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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 2, 2003 as doi:10.1096/fj.02-0579fje. |
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* Department of Environmental Biology, University of Adelaide, Adelaide SA 5005, Australia;
Department of Nuclear Medicine, Royal Adelaide Hospital, Adelaide SA 5000, Australia;
Ludwig Institute for Cancer Research, Royal Melbourne Hospital, Victoria, Australia; and
Department of Plastic and Reconstructive Surgery, Royal Adelaide Hospital and University of Adelaide, Adelaide SA 5005, Australia
2Correspondence: Department of Environmental Biology, University of Adelaide, Adelaide SA 5005. E-mail: chris.daniels{at}adelaide.edu.au
SPECIFIC AIMS
The Australian gecko lizard Christinus marmoratus can shed its tail voluntarily (tail autotomy) and regenerate a new complex tail consisting of skin, muscle, fat, cartilage, and neural tissues. Whether lymphatic vessels are restored in a regenerating lizard tail has not been established. Nevertheless, the tail never appears lymphedematous. Here we characterize tail regeneration by describing the formation and function of new lymphatics and identify growth factors involved in the control of lymphangiogenesis. We demonstrate that vascular endothelial growth factor (VEGF) -like molecule(s) exist in the gecko and may have important roles in regulating lymphatic vessel formation throughout tail regeneration.
PRINCIPAL FINDINGS
1. Morphology and histology
After controlled autotomy of the tails, regenerates were sampled 3, 6, 9, 12, 15, and 18 wk later. Original tails have a dense connective tissue band rich in adipose tissue surrounding the central spinal cord. The spine is sheathed with a thick layer of highly organized longitudinal muscle bundles. An organized network of densely packed lymphatic vessels exists in the connective tissue between the muscle bands and dermal layer, as well as in the central connective tissue band. Regenerated tails have a central cartilage tube as opposed to vertebrae. Throughout the regeneration process the size, density, and level of organization of the longitudinal muscle bands increase, as does the extent of connective and adipose tissue. Image analyses demonstrated that original tails have a greater number (Student Newman Keuls multiple comparison test, P<0.05) of wider (P<0.01) lymphatic vessels than full regenerates (Fig. 1
a, b). Three weeks after autotomy, the regenerating tail contains a smaller number (P<0.01) of wide lymphatic vessels than original tails (Fig. 1a, b
). The numbers of lymphatic (P<0.01) and blood vessels (P<0.05) increase significantly between 3 and 6 wk of regeneration (Fig. 1a
). The number of blood vessels far exceeds (>5-fold) that of lymphatic vessels at every stage (Fig. 1a
). Lymphatic vessel diameter decreases substantially from 21 to 12 µm (P<0.001) between 3 and 6 wk of regeneration (Fig. 1b
). Although the diameter of lymphatic vessels in fully regenerated tails is significantly smaller than those in original tails (P<0.01), an organized lymphatic network is apparent.
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2. Lymphatic function
Subdermal administration of the radiocolloid 99mTc-antimony trisulfide into the tip of the gecko tail demonstrated that geckos with original tails show a faster migration of the tracer along the ventral trunk than geckos with fully regenerated tails. By 60 min, the tracer has entered the blood circulation at the cloaca in original and fully regenerated tails. However, it localized with a much higher intensity in the heart and liver in the geckos with original tails. Fully regenerated tails have significantly slower lymph velocity (Dunnett, P<0.01) than comparably sized original tails. Similarly, radiocolloid migration after 60 min is significantly greater in original tails compared with regenerated tails at all times (Dunnett, P<0.05).
3. Western blot analysis
Western blot analysis, using a human, mouse, and rat reactive VEGF-C specific antibody raised to a polypeptide sequence of 20 amino acids of the carboxyl terminus of human VEGF-C, indicated the presence of a lizard protein with homology to mammalian VEGF-C. Two immunogenic peptides (58 and 43 kDa) were identified (Fig. 2
a). Absorbance analysis of relative quantities of the 43 kDa peptide demonstrated that the protein has a basal level of expression in tissue with functional lymphatics in both original and fully regenerated tails (Fig. 2b
). After complete disruption of the lymphatics following autotomy, protein expression in the regenerating stump was undetectable at 3 wk (Fig. 2)
but increased at 6 wk, to peak at 9 and 12 wk after regeneration commenced (Fig. 2)
. Levels at 9, 12, and 15 wk were significantly up-regulated over basal levels in original tails (Dunnett, P<0.05).
