Amphibian Limb Regeneration Research Articles

The apical ectodermal ridge (AER) can be re-induced by wounding, wnt-2b, and fgf-10 in the chicken limb bud

Little effort has been made to apply the insights gained from studies of amphibian limb regeneration to higher vertebrates. During amphibian limb regeneration, a functional epithelium called the apical ectodermal cap (AEC) triggers a regenerative response. As long as the AEC is induced, limb regeneration will take place. Interestingly, similar responses have been observed in chicken embryos. The AEC is an equivalent structure to the apical ectodermal ridge (AER) in higher vertebrates. When a limb bud is amputated it does not regenerate; however, if the AER is grafted onto the amputation surface, damage to the amputated limb bud can be repaired. Thus, the AER/AEC is able to induce regenerative responses in both amphibians and higher vertebrates. It is difficult, however, to induce limb regeneration in higher vertebrates. One reason for this is that re-induction of the AER after amputation in higher vertebrates is challenging. Here, we evaluated whether AER re-induction was possible in higher vertebrates. First, we assessed the sequence of events following limb amputation in chick embryos and compared the features of limb development and regeneration in amphibians and chicks. Based on our findings, we attempted to re-induce the AER. When wnt-2b/ fgf-10-expressing cells were inserted concurrently with wounding, successful re-induction of the AER occurred. These results open up new possibilities for limb regeneration in higher vertebrates since AER re-induction, which is considered a key factor in limb regeneration, is now possible.

Adult Stem Cells and Mammalian Epimorphic Regeneration-Insights from Studying Annual Renewal of Deer Antlers

Mammalian organ regeneration is the "Holy Grail" of modern regenerative biology and medicine. The most dramatic organ replacement is known as epimorphic regeneration. To date our knowledge of epimorphic regeneration has come from studies of amphibians. Notably, these animals have the ability to reprogram phenotypically committed cells at the amputation plane toward an embryonic-like cell phenotype (dedifferentiation). The capability of mammals to initiate analogous regeneration, and whether similar mechanisms would be involved if it were to occur, remain unclear. Deer antlers are the only mammalian appendages capable of full renewal, and therefore offer a unique opportunity to explore how nature has solved the problem of mammalian epimorphic regeneration. Following casting of old hard antlers, new antlers regenerate from permanent bony protuberances, known as pedicles. Studies through morphological and histological examinations, tissue deletion and transplantation, and cellular and molecular techniques have demonstrated that antler renewal is markedly different from that of amphibian limb regeneration (dedifferentiation-based), being a stem cell-based epimorphic process. Antler stem cells reside in the pedicle periosteum. We envisage that epimorphic regeneration of mammalian appendages, other than antler, could be made possible by recreating comparable milieu to that which supports the elaboration of that structure from the pedicle periosteum.

Immunolocalization of FGF1 and FGF2 in the regenerating tail of the lizard Lampropholis guichenoti: Implications for FGFs as trophic factors in lizard tail regeneration

A role for fibroblast growth factors in stimulating limb and tail regeneration in amphibians has been shown; however, it is unknown whether these growth factors are also involved in the regeneration of the tail of lizard, an amniote model for studies on tissue regeneration. The presence of fibroblast growth factor-1 (FGF1) and -2 (FGF2) in the regenerating tail of the lizard Lampropholis guichenoti has been studied using immunofluorescence labeling. The study reveals that FGF2 is mainly localized in the wound and scaling epidermis, in differentiating muscles, in spinal ganglia, regenerating nerves and spinal cord. FGF1 is also present in the wound and differentiating epidermis, but is detectable at lower levels in the regenerating muscles and spinal cord. FGF1 is present in blastema cells, while FGF2 labeling is relatively low in these cells. Fibroblasts of the forming dermis are rich in FGF1 but not in FGF2. Developing blood vessels label for both FGF1 and FGF2 while the cartilaginous, bone and fat tissues are poorly labeled or unlabeled for FGFs. The present study suggests that most FGFs in the regenerating tail are located in the nervous system, in the epidermis and muscles, and these tissues most likely require these growth factors for their differentiation and growth. The present study suggests that FGFs produced in the regenerating epidermis, spinal cord and nerves can stimulate tail regeneration in lizards.

