observed under a fluorescent microscope. For western blot analysis, three regenerating tail blastemas of 2-3mm in length, and three normal tails were collected and immediately homogenized as indicated below. For the separation of the epidermis from the dermis and inner tissues, other five normal and five regenerating tails of 2-3mm in length were utilized for protein extraction. On this purpose, small pieces of...

A BRIEF REVIEW OF LIMB ANOMALIES IN LIZARDS AND PRESENCE OF ECTRODACTILY IN Aspidoscelis costatus (SQUAMATA: TEIIDAE)

“…However, we would argue that the former is more likely based on the presence of asymmetric, grey-black scales that covered the stump. It was similar to the outgrowths described by Alibardi (2017), seen after the amputation of a lizard's limb, and characterized by being short or flat as well as having a dense, hard, and scaled structure. It is also difficult to determine what may have caused the possible injury.…”

The Review of the Autotomy of Agamid Lizards with Considerations about the Types of Autotomy and Regeneration

“…It includes wound healing, blastema formation, differentiation and growth. It is known that some lizards are capable of imperfect regeneration of limbs [2,10,18], from regenerative buds to tail-like outgrowths. The regenerative abilities of tissues, preserved in the evolutionary history of reptiles, are expressed in the regeneration of the autotomized tail.…”

“…Most information about reptilian tissue regeneration derives from the study of the regenerating tail of lizards, an organ that measures a few centimeters in length in most of the species studied. A few other examples of organ regeneration among reptiles include the regeneration of the tail and jaws of crocodilians and the shell of turtles [2,10,18]. Fracture planes are registered in the lepidosaurian the most and can be considered as a common primitive rhynchocephalian and saurian character [2].…”