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CONCLUSIONS AND SIGNIFICANCE
We have demonstrated that regeneration of tail tissue was substantially completed within the first 9 wk after autotomy. The major proliferative phase of lymph angiogenesis appeared to occur between 3 and 6 wk of regeneration. Angiogenesis preceded lymphangiogenesis, and there was an 11:1 ratio of blood to lymph vessels 3 wk after autotomy. It appears that lymphatic regeneration proceeds in the first 3 wk via formation of a few large lymphatic vessels located within the central connective tissue band of the lizard tail, which subsequently proliferate into a network of subdermal small diameter lymph vessels (Fig. 3
).
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Using lymphoscintigraphy, we demonstrated that a functional lymphatic network is established after 6 wk of regeneration. Both lymph velocity and lymphatic migration remained identical thereafter. Original tails had a faster lymph velocity and greater lymph migration than fully regenerated tails (Fig. 3)
. The greater migration is most likely related to the greater number of larger lymphatic vessels in original tails compared with full regenerates. Greater lymph velocity, on the other hand, may be related to the more developed muscle bundles in the original tail, which may act to propel lymph fluid more efficiently.
Using Western blot analysis and antibodies raised to human VEGFs, we are the first to determine that reptiles have a protein growth factor homologous to human VEGF-C at the carboxyl terminus. However, it is possible that the detected molecule has homology to both VEGF-C and VEGF-D molecules or that there is only one such molecule in the reptile, which is functionally related to both VEGF-C and VEGF-D. Until further characterization is undertaken, the reptilian molecule is best described as a VEGF-C/D homologue and termed reptilian (r) VEGF-C/D.
In the 3 wk after autotomy, rVEGF-C/D was not detectable in the regenerated tail tissue. Hence, there appears to be a lag time before lymph angiogenesis is triggered. rVEGF-C/D expression increased dramatically between 3 and 6 wk after autotomy and was significantly up-regulated above levels in original tails at 9, 12, and 15 wk. The up-regulation of rVEGF-C/D correlated with lymph vessel formation. This temporal delay in lymphatic proliferation may reflect an inherently pre-programmed sequence of regeneration events. Alternatively, the major phase of lymphatic proliferation may be triggered by a physical parameter such as an increase in interstitial fluid pressure. It is also possible that the up-regulation in the number of rVEGF-C receptors is delayed.
How lymphangiogenesis is promoted in 3–6 wk is unknown, but this period may hold the key to establishing complete lymphangiogenesis in humans where lymphatic vessels are damaged or absent. This natural tail-regenerating model capable of rapidly regenerating a functional lymphatic system allows a study of the molecular mechanisms responsible for creating a functional lymphatic system. Using this model, it may be possible to eventually determine why only transient lymphangiogenesis occurs in humans throughout wound healing.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0579fje; to cite this article, use FASEB J. (January 2, 2003) 10.1096/fj.02-0579fje 
3 Present address: Department of Clinical Pharmacology, Flinders Medical Center, Flinders University School of Medicine, Bedford Park, South Australia.
4 Present address: Department of Ecology and Evolutionary Biology, University of California, Irvine, CA 92697, USA.
This article has been cited by other articles:
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  C. Cursiefen, S. Ikeda, P. M. Nishina, R. S. Smith, A. Ikeda, D. Jackson, J.-S. Mo, L. Chen, M. R. Dana, B. Pytowski, et al. Spontaneous Corneal Hem- and Lymphangiogenesis in Mice with Destrin-Mutation Depend on VEGFR3 Signaling Am. J. Pathol., May 1, 2005; 166(5): 1367 - 1377. [Abstract] [Full Text] [PDF]
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