Proteomics analysis of regenerating amphibian limbs: changes during the onset of regeneration

During amphibian epimorphic limb regeneration, local injury produces metabolic changes that lead to cellular dedifferentiation and formation of a blastema, but few details of these changes have been elucidated. Here we report the first global proteomic analysis of epimorphic regeneration comparing the profiles of abundant proteins in larval limbs of the anuran Xenopus laevis (stage 53) at the time of amputation (0dPA) and 3 days post-amputation when the regeneration blastema is developing (3dPA). We identified and quantified 1517 peptides, of which 1067 were identified with high peptide ID confidence. Of these 1067 proteins, 489 showed significant changes in quantity between the two groups. Taking into account identical peptides whose fold changes were within 20%, and not including peptides whose fold changes were below the observed fold changes of peptides for the internal standard (chicken lysozyme), we were able to identify 145 peptides elevated in 3dPA relative to 0dPA and 220 peptides in 0dPA relative to 3dPA. In this report, we focus on those proteins that were elevated in the 3dPA tissue relative to 0dPA. In this class were members of the annexin family (e.g. ANXA1, ANXA2, ANXA5) and the ANXA2-binding partner S100A10, which have important immunoregulatory roles in other systems and were also shown to be differentially expressed in stage 53 and 57 3dPA and 5dPA blastemas in our previous microarray studies. Besides elucidating the possible modulation of inflammation during amphibian limb regeneration, our proteomic study also provides insight into dedifferentiation by revealing up-regulation of proteins known to characterize many stem cells.

A tribute to Elizabeth D. Hay, 1927–2007

A tribute to Elizabeth D. Hay, 1927–2007

Embryonic chick corneal epithelium: a model system for exploring cell-matrix interactions.

In her initial research, Elizabeth D. Hay studied amphibian limb regeneration, but later switched her focus, and for the remainder of her career addressed the role of the extracellular matrix (ECM) in regulating embryonic morphogenesis. Much of that work used the embryonic chick corneal epithelial model. This review highlights many of the discoveries that she made using this model. Hay was the first to show that embryonic corneal epithelial cells produce fibrillar collagen. Her lab was among the first to demonstrate that corneal epithelial cells respond to a collagenous substrate by increasing ECM production, and that purified ECM molecules, added to cultures of epithelial sheets, induce a reorganization of the actin cytoskeleton. These data led to the first theories of cell-matrix interactions, illustrated in a 'hands across the membrane' sketch drawn by Hay. Recent work with the epithelial sheet model system has elucidated many of the signal transduction pathways required for actin reorganization in response to the ECM. In all, this body of work has amply supported Hay's belief that the embryonic corneal epithelium is a powerful model system for exploring the role of the ECM in regulating the cytoskeleton, in directing cell migration, and in profoundly influencing cell growth and differentiation during development.

Proteomic analysis of changes during the onset of amphibian limb regeneration

Synthesis of specific proteins has been examined during the early phase of limb regeneration, but an overall analysis of protein changes in this period has been lacking. We report the first global proteomic analysis of this system, utilizing LC/MS‐based identification and quantification methods to compare abundant proteins in hindlimbs of the larval anuran Xenopus laevis (stage 53) at the time of amputation (0dPA) and 3 days later when the regeneration blastema is developing (3dPA). We identified and quantified 1517 peptides, of which 1067 were identified with high peptide ID confidence (555 with multiple peptides and 512 with a single peptide). Of these 1067 proteins, 489 showed significant changes in quantity between the two groups. Among those proteins that were elevated in the tissue relative to 0dPA were members of the annexin family (e.g. ANXA1, ANXA2, ANXA5) and the ANXA2‐binding partner S100A10, which have important immunoregulatory roles in other systems and which were also shown to be differentially expressed in stage 53 and 57 3dPA and 5dPA tissues in our previous microarray studies. Quantitative PCR (qPCR) revealed that the differences in annexins and S100A10 protein levels were consistent with the differences in levels of gene expression. Besides elucidating the possible modulation of inflammation during amphibian limb regeneration, the results also provide insight into the dedifferentiation process by revealing up‐regulation of various proteins known to characterize many stem cells. This work was supported by a pilot grant from the Indiana University School of Medicine.

