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

This review elaborates the idea that organ regeneration derives from specific evolutionary histories of vertebrates. Regenerative ability depends on genomic regulation of genes specific to the life-cycles that have differentially evolved in anamniotes and amniotes. In aquatic environments, where fish and amphibians live, one or multiple metamorphic transitions occur before the adult stage is reached. Each transition involves the destruction and remodeling of larval organs that are replaced with adult organs. After organ injury or loss in adult anamniotes, regeneration uses similar genes and developmental process than those operating during larval growth and metamorphosis. Therefore, the broad presence of regenerative capability across anamniotes is possible because generating new organs is included in their life history at metamorphic stages. Soft hyaluronate-rich regenerative blastemas grow in submersed or in hydrated environments, that is, essential conditions for regeneration, like during development. In adult anamniotes, the ability to regenerate different organs decreases in comparison to larval stages and becomes limited during aging. Comparisons of genes activated during metamorphosis and regeneration in anamniotes identify key genes unique to these processes, and include thyroid, wnt and non-coding RNAs developmental pathways. In the terrestrial environment, some genes or developmental pathways for metamorphic transitions were lost during amniote evolution, determining loss of regeneration. Among amniotes, the formation of soft and hydrated blastemas only occurs in lizards, a morphogenetic process that evolved favoring their survival through tail autotomy, leading to a massive although imperfect regeneration of the tail. Deciphering genes activity during lizard tail regeneration would address future attempts to recreate in other amniotes regenerative blastemas that grow into variably completed organs.     

The regenerating tail of lizard transits through a tumour-like stage represented by the regenerative blastema

Review. The regenerating tail of lizard transits through a tumour-like stage represented by the regenerative blastema. Acta Zoologica (Stockolm). Molecular studies on lizard tail regeneration indicate that the blastema stage is a tumour-like outgrowth capable of self-regulate to produce a new tail. Various oncogenes and tumour suppressors are expressed, and their proteins are localized in specific regions of the growing blastema. SnoRNAs are exclusively overexpressed in the tail blastema suggesting changes in ribosome translation efficiency in blastema cells, like in cancer. Blastema cells secrete high levels of hyaluronate and adopt an anaerobic metabolism (Warburg effect). These studies indicate that the lizard blastema represents a unique case among terrestrial vertebrates of physiological tumour remission. Mesenchymal cells and fibroblasts forming the blastema are turned within 1–2 months into a functional organ, the tail. In vitro studies on isolated mesenchymal cells from the regenerative blastema shows that these cells do not undergo contact inhibition but continue proliferation after confluence, and contain nestin, vimentin and K17. After 2–3 weeks they stratify into 5–7 layers forming a pellicle of loose connective tissue. Future molecular studies on genes and proteins that allow the control of growth in the lizard blastema may help to determine how lizards turn a tumour into a new organ with numerous differentiated and functional tissues, providing clues on cancer growth regulation.     

Limb regeneration in axolotl: is it superhealing?

The ability of axolotls to regenerate their limbs is almost legendary. In fact, urodeles such as the axolotl are the only vertebrates that can regenerate multiple structures like their limbs, jaws, tail, spinal cord, and skin (the list goes on) throughout their lives. It is therefore surprising to realize, although we have known of their regenerative potential for over 200 years, how little we understand the mechanisms behind this achievement of adult tissue morphogenesis. Many observations can be drawn between regeneration and other disciplines such as development and wound healing. In this review, we present new developments in functional analysis that will help to address the role of specific genes during the process of regeneration. We also present an analysis of the resemblance between wound healing and regeneration, and discuss whether axolotls are superhealers. A better understanding of these animals' regenerative capacity could lead to major benefits by providing regenerative medicine with directions on how to develop therapeutic approaches leading to regeneration in humans.     

TGF-beta signaling is required for multiple processes during Xenopus tail regeneration

Xenopus tadpoles can fully regenerate all major tissue types following tail amputation. TGF-β signaling plays essential roles in growth, repair, specification, and differentiation of tissues throughout development and adulthood. We examined the localization of key components of the TGF-β signaling pathway during regeneration and characterized the effects of loss of TGF-β signaling on multiple regenerative events. Phosphorylated Smad2 (p-Smad2) is initially restricted to the p63+ basal layer of the regenerative epithelium shortly after amputation, and is later found in multiple tissue types in the regeneration bud. TGF-β ligands are also upregulated throughout regeneration. Treatment of amputated tails with SB-431542, a specific and reversible inhibitor of TGF-β signaling, blocks tail regeneration at multiple points. Inhibition of TGF-β signaling immediately following tail amputation reversibly prevents formation of a wound epithelium over the future regeneration bud. Even brief inhibition immediately following amputation is sufficient, however, to irreversibly block the establishment of structures and cell types that characterize regenerating tissue and to prevent the proper activation of BMP and ERK signaling pathways. Inhibition of TGF-β signaling after regeneration has already commenced blocks cell proliferation in the regeneration bud. These data reveal several spatially and temporally distinct roles for TGF-β signaling during regeneration: (1) wound epithelium formation, (2) establishment of regeneration bud structures and signaling cascades, and (3) regulation of cell proliferation.     

Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

Background

Epimorphic regeneration results in the restoration of lost tissues and structures from an aggregation of proliferating cells known as a blastema. Among amniotes the most striking example of epimorphic regeneration comes from tail regenerating lizards. Although tail regeneration is often studied in the context of ecological costs and benefits, details of the sequence of tissue-level events are lacking. Here we investigate the anatomical and histological events that characterize tail regeneration in the leopard gecko, Eublepharis macularius.

Results

Tail structure and tissue composition were examined at multiple days following tail loss, revealing a conserved pattern of regeneration. Removal of the tail results in a consistent series of morphological and histological events. Tail loss is followed by a latent period of wound healing with no visible signs of regenerative outgrowth. During this latent period basal cells of the epidermis proliferate and gradually cover the wound. An additional aggregation of proliferating cells accumulates adjacent to the distal tip of the severed spinal cord marking the first appearance of the blastema. Continued growth of the blastema is matched by the initiation of angiogenesis, followed by the re-development of peripheral axons and the ependymal tube of the spinal cord. Skeletal tissue differentiation, corresponding with the expression of Sox9, and muscle re-development are delayed until tail outgrowth is well underway.

Conclusions

We demonstrate that tail regeneration in lizards involves a highly conserved sequence of events permitting the establishment of a staging table. We show that tail loss is followed by a latent period of scar-free healing of the wound site, and that regeneration is blastema-mediated. We conclude that the major events of epimorphic regeneration are highly conserved across vertebrates and that a comparative approach is an invaluable biomedical tool for ongoing regenerative research.     

Developmental and adult-specific processes contribute to de novo neuromuscular regeneration in the lizard tail

Peripheral nerves exhibit robust regenerative capabilities in response to selective injury among amniotes, but the regeneration of entire muscle groups following volumetric muscle loss is limited in birds and mammals. In contrast, lizards possess the remarkable ability to regenerate extensive de novo muscle after tail loss. However, the mechanisms underlying reformation of the entire neuromuscular system in the regenerating lizard tail are not completely understood. We have tested whether the regeneration of the peripheral nerve and neuromuscular junctions (NMJs) recapitulate processes observed during normal neuromuscular development in the green anole, Anolis carolinensis. Our data confirm robust axonal outgrowth during early stages of tail regeneration and subsequent NMJ formation within weeks of autotomy. Interestingly, NMJs are overproduced as evidenced by a persistent increase in NMJ density 120 and 250 days post autotomy (DPA). Substantial Myelin Basic Protein (MBP) expression could also be detected along regenerating nerves indicating that the ability of Schwann cells to myelinate newly formed axons remained intact. Overall, our data suggest that the mechanism of de novo nerve and NMJ reformation parallel, in part, those observed during neuromuscular development. However, the prolonged increase in NMJ number and aberrant muscle differentiation hint at processes specific to the adult response. An examination of the coordinated exchange between peripheral nerves, Schwann cells, and newly synthesized muscle of the regenerating neuromuscular system may assist in the identification of candidate molecules that promote neuromuscular recovery in organisms incapable of a robust regenerative response.     

Cytochrome oxidase and ascorbic acid in the normal and regenerating tail of the scincid lizard, Mabuya carinata. A histophysiological study.

The concentration of ascorbic acid (AA) and the histochemical distribution of this vitamin together with cytochrome oxidase have been investigated in the normal and regenerating tail of the Scincid lizard, Mabuya carinata. An interesting aspect of this investigation is the observation of a total lack of cytochrome oxidase in both the normal and regenerating tail of the lizard, except for the differentiating phase. On the other hand, AA has been found to be present in the normal and regenerating tail with above normal levels during wound healing (twofold) and differentiation (fivefold). In the light of the poor cytochrome oxidase activity, the higher content of AA noted during regeneration has been construed to play a possible role in the respiratory mechanics of the regenerating lizard tail. Further, the importance of AA in cellular metabolism and the wound healing and differentiative processes have also been discussed.     

