Amphibians as research models for regenerative medicine

The ability to regenerate bone across a critical size defect would be a marked clinical advance over current methods for dealing with such structural gaps. Here, we briefly review the development of limb bones and the mandible, the regeneration of urodele limbs after amputation, and present evidence that urodele and anuran amphibians represent a valuable research model for the study of segment defect regeneration in both limb bones and mandible.     

Co-operative Bmp- and Fgf-signaling inputs convert skin wound healing to limb formation in urodele amphibians

Urodele amphibians have remarkable organ regeneration capability, and their limb regeneration capability has been investigated as a representative phenomenon. In the early 19th century, nerves were reported to be an essential tissue for the successful induction of limb regeneration. Nerve substances that function in the induction of limb regeneration responses have long been sought. A new experimental system called the accessory limb model (ALM) has been established to identify the nerve factors. Skin wounding in urodele amphibians results in skin wound healing but never in limb induction. However, nerve deviation to the wounded skin induces limb formation in ALM. Thus, nerves can be considered to have the ability to transform skin wound healing to limb formation. In the present study, co-operative Bmp and Fgf application, instead of nerve deviation, to wounded skin transformed skin wound healing to limb formation in two urodele amphibians, axolotl (Ambystoma mexicanum) and newt (Pleurodeles waltl). Our findings demonstrate that defined factors can induce homeotic transformation in postembryonic bodies of urodele amphibians. The combination of Bmp and Fgf(s) may contribute to the development of novel treatments for organ regeneration.     

Deer antler regeneration: cells, concepts, and controversies

The periodic replacement of antlers is an exceptional regenerative process in mammals, which in general are unable to regenerate complete body appendages. Antler regeneration has traditionally been viewed as an epimorphic process closely resembling limb regeneration in urodele amphibians, and the terminology of the latter process has also been applied to antler regeneration. More recent studies, however, showed that, unlike urodele limb regeneration, antler regeneration does not involve cell dedifferentiation and the formation of a blastema from these dedifferentiated cells. Rather, these studies suggest that antler regeneration is a stem-cell-based process that depends on the periodic activation of, presumably neural-crest-derived, periosteal stem cells of the distal pedicle. The evidence for this hypothesis is reviewed and as a result, a new concept of antler regeneration as a process of stem-cell-based epimorphic regeneration is proposed that does not involve cell dedifferentiation or transdifferentiation. Antler regeneration illustrates that extensive appendage regeneration in a postnatal mammal can be achieved by a developmental process that differs in several fundamental aspects from limb regeneration in urodeles.     

Expression of complement 3 and complement 5 in newt limb and lens regeneration

Some urodele amphibians possess the capacity to regenerate their body parts, including the limbs and the lens of the eye. The molecular pathway(s) involved in urodele regeneration are largely unknown. We have previously suggested that complement may participate in limb regeneration in axolotls. To further define its role in the regenerative process, we have examined the pattern of distribution and spatiotemporal expression of two key components, C3 and C5, during limb and lens regeneration in the newt Notophthalmus viridescens. First, we have cloned newt cDNAs encoding C3 and C5 and have generated Abs specifically recognizing these molecules. Using these newt-specific probes, we have found by in situ hybridization and immunohistochemical analysis that these molecules are expressed during both limb and lens regeneration, but not in the normal limb and lens. The C3 and C5 proteins were expressed in a complementary fashion during limb regeneration, with C3 being expressed mainly in the blastema and C5 exclusively in the wound epithelium. Similarly, during the process of lens regeneration, C3 was detected in the iris and cornea, while C5 was present in the regenerating lens vesicle as well as the cornea. The distinct expression profile of complement proteins in regenerative tissues of the urodele lens and limb supports a nonimmunologic function of complement in tissue regeneration and constitutes the first systematic effort to dissect its involvement in regenerative processes of lower vertebrate species.     

Initiation of limb regeneration: the critical steps for regenerative capacity

While urodele amphibians (newts and salamanders) can regenerate limbs as adults, other tetrapods (reptiles, birds and mammals) cannot and just undergo wound healing. In adult mammals such as mice and humans, the wound heals and a scar is formed after injury, while wound healing is completed without scarring in an embryonic mouse. Completion of regeneration and wound healing takes a long time in regenerative and non-regenerative limbs, respectively. However, it is the early steps that are critical for determining the extent of regenerative response after limb amputation, ranging from wound healing with scar formation, scar-free wound healing, hypomorphic limb regeneration to complete limb regeneration. In addition to the accumulation of information on gene expression during limb regeneration, functional analysis of signaling molecules has recently shown important roles of fibroblast growth factor (FGF), Wnt/beta-catenin and bone morphogenic protein (BMP)/Msx signaling. Here, the routine steps of wound healing/limb regeneration and signaling molecules specifically involved in limb regeneration are summarized. Regeneration of embryonic mouse digit tips and anuran amphibian (Xenopus) limbs shows intermediate regenerative responses between the two extremes, those of adult mammals (least regenerative) and urodele amphibians (more regenerative), providing a range of models to study the various abilities of limbs to regenerate.     

