Spinal Cord Regeneration in Amphibians: A Historical Perspective

In some vertebrates, a grave injury to the central nervous system (CNS) results in functional restoration, rather than in permanent incapacitation. Understanding how these animals mount a regenerative response by activating resident CNS stem cell populations is of critical importance in regenerative biology. Amphibians are of a particular interest in the field because the regenerative ability is present throughout life in urodele species, but in anuran species it is lost during development. Studying amphibians, who transition from a regenerative to a nonregenerative state, could give insight into the loss of ability to recover from CNS damage in mammals. Here, we highlight the current knowledge of spinal cord regeneration across vertebrates and identify commonalities and differences in spinal cord regeneration between amphibians.     

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.     

EvoRegen in animals: Time to uncover deep conservation or convergence of adult stem cell evolution and regenerative processes

How do animals regenerate specialised tissues or their entire body after a traumatic injury, how has this ability evolved and what are the genetic and cellular components underpinning this remarkable feat? While some progress has been made in understanding mechanisms, relatively little is known about the evolution of regenerative ability. Which elements of regeneration are due to lineage specific evolutionary novelties or have deeply conserved roots within the Metazoa remains an open question. The renaissance in regeneration research, fuelled by the development of modern functional and comparative genomics, now enable us to gain a detailed understanding of both the mechanisms and evolutionary forces underpinning regeneration in diverse animal phyla. Here we review existing and emerging model systems, with the focus on invertebrates, for studying regeneration. We summarize findings across these taxa that tell us something about the evolution of adult stem cell types that fuel regeneration and the growing evidence that many highly regenerative animals harbor adult stem cells with a gene expression profile that overlaps with germline stem cells. We propose a framework in which regenerative ability broadly evolves through changes in the extent to which stem cells generated through embryogenesis are maintained into the adult life history.     

Phylogenetic distribution of regeneration and asexual reproduction in Annelida: regeneration is ancestral and fission evolves in regenerative clades

Regeneration, the ability to replace lost body structures, and agametic asexual reproduction, such as fission and budding, are post‐embryonic developmental capabilities widely distributed yet highly variable across animals. Regeneration capabilities vary dramatically both within and across phyla, but the evolution of regeneration ability has rarely been reconstructed in an explicitly phylogenetic context. Agametic reproduction appears strongly associated with high regenerative abilities, and there are also extensive developmental similarities between these two processes, suggesting that the two are evolutionarily related. However, the directionality leading to this relationship remains unclear: while it has been proposed that regeneration precedes asexual reproduction, the reverse hypothesis has also been put forward. Here, we use phylogenetically explicit methods to reconstruct broad patterns of regeneration evolution and formally test these hypotheses about the evolution of fission in the phylum Annelida (segmented worms). We compiled from the literature a large dataset of information on anterior regeneration, posterior regeneration, and fission abilities for 401 species and mapped this information onto a phylogenetic tree based on recent molecular studies. We used Markovian maximum likelihood and Bayesian MCMC methods to evaluate different models for the evolution of regeneration and fission and to estimate the likelihood of each of these traits being present at each node of the tree. Our results strongly support anterior and posterior regeneration ability being present at the basal node of the annelid tree and being lost 18 and 5 times, respectively, but never regained. By contrast, the ability to fission is reconstructed as being absent at the basal node and being gained at least 19 times, with several possible losses. Models assuming independent evolution of regeneration and fission yield significantly lower likelihoods. Our findings suggest that anterior and posterior regeneration are ancestral for Annelida and are consistent with the hypothesis that regenerative ability is required to evolve fission.     

The costs of autotomy and regeneration in animals: a review and framework for future research

Many organisms have the ability to shed an appendage (autotomy)to escape a predator or fouled molting event. Despite its immediateadvantage on survivorship, autotomy can have important consequencesfor locomotion, foraging, survivorship, and/or reproduction.Thus, regeneration is a way that animals alleviate some of thecosts associated with losing an appendage. Like autotomy, however,appendage regeneration can have important consequences for avariety of aspects of fitness; in a wide range of amphibians,reptiles, fishes, and arthropods, the allocation of resourcesto regenerate a lost appendage negatively affects somatic orreproductive growth. Previous research into the costs associatedwith regeneration has provided a strong framework to explorehow trade-offs associated with regeneration may have influencedits evolution. However, all research to date describing thecosts and benefits associated with autotomy and regenerationhave compared individuals autotomizing and regenerating an appendagewith individuals that have never lost an appendage. I suggestthat for studies of the evolutionary significance of regeneration,an alternative comparison is between individuals experiencingautotomy without regeneration and individuals experiencing autotomywith regeneration. Future work in this direction promises newinsights into the evolution of regenerative tendencies, as wellas how regeneration may be influencing animal form and function.     