Msx1‐2 immunolocalization in the regenerating tail of a lizard but not in the scarring limb suggests its involvement in the process of regeneration

The immunolocalization of the muscle segmental homoeobox protein Msx1‐2 of 27–34 kDa in the regenerating tail blastema of a lizard shows prevalent localization in the apical ependyma of the regenerating spinal cord and less intense labelling in the wound epidermis, in the apical epidermal peg (AEP), and in the regenerating segmental muscles. The AEP is a micro‐region of the regenerating epidermis located at the tail tip of the blastema, likely corresponding to the AEC of the amphibian blastema. No immunolabelling is present in the wound epidermis and scarring blastema of the limb at 18–21 days of regeneration, except for sparse repairing muscles. The presence of a proximal–distal gradient of Msx1‐2 protein, generated from the apical ependyma, is suggested by the intensity of immunolabelling. The AEP and the ependyma are believed to induce and maintain tail regeneration, and this study suggests that Msx1‐2 proteins are components of the signalling system that maintains active growth of the tail blastema. The lack of activation and production of Msx1‐2 protein in the limb are likely due to the intense inflammatory reaction following amputation. This study confirms that, like during regeneration in fishes and amphibians, also the blastema of lizards utilizes common signalling pathways for maintaining regeneration.     

Mammalian regeneration and regenerative medicine

Mammals are generally considered to be poor regenerators, yet there are a handful of mammalian models that display a robust ability to regenerate. One such system is the regenerating tips of digits in both humans and mice. In vitro studies of regenerating fetal human and mouse digit tips display both anatomical and molecular similarities, indicating that the mouse digit is a clinically relevant model. At the same time, genetic studies on mouse digit tip regeneration have identified signaling pathways required for the regeneration response that parallel those known to be important for regeneration in lower vertebrates. In addition, recent studies establish that digit tip regeneration involves the formation of a blastema that shares similarities with the amphibian blastema, thus establishing a conceptual bridge between clinical application and basic research in regeneration. In this review we discuss how the study of endogenous regenerating mammalian systems is enhancing our understanding of regenerative mechanisms and helping to shed light on the development of therapeutic strategies in regenerative medicine. Birth Defects Research (Part C) 84:265–280, 2008. © 2008 Wiley‐Liss, Inc.     

Immunolocalization of Wnts in the lizard blastema supports a key role of these signaling proteins for tail regeneration

A highly upregulated gene during tail regeneration in lizards is Wnt2b, a gene broadly expressed during development. The present study examines the distribution of Wnt proteins, most likely wnt2b, by western blotting and immunofluorescence in the blastema-cone of lizards using a specific antibody produced against a lizard Wnt2b protein. Immunopositive bands at 48–50 and 18 kDa are present in the regenerative blastema, the latter likely as a degradation product. Immunofluorescence is mainly observed in the wound epidermis, including in the Apical Epidermal Peg where the protein appears localized in intermediate and differentiating keratinocytes. Labeling is more intense along the perimeter of keratinocytes, possibly as a secretory product, and indicates that the high epidermal proliferation of the regenerating epidermis is sustained by Wnt proteins. The regenerating spinal cord forms an ependymal tube within the blastema and shows immunolabeling especially in the cytoplasm of ependymal cells contacting the central canal where some secretion might occur. Also, regenerating nerves and proximal spinal ganglia innervating the regenerating blastema contain this signaling protein. In contrast, the blastema mesenchyme, muscles and cartilage show weak immunolabeling that tends to disappear in tissues located in more proximal regions, close to the original tail. However, a distal to proximal gradient of Wnt proteins was not detected. The present study supports the hypothesis that Wnt proteins, in particular Wnt2b, are secreted by the apical epidermis covering the blastema and released into the mesenchyme where they stimulate cell multiplication.     

Immunolocalization of c‐myc‐positive cells in lizard tail after amputation suggests cell activation and proliferation for tail regeneration

After tail amputation in lizard, a regenerative response is elicited leading to the formation of a new tail. The stimulation of the proliferation process may involve the proto‐oncogene c‐myc. The immunocytochemical analysis detects the c‐myc protein few days after wound in free cells accumulating over the injured tissues of the tail stump. Western blot detects a protein band at 68–70 kDa that is more intense in the regenerating blastema than in normal tail tissues. Nuclei positive for the c‐myc protein are seen in mesenchymal‐like cells located among muscles, connectives and fat tissues of the tail stump 4 days postamputation. Proliferating cells labelled for 5BrdU are seen at 4 days postamputation and are sparse in the mesenchyme of the regenerating blastema formed at 12 days postamputation. Fine immunolocalization of the c‐myc protein shows it is mainly located over euchromatin or poorly condensed chromatin to indicate gene activation. The study correlates the detection of the c‐myc protein with activation of cell division in the injured tissues leading to the formation of the regenerative blastema. The lizard c‐myc protein probably activates a controlled proliferation process through a mechanism that can give information on the uncontrolled process occurring in cancer.