Limb regeneration in Xenopus laevis froglet

Limb regeneration in amphibians is a representative process of epimorphosis. This type of organ regeneration, in which a mass of undifferentiated cells referred to as the "blastema" proliferate to restore the lost part of the amputated organ, is distinct from morphallaxis as observed, for instance, in Hydra, in which rearrangement of pre-existing cells and tissues mainly contribute to regeneration. In contrast to complete limb regeneration in urodele amphibians, limb regeneration in Xenopus, an anuran amphibian, is restricted. In this review of some aspects regarding adult limb regeneration in Xenopus laevis, we suggest that limb regeneration in adult Xenopus, which is pattern/tissue deficient, also represents epimorphosis.     

Limb regeneration in amphibians: immunological considerations

We review key aspects of what is known about limb regeneration in urodele and anuran amphibians, with a focus on the early events of the process that lead to formation of the regeneration blastema. This includes the role of the nerves and wound epithelium, but also covers the inflammatory effects of the amputation trauma and their importance for regenerative growth. We propose that immunotolerance is important for limb regeneration and changes in its regulation may underlie the loss of regenerative capacity during anuran metamorphosis.     

Regeneration inducers in limb regeneration

Limb regeneration ability, which can be observed in amphibians, has been investigated as a representative phenomenon of organ regeneration. Recently, an alternative experimental system called the accessory limb model was developed to investigate early regulation of amphibian limb regeneration. The accessory limb model contributed to identification of limb regeneration inducers in urodele amphibians. Furthermore, the accessory limb model may be applied to other species to explore universality of regeneration mechanisms. This review aims to connect the insights recently gained to emboss universality of regeneration mechanisms among species. The defined molecules (BMP7 (or2) + FGF2 + FGF8) can transform skin wound healing to organ (limb) regeneration responses. The same molecules can initiate regeneration responses in some species.     

Quantitative Estimation of HRP-Labeled Sensory and Motor Neurons during Nerve-Dependent and Nerve-Independent Periods of Urodele Limb Regeneration

The relationship between urodele regeneration and the possibility of regeneration in mammals is unclear, but the idea of possible regeneration of neural elements in man is being studied because of its potential clinical importance. One of the great challenges is to gain sufficient knowledge about the basic biology of animal regeneration and to use it for the betterment of the mankind. It is known that the initial stages of urodele limb regeneration depend on the presence of intact nerve fibers connected to their cell bodies. The nerve fibers severed at the level of limb amputation regrow and penetrate the blastema, providing blastema cells with indispensable factors. These factors are produced in the perikarya of neurons and transported via their axons to the blastema. Numerous studies have been performed to elucidate the quantitative relationships between nerve fibers and limb regeneration. However, there are no reports dealing with the individual nerve cells at work. The aim of this investigation was to analyze the quantitative participation and qualitative distinctions of different nerve cells innervating the regenerating parts of the urodele limb and their possible roles in the nerve-dependent and nerve-independent periods of regeneration. The cells under study are housed in the dorsal ganglia (sensory neurons) and in the ventral part of the spinal cord gray matter (motor neurons). The direct involvement of these neurons in different regeneration periods was visualized by means of horseradish peroxidase (HRP) labeling. A total of 34 animals (21 experimental and 13 control) were used to study fluctuations in the numbers of labeled nerve cells. The results are summarized as follows: (a) the first nerve cells incorporating HRP within 5 days after amputation are found in the dorsal ganglia, whereas motor neurons in the gray matter are labeled within 7 days; (b) the number of labeled perikarya increases during the nerve-dependent regeneration period (0–21 days after amputation), with the percentage of implicated sensory neurons exceeding that found in the control series; and (c) during the next, nerve-independent period, the number of participating labeled neurons decreases gradually. Such fluctuations in the number of labeled neurons might represent the metabolic status of these cells in their effort to provide the blastema cells with the factors needed at the appropriate time. The current findings support previous observations that the periods of dependence and independence of urodele limb regeneration on the integrated control of brachial nerves reflect changes in the metabolism of individual sensory and motor neurons.     