Regeneration and clonality in Metazoa. The price to pay for evolving complexity

Explaining the high variability of regenerative ability across metazoan taxa is one of the major challenges in modern biology. Although common and widespread, regeneration shows a heterogeneous distribution and most authors consider regeneration capacity to be an ancestral trait that has been restricted or completely lost over the course of metazoan evolution. Basal Metazoans show the highest capacity for regeneration. By contrast, this feature is highly variable within bilaterians, with many taxa limited in their capacity for regeneration or not regenerating at all. The causes of the loss and/or maintenance of regeneration remain poorly understood, with most explanations invoking adaptive mechanisms. In the present study Metazoan regeneration is discussed with reference to stem cell biology, tissue plasticity, evolution of tissue complexity, cell turnover and lifespan. The presence or absence of regenerative ability cannot be seen only as an adaptation to a particular environment but can also be a consequence of body plan and developmental constraints such as may arise from the evolution of an adaptive immune system.     

Investigating regeneration and functional integration of CNS neurons: Lessons from zebrafish genetics and other fish species

Zebrafish possess a robust, innate CNS regenerative ability. Combined with their genetic tractability and vertebrate CNS architecture, this ability makes zebrafish an attractive model to gain requisite knowledge for clinical CNS regeneration. In treatment of neurological disorders, one can envisage replacing lost neurons through stem cell therapy or through activation of latent stem cells in the CNS. Here we review the evidence that radial glia are a major source of CNS stem cells in zebrafish and thus activation of radial glia is an attractive therapeutic target. We discuss the regenerative potential and the molecular mechanisms thereof, in the zebrafish spinal cord, retina, optic nerve and higher brain centres. We evaluate various cell ablation paradigms developed to induce regeneration, with particular emphasis on the need for (high throughput) indicators that neuronal regeneration has restored sensory or motor function. We also examine the potential confound that regeneration imposes as the community develops zebrafish models of neurodegeneration. We conclude that zebrafish combine several characters that make them a potent resource for testing hypotheses and discovering therapeutic targets in functional CNS regeneration. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.     

Human regeneration: An achievable goal or a dream?

The main objective of regenerative medicine is to replenish cells or tissues or even to restore different body parts that are lost or damaged due to disease, injury and aging. Several avenues have been explored over many decades to address the fascinating problem of regeneration at the cell, tissue and organ levels. Here we discuss some of the primary approaches adopted by researchers in the context of enhancing the regenerating ability of mammals. Natural regeneration can occur in different animal species, and the underlying mechanism is highly relevant to regenerative medicine-based intervention. Significant progress has been achieved in understanding the endogenous regeneration in urodeles and fishes with the hope that they could help to reach our goal of designing future strategies for human regeneration.     

Evolutionary modification of regenerative capability in vertebrates: a comparative study on teleost pectoral fin regeneration.

The regenerative ability of the pectoral fins of 14 species from 6 euteleostean families was tested. Blastema formation and distal outgrowth was observed in all species, indicating the initiation of regeneration in all species tested. Interspecific variation exists with respect to the frequency of malformations and the patterns produced by heteromorphic regeneration. Taking into account published reports on pectoral fin regeneration, the systematic distribution of homo- and heteromorphic regeneration leads to the following conclusions: 1) regenerative ability of pectoral fins is a property inherited from the common ancestor of euteleosteans. Whether it is also the ancestral condition for the whole teleostean group cannot be determined, because reports on more primitive teleosteans like the herring and the osteoglossimorphs are missing. 2) A propensity to produce high frequencies of heteromorphic regenerates originated independently at least three times in Cypriniformes, Scorpaeniformes, and Perciformes. 3) Impaired regeneration is most commonly found in bottom fishes, although not all ground fish groups show heteromorphic regeneration. This suggests that impaired regeneration is not directly related to bottom dwelling, but most probably originated as a side effect of other adaptive changes. Hence, neither the presence nor the loss of faithful regeneration can be associated with particular adaptive scenarios in this group, since regeneration seems to be ancestral to all major euteleost groups and its loss has no clear adaptive significance. Whether there are adaptive reasons to maintain regenerative capability or whether there are cases of reestablishment of regeneration after it was lost cannot be decided on the basis of recent evidence. More observations on phylogenetically closely related species with variable regenerative capability are necessary to assess adaptive explanations of regeneration.     

