Tail Regeneration in Lizards

Introduction

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, Eublepharis macularius. Lateral view of an adult with a regenerate tail. Lines identify the proximal extent of the regenerate portion of the tail. (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).

Video:

Time-Lapse Tail Regeneration

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.’

I

  • exposed tissues
  • spinal cord retracts and is capped by small blood clot
  • integument begins to collapse around the wound site

II

  • adjacent muscles and connective tissues retracted
  • blastema appears
  • first appearance of osteoclasts and chondroclasts

III

  • angiogenesis begins within blastema
  • axons from dorsal root ganglia of original tail begin to grow into blastema

IV

  • regenerating tail is dome-shaped and wider than long

V early

  • earliest appearance of presumptive cartilaginous skeleton around ependymal tube

V late

  • keratinization and the formation of scales within the epidermis
  • first expression of Sox9 in cells of regenerating cartilage

VI

  • tail is a tapering cone
  • differentiated dermis
  • differentiation of cartilage, muscle, and adipose tissue nearing completion

VII

  • tail is longer than wide
  • pigmentation begins

(McLean et al., 2011)

Summary of the onset and duration of major events during tail regeneration in Eublepharis macularius. (McLean et al., 2011)

Pathways Involved in Lizard Tail Regeneration:

Sox9

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.

Stage V of tail regeneration. Eublepharis macularius. (A,B) Gross morphology of the regenerating tail in caudal (A) and dorsal view (B). The regenerating tail is an elevated dome with a length: diameter between 0.5 and 1.0. Serial sections (dorsal towards the top) stained with hematoxylin and eosin (C), Masson's trichrome (D,G,F), Alcian blue (E,H) or immunostained with Sox9 (I). (C-F) Early stage V. (C) Sagittal section of tail with the developing blastema (right side of image) shaped like an elevated dome. (D) Closer view of the region identified in panel (C) taken from a different section. Note the wound epithelium and underlying blastema. (E) Closer view of the region identified in panel (C) taken from a different section. In this image the precartilaginous mesenchymal condensation is visualized by weakly positive staining for Alcian blue. (F) Transverse section through the regenerate tail depicting early muscle formation (stained red) and precartilaginous mesenchymal condensation surrounding the ependymal tube. (G-I) Late stage V. (G) Closer view of epidermal ingrowths, the first evidence of scalation. (H) Parasagittal section through developing cartilaginous cone visualized by strong, positive staining for Alcian blue. (I) Closer view of cartilage cone immunostained for Sox9 (brown), to identity differentiating chondrocytes, and counterstained with hematoxylin (blue). bl, blastema; bv, blood vessel; cc, cartilage cone; co, mesenchymal condensation; em, epaxial muscle; et, ependymal tube; hm, hypaxial muscle; no, notochord; rm, regenerated muscle; sc, spinal cord; we, wound epithelium. Scale bars: c = 20 μm; d = 500 μm; e-g = 100 μm. (McLean et al., 2011)

CD59

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).

(A) Quantitative results for RT-PCR amplification of CD59 for the spinal cord from L13 to the 6th caudal vertebra for the controls (Nor) and following tail amputation at 1 day, 3 days, 1 week and 2 weeks. Quantities were normalized to endogenous EF-1α expression. Error bars represent the standard deviation (P<0.01). (B) Localization of CD59 mRNA in the spinal cord by in situ hybridization using CD59 antisense RNA probes. L, M, O and P indicate sections of spinal cord segments at 1 day, 3 days, 1 week and 2 weeks post amputation. J indicates control section with sense probe. Scale bars: J-P, 50 µm. (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).

Helpful websites:

http://en.wikipedia.org/wiki/Leopard_gecko

http://en.wikipedia.org/wiki/Regeneration_(biology)



Immunohistochemistry tutorial

References

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

6 Responses to Tail Regeneration in Lizards

  1. Waseem Ullah says:

    Hello Sir, can i use these information in my research? can i give reference to to this website if it is valid for the future?

  2. Mahesh Patil says:

    you send me new research on lizards & other genetic research.

  3. Sonny says:

    Hello, a lot of people said that the myoseverin is the molecule who allows regeneration of lizard’stail, so could you give me some informations about this molecule please!

  4. Zachary Barnett says:

    School project. This info helpedalot. Thanks

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