Treatment of brachial nerves with colchicine inhibits limb regeneration in the newt Notophthalmus viridescens

In urodele amphibians, limb regeneration is dependent on innervation and is blocked by the administration of colchicine. The objective of this experiment was to determine if colchicine blocks limb regeneration by a direct action on the blastema cells or by an indirect action on the nerves, specifically, if colchicine treatment of the brachial nerves would inhibit limb regeneration in the newt Notophthalmus viridescens. Colchicine was applied to the nerves by implanting a colchicine-loaded silastin block adjacent to the brachial nerves of an amputated newt limb. With appropriate dose levels of colchicine, limb regeneration was completely inhibited. Contralateral control limbs, carrying unloaded silastin blocks, and control limbs with colchicine-loaded blocks implanted equidistant from the blastema, but not adjacent to the brachial nerves, regenerated normally. Thus, the results indicate that the colchicine inhibition of limb regeneration is mediated by colchicine effects on the nerves. The possible mechanism of colchicine action on nerves may involve either wallerian degeneration, or inhibition of axoplasmic transport, or both.     

Transforming growth factor: beta signaling is essential for limb regeneration in axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-beta). In the present study, the full length sequence of the axolotl TGF-beta1 cDNA was isolated. The spatio-temporal expression pattern of TGF-beta1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-beta signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-beta type I receptor, SB-431542, we show that TGF-beta signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-beta signaling are down-regulated. These data directly implicate TGF-beta signaling in the initiation and control of the regeneration process in axolotls.     

The role of stem cells in limb regeneration

Limb regeneration is a complex yet fascinating process observed to some extent in many animal species, though seen in its entirety in urodele amphibians. Accomplished by formation of a morphologically uniform intermediate, the blastema, scientists have long attempted to define the cellular constituents that enable regrowth of a functional appendage. Today, we know that the blastema consists of a variety of multipotent progenitor cells originating from a variety of tissues, and which contribute to limb tissue regeneration in a lineage-restricted manner. By continuing to dissect the role of stem cells in limb regeneration, we can hope to one day modulate the human response to limb amputation and facilitate regrowth of a working replacement.     

New regulators of vertebrate appendage regeneration

Appendage regeneration is a complex and fascinating biological process exhibited in vertebrates by urodele amphibians and teleost fish. A current focus in the field is to identify new molecules that control formation and function of the regeneration blastema, a mass of proliferative mesenchyme that emerges after limb or fin amputation and serves as progenitor tissue for lost structures. Two studies published recently have illuminated new molecular regulators of blastemal proliferation. After amputation of a newt limb, the nerve sheath releases nAG, a blastemal mitogen that facilitates regeneration. In amputated zebrafish fins, regeneration is optimized through depletion of the microRNA miR-133, a mechanism that requires Fgf signaling. These discoveries establish research avenues that may impact the regenerative capacity of mammalian tissues.     

Regeneration: The origin of cancer or a possible cure?

A better understanding of the forces controlling cell growth will be essential for developing effective therapies in regenerative medicine and cancer. Historically, the literature has linked cancer and tissue regeneration—proposing regeneration as both the source of cancer and a method to inhibit tumorigenesis. This review discusses two powerful regeneration models, the vertebrate urodele amphibians and invertebrate planarians, in light of cancer regulation. Urodele limb and eye lens regeneration is described, as well as the planarian's emergence as a molecular and genetic model system in which recent insights begin to molecularly dissect cancer and regeneration in adult tissues.     

Transforming Growth Factor: β Signaling Is Essential for Limb Regeneration in Axolotls

Axolotls (urodele amphibians) have the unique ability, among vertebrates, to perfectly regenerate many parts of their body including limbs, tail, jaw and spinal cord following injury or amputation. The axolotl limb is the most widely used structure as an experimental model to study tissue regeneration. The process is well characterized, requiring multiple cellular and molecular mechanisms. The preparation phase represents the first part of the regeneration process which includes wound healing, cellular migration, dedifferentiation and proliferation. The redevelopment phase represents the second part when dedifferentiated cells stop proliferating and redifferentiate to give rise to all missing structures. In the axolotl, when a limb is amputated, the missing or wounded part is regenerated perfectly without scar formation between the stump and the regenerated structure. Multiple authors have recently highlighted the similarities between the early phases of mammalian wound healing and urodele limb regeneration. In mammals, one very important family of growth factors implicated in the control of almost all aspects of wound healing is the transforming growth factor-beta family (TGF-β). In the present study, the full length sequence of the axolotl TGF-β1 cDNA was isolated. The spatio-temporal expression pattern of TGF-β1 in regenerating limbs shows that this gene is up-regulated during the preparation phase of regeneration. Our results also demonstrate the presence of multiple components of the TGF-β signaling machinery in axolotl cells. By using a specific pharmacological inhibitor of TGF-β type I receptor, SB-431542, we show that TGF-β signaling is required for axolotl limb regeneration. Treatment of regenerating limbs with SB-431542 reveals that cellular proliferation during limb regeneration as well as the expression of genes directly dependent on TGF-β signaling are down-regulated. These data directly implicate TGF-β signaling in the initiation and control of the regeneration process in axolotls.     