Extracellular control of muscle regeneration

In urodele amphibian limb regeneration, the muscle provides a reservoir for uncommitted stem‐like blastema cells that will participate in the replacement of the lost structure. However, it is currently not clear what signals activate muscle‐associated satellite cells and/or dedifferentiation of multinucleate myofibers. Cultures of newt muscle fibers allow us to follow these physiological changes at the cellular level using time‐lapse video microscopy.Our studies on extracellular matrix (ECM) distribution during blastema formation demonstrate upregulation of hyaluronic acid (HA), fibronectin (FN), and Tenascin C, suggesting dynamic changes in the ECM composition. Contemporaneous with ECM reorganization, histological analyses of regenerating blastemas reveal increased satellite cell numbers and migration along with myofiber fragmentation. In order to gain a better understanding of the signaling properties of the ECM in regeneration, we are investigating various ECMs and their effects on cell physiology and morphology. Our data indicate that HA induces fragmentation of myotubes and limits adhesion whereas FN or laminin‐containing matrigel stimulates formation of myotubes.The establishment of a physiologically relevant environment and its manipulation in vitro will provide new insights into the control of the differentiated state of the cell by its ECM.Supported by DARPA, NIH, Searle Funds

Wound epidermis formation and function in urodele amphibian limb regeneration.

Upon amputation of the urodele limb, the epidermal cells surrounding the amputation plane migrate to heal the wound. The resulting wound epidermis (WE) induces the regeneration process, resulting in blastema formation, cell division, and ultimately repatterning into a new limb. Despite its central role in the initiation of limb regeneration, little is known about how the WE forms. Here we discuss various models of WE formation and the experimental data in support of each.

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.

Correlation between Shh expression and DNA methylation status of the limb-specific Shh enhancer region during limb regeneration in amphibians

The Xenopus adult limb has very limited regeneration ability, and only a simple cartilaginous spike structure without digits is formed after limb amputation. We found that expression of Shh and its downstream genes is absent from the regenerating blastema of the Xenopus froglet limb. Moreover, we found that a limb enhancer region of the Shh gene is highly methylated in the froglet, although the sequence is hypomethylated in the Xenopus tadpole, which has complete limb regeneration ability. These findings, together with the fact that the promoter region of Shh is hardly methylated in Xenopus, suggest that regenerative failure (deficiency in repatterning) in the Xenopus adult limb is associated with methylation status of the enhancer region of Shh and that a target-specific epigenetic regulation is involved in gene re-activation for repatterning during the Xenopus limb regeneration process. Because the methylation level of the enhancer region was low in other amphibians that have Shh expression in the blastemas, a low methylation status may be the basic condition under which transcriptional regulation of Shh expression can progress during the limb regeneration process. These findings provide the first evidence for a relationship between epigenetic regulation and pattern formation during organ regeneration in vertebrates.

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.

Stem/progenitor cells in amphibian limb and spinal cord regeneration

Amphibian regeneration may replace tissue via homotypic cells, dedifferentiation, transdifferentiation or stem cells. The mechanism may vary with life cycle stage. Expression of Notch signaling components suggests a role for stem cells. We examine the role of stem cells in regeneration-competent and incompetent stages of Xenopus laevis (clawed frog) tadpoles, juvenile and adult axolotls (the Mexican salamander Ambystoma mexicanum) and the regeneration-deficient axolotl mutant short-toes. Notch1 signaling is a key pathway in maintaining stem cell populations. The stem cell marker Musashi (Msi) is an RNA-binding protein inhibiting translation of Numb to maintain Notch1 signaling. Isoforms of Msi1 and Msi2 are expressed in temporal and spatial patterns that correlate with regeneration behavior. Msi-1 is expressed in specific tissue compartments in regeneration-competent Xenopus limb and spinal cord and decreases in incompetent tadpole and adult Xenopus tissues. Msi2 isoforms change between competent and incompetent Xenopus stages. Rapidly growing juvenile axolotls show high levels of Msi1 in both cord and limb, while intact adult axolotl tissues shows low levels of Msi1 that are upregulated following injury. Msi expression is also lost in limbs of the axolotl limb regeneration mutant short-toes, but maintained in the regeneration competent mutant spinal cord. Support: Indiana 21st Century Research and Technology Fund ; W.M. Keck Foundation.

