The leopard gecko, like many other lizards, is able to voluntarily shed its tail as a strategy to escape predation. These lizards are able to develop a replacement appendage through epimorphic regeneration that resembles the original, complete with nerves, blood vessels, and skeletal support.
Epimorphic regeneration is the restoration of lost tissues and structures from an aggregation of proliferating cells known as a blastema. To date, most research on naturally evolved epimorphic regeneration has focused on non-amniotes including zebrafish and newts. Although explored in context of ecological costs and benefits, less is known about the sequence of cellular and tissue level events of lizard tail regeneration (McLean et al., 2011).
The leopard gecko, E. macularius, is a good model for regeneration, with a tail that is able to detach and regenerate naturally. The tail represents approximately 41% of total body length and is composed of multiple tissue types including striated muscle, vasculature, adipose tissue, a bony vertebral column, and a spinal cord. Unlike other lizards, geckos also retain the notochord into skeletal maturity (McLean et al., 2011).
A regenerated tail is superficially similar to the original, but not a perfect replica. The bony vertebrae and notochord are replaced by a hollow cartilaginous cone. The spinal cord is incompletely restored, and the scalation pattern of the regenerate tail is slightly different from the original (Alibardi, L., 2010).
Stages of Regeneration
McLean et al. did an excellent job describing the stages of tail regeneration in the leopard gecko. This was a major strength of the paper, ‘A novel amniote model of epimorphic regeneration: the leopard gecko, Eulepharis macularius.’
- exposed tissues
- spinal cord retracts and is capped by small blood clot
- integument begins to collapse around the wound site
- adjacent muscles and connective tissues retracted
- blastema appears
- first appearance of osteoclasts and chondroclasts
- angiogenesis begins within blastema
- axons from dorsal root ganglia of original tail begin to grow into blastema
- regenerating tail is dome-shaped and wider than long
- earliest appearance of presumptive cartilaginous skeleton around ependymal tube
- keratinization and the formation of scales within the epidermis
- first expression of Sox9 in cells of regenerating cartilage
- tail is a tapering cone
- differentiated dermis
- differentiation of cartilage, muscle, and adipose tissue nearing completion
- tail is longer than wide
- pigmentation begins
(McLean et al., 2011)
Pathways Involved in Lizard Tail Regeneration:
To identify early evidence of cartilage formation, McLean et al. used Sox9 immunohistochemistry. Proliferating cell nuclear antigen (PCNA) immunohistochemistry is a method that uses a nuclear protein that is required for chromosomal DNA replication and is commonly used as a marker of the synthesis (S) phase of the cell cycle (Bravo et al., 1987).
Sox9 is a transcription factor that is involved in the regulation of chondrogenesis, or cartilage development. There were no Sox9 positive cells in the original tail, and Sox9 immunopositive cells were not detected in the regenerating lizard tails until late stage V. This shows that cartilage does not start to develop until that time in the regenerating tail (McLean et al., 2011).
One weakness of the McLean et al. paper was that Sox9 was the only genetic marker of regeneration that was researched. Although the stages of regeneration were very thoroughly described, more genetic regulators of the developing tail could have been explored.
Wang et al. identified the regulatory protein CD59 as a determinant of proximal-distal cell identity in Gekko japonicus. Quantitative Real Time PCR (qRT-PCR) was used to quantify expression of CD59 and measure the amount of protein present at a given time in the regenerating tail.
Strong CD59 signals were detected in cells anterior to the blastema, with a gradual decrease along the proximodistal axis. Overexpression of CD59 during tail regeneration causes distal blastemal cells to translocate to a more proximal location. This suggests that position identity has already been established in tail blastema of reptiles (Wang et al., 2011).
FGF1 and FGF2
Fibroblast growth factors have been shown to stimulate limb and tail regeneration in amphibians. Immunofluorescence was used to visualize FGF1 and FGF2 expression in the lizard Lampropholis guichenoti. Immunofluorescence is a technique that uses a specific antibody and fluorescent dye to visualize target molecules under a fluorescent microscope.
Alibardi et al. have shown that FGF1 and FGF2 play roles in tail regeneration in the lizard Lampropholis guichenoti. FGF1 is present in blastema cells and differentiating epidermis. FGF2 is mainly localized in the wound and scaling epidermis, muscles, spinal ganglia, and regenerating nerves and spinal cord. Cartilaginous, bone, and fat tissues expressed FGFs poorly (Alibardi et al., 2010).
McLean et al. A novel amniote model of epimorphic regeneration: the leopard gecko, Eulepharis macularius. BMC Developmental Biology 2011, 11:50. http://www.biomedcentral.com/1471-213X/11/50
Bravo R, Macdonald-Bravo H: Existence of two populations of cyclin/proliferating cell nuclear antigen during the cell cycle: association with DNA replication sites. J Cell Biol 1987, 105:1549-1554.
Alibardi L: Morphological and cellular aspects of tail and limb regeneration in lizards: A model system with implications for tissue regeneration in mammals. Adv Anat Embryol Cell Biol 2010, 207:1-109.
Wang Y, Wang R, Jiang S, Zhou W, Liu Y, et al. (2011) Gecko CD59 Is Implicated in Proximodistal Identity during Tail Regeneration. PLoS ONE 6(3):e17878. doi:10.1371/journal.pone.0017878. http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017878
Alibardi L, Lovicu FJ. Immunolocalization of FGF1 and FGF2 in the regenerating tail of the Lampropholis guichenoti: Implications for FGFs as trophic factors in lizard tail regeneration. Acta histochemica 112 (2010) 459-473.http://www.sciencedirect.com/science/article/pii/S0065128109000488