Identification of newt connective tissue growth factor as a target of retinoid regulation in limb blastemal cells

In order to analyse target genes regulated by retinoic acid in urodele limb regeneration, we have used pseudotyped retroviruses to obtain stably transfected newt limb blastemal (progenitor) cells in culture which express chimeric retinoic acid/thyroid hormone receptors δ1 or δ2. After treatment with thyroid hormone to activate the chimeric receptors, we used a polymerase chain reaction (PCR)-based subtraction method to identify target genes which are retinoid regulated. Newt connective tissue growth factor, a secreted protein recognised in several vertebrates, has been identified in this way and found to be expressed in the limb blastema and regulated by retinoic acid. This approach should permit a systematic analysis of retinoid target genes in limb regeneration.     

Distinctive expression of Myf5 in relation to differentiation and plasticity of newt muscle cells

Regeneration in urodele amphibians such as the newt reflects the local plasticity of differentiated cells. Newt myotubes and myofibres undergo S phase re-entry and cellularisation in the limb blastema, and we have analysed the regulation of Myf5 in relation to these events. Surprisingly, Myf5 was expressed after fusion in cultured newt myotubes and in myofibers of the adult limb, in contrast to its familiar expression in myoblasts in other vertebrates. Its expression was markedly down regulated in cultured newt myotubes after S phase re-entry induced by serum stimulation, as well as by exposure to the trisubstituted purine called myoseverin which induces cellularisation. We have attempted to relate this striking difference from other vertebrates to the requirement for multinucleate urodele muscle cells to contribute to the regeneration blastema.     

Some current problems in amphibian limb regeneration.

Limb regeneration in adult urodele amphibians proceeds by formation of a blastema at the amputation plane. This paper discusses how the blastema forms, and how its positional identity on the proximodistal axis is manifest. Retinoic acid is able to reset axial specification and there is particular interest in determining how it acts. Although limb regeneration is restricted among vertebrates to the urodeles, its mechanism poses fundamental questions in development biology.     

Higher Vertebrates Do Not Regenerate Digits and Legs Because the Wound Epidermis Is Not Functional

The necessity of injury, nerves, and wound epidermis for urodele limb regeneration is well accepted. Whether one or more of these three factors is limiting in amputated nonregenerating limbs of other vertebrates is a problem area in need of resolution. One view, that higher vertebrates possess inadequate innervation for limb regeneration to occur, is not strongly supported by experimental results. Superinnervation of lizard and mammalian limbs fails to elicit limb regeneration. Furthermore, in the well-known cases of mammalian regeneration, deer antlers and rabbit ears, a nerve requirement has not been demonstrated.
In urodeles, the wound epidermis has recently been shown to have the role of maintaining dedifferentiated cells of the amputated limb stump in the cell cycle. The result of this wound epidermal stimulus is a sufficient number of cell divisions such that blastema formation occurs.
We postulate that in amputated limbs of higher vertebrates, the wound epidermis is nonfunctional. Dedifferentiated or undifferentiated cells are not maintained in the cell cycle and blastema formation therefore does not occur. Instead, tissue regeneration occurs precociously due to lack of a cycling stimulus. The scar tissue which forms at the limb tips of nonregenerating vertebrates is the result of a nonfunctional wound epidermis.     

Acetazolamide does not disrupt limb regenerate morphogenesis in the salamander, Plethodon cinereus

Acetazolamide, a potent and highly specific inhibitor of carbonic anhydrase, is teratogenic in mammalian embryos and when administered during early limb development causes unique limb defects in a time- and dose-dependent manner. The regenerating urodele limb is often considered to be a good experimental analog of limb development and, if it employs the same mechanisms of tissue interactions during pattern formation, should be susceptible to teratogens which selectively disrupt developmental limb patterning. This study demonstrates that while carbonic anhydrase inhibition is toxic to the red-backed salamander, Plethodon cinereus, it does not have the same teratogenic effect on limb regeneration as seen in mammalian limb development. Several points are considered as to why the regenerating limb, at least in this salamander species, may not be suitable for studying this class of teratogen.