Transgenic Xenopus with prx1 limb enhancer reveals crucial contribution of MEK/ERK and PI3K/AKT pathways in blastema formation during limb regeneration

Understanding the mechanisms that control amphibian limb regeneration should allow us to decipher the critical differences between amphibians and humans, which have the limited ability of organ regeneration. However, many issues at the cellular and molecular levels still remain unresolved. We have generated a transgenic Xenopus laevis line that expresses green fluorescent protein (GFP) under the control of mouse prx1 limb enhancer, which directs reporter gene expression in limb mesenchyme in mice, and found that GFP accumulated in blastemal mesenchymal cells of the transgenic froglets after limb amputation. Thus, this transgenic line should provide a new approach to gain insights into the cellular dynamics and signaling pathways involved in limb blastema formation. We have also developed a culture system for forelimb explants of froglets and found that treatment with inhibitors of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK) kinase 1/2 (MEK1/2) and phosphatidylinositol 3-kinase (PI3K) repressed GFP expression. These effects were partially reversible, and down-regulation of GFP was associated with inhibition of cell-cycle progression and induction of ectopic apoptosis. In addition, we found that ERK1/2 and AKT, downstream mediators of MEK1/2 and PI3K pathways, were activated in amputated forelimb stumps. These results demonstrate that MEK/ERK and PI3K/AKT pathways regulate limb blastema formation in the X. laevis froglet.

Publication of a Special Issue: Limb and Fin Regeneration

• Limb regeneration in amphibians: Immunological considerations • Limb regeneration in Axolotl: Is it superhealing? • Limb regeneration in Xenopus laevis froglet • Transcriptional profiling of caudal fin regeneration in Zebrafish • Newt opportunities for understanding the dedifferentiation process • Comparative experimental analysis of fin lepidotrichia in fishes and several skeletal tissues in vertebrates including humans

Limb Regeneration in Amphibians: Immunological Considerations

Limb Regeneration in Amphibians: Immunological Considerations

Prospects for Limb Regeneration

Salamanders are able to regenerate a lost limb, a feature of ongoing development sadly lost to humans. Brockes and Kumar review what is known about amphibian limb regeneration and speculate on how these observations could inform application of stem cell and regenerative medicine to mammalian cases. Zebrafish, as well, can regenerate their fins. Regeneration occurs through initial formation of a clump of undifferentiated cells, the blastema, which through growth and differentiation elaborates a replacement fin. Whitehead et al. (see the Perspective by Antebi) have now identified one of the signaling factors critical to formation of the blastema. In zebrafish, the dob mutation affects a gene that encodes signaling factor Fgf20, which seems to be used specifically for regeneration rather than for normal embryonic development. J. P. Brockes, A. Kumar, Appendage regeneration in adult vertebrates and implications for regenerative medicine. Science 310 , 1919-1923 (2005). [Abstract] [Full Text] G. G. Whitehead, S. Makino, C.-L. Lien, M. T. Keating, fgf20 is essential for initiating zebrafish fin regeneration. Science 310, 1957-1960 (2005). [Abstract] [Full Text] A. Antebi, The tick-tock of aging? Science 310 , 1911-1913 (2005). [Summary] [Full Text]

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.

Limb Regeneration inXenopus laevisFroglet

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 higher vertebrates: Developing a roadmap

We review what is known about amphibian limb regeneration from the prospective of developing strategies for the induction of regeneration in adult mammals. Prominent in urodele amphibian limb regeneration is the formation of a blastema of undifferentiated cells that goes on to reform the limb. The blastema shares many properties with the developing limb bud; thus, the outgrowth phase of regeneration can be thought of as cells going through development again, i.e., redevelopment. Getting to a redevelopment phase in mammals would be a major breakthrough given our extensive understanding of limb development. The formation of the blastema itself represents a transition phase in which limb cells respond to injury by dedifferentiating to become embryonic limb progenitor cells that can undergo redevelopment. During this phase, rapid wound closure is followed by the dedifferentiation of limb cells to form the blastema. Thus, the regeneration process can be divided into a wound-healing/dedifferentiation phase and a redevelopment phase, and we propose that the interface between the wound-healing response and gaining access to developmentally regulated programs (dedifferentiation) lies at the heart of the regeneration problem in mammals. In urodele amphibians, dedifferentiation can occur in all of the tissues of the limb; however, numerous studies lead us to focus on the epidermis, the dermis, and muscle as key regulators of regeneration. Among higher vertebrates, the digit tip in mammals, including humans, is regeneration-competent and offers a unique mammalian model for regeneration. Recent genetic studies in mice identify the Msx1 gene as playing a critical role in the injury response leading to digit tip regeneration. The results from regeneration studies ranging from amphibians to mammals can be integrated to develop a roadmap for mammalian regeneration that has as its focus understanding the phenomenon of dedifferentiation.