Distribution of segment regeneration ability in the Annelida

The annelids are an excellent group in which to investigatethe evolution of regeneration abilities. They exhibit qualitativeand quantitative variation in regeneration ability, includingamong closely related species, and their segmental body organizationmakes comparing results among species relatively straightforward.Here, I compile information on the presence/absence of segmentregeneration ability across the annelids. The ability to regenerateposteriorly appears to be nearly universal in the annelids.It is almost certainly ancestral for the phylum and may havebeen lost only a few times. The ability to regenerate anteriorlyis common but less widespread. It is absent in about a dozengroups, almost surely representing multiple independent lossesof this ability. Several non-regenerating species are closelyrelated to regenerating species, indicating very recent losses(or gains). Despite the fact that lack of this ability is unusual,there is a publication bias against reporting the lack of regenerationability, and in many cases the judgment that a particular speciesis unable to regenerate is based on incomplete or unpublisheddata. Thus, in order to build rigorous frameworks for futurecomparative studies of annelid regeneration, there is a needfor published studies clearly documenting the lack of regenerationabilities in annelid species. The review of regeneration datapresented here is especially useful in highlighting annelidgroups that possess both regenerating and non-regenerating representatives.Investigations of these groups may be particularly useful forelucidating the mechanisms leading to the loss (or perhaps gain)of segment regeneration ability.     

Survey of the differences between regenerative and non-regenerative animals

To investigate the boundaries between regenerative and non-regenerative animals, we first survey regenerative ability across animal phyla from sponges to chordates (including mammals). There are both regenerative and non-regenerative animals in each phylum. The cells participating in regeneration also vary among different species. Thus, it is hard to find clear rules concerning regeneration ability across the animal kingdom, suggesting that it is not useful to compare the difference of regenerative ability across phyla to seek the boundary between regenerative and non-regenerative animals. Instead, if we carefully compare the differences of regenerative ability between closely related species within each phylum and accumulate these differences at the cellular molecular levels, we may be able to clarify the boundary between regenerative and non-regenerative animals. Here we introduce our comparative analysis of cellular events after amputation of lower jaws between frogs and newts. Then we propose that such comparative analyses using closely related species within the same phylum should be accumulated to understand the boundary between regenerative and non-regenerative animals in order to apply this understanding for realizing regenerative medicine in the future.     

Dynamic gene expression profiles during arm regeneration in the brittle star Amphiura filiformis

Echinoderms and in particular brittle stars display a remarkable ability to regenerate lost or damaged tissues. They offer an excellent model in which to study regeneration displaying extensive regenerative ability and close relationship to vertebrates providing the opportunity for comparative studies. Previous studies of gene expression during arm regeneration in brittle stars have focused on single genes commonly associated with the regenerative process. In this study we present the first microarray investigation of gene expression during arm regeneration in the brittle star Amphiura filiformis. We show the large-scale gene expression changes associated with the complex process of regeneration with over 50% of the clones measured showing a significant change at some point during the process when compared to non-regenerating arms. Particular attention is paid to genes associated with Hox gene expression regulation, neuronal development and the bone morphogenic protein BMP-1. Our data give an insight into the molecular control required during the various stages of regeneration from the stem cell rich blastema stage through to the highly differentiated regenerate. This work also forms an important basis for future gene expression investigations in this emerging model of limb regeneration.     

Newt opportunities for understanding the dedifferentiation process

Urodele amphibians, such as the newt Notophthalmus viridescens, have the unique ability to regenerate limbs, spinal cord, eye structures, and many vital organs through a process called epimorphic regeneration. Although the cellular basis of regeneration has been studied in detail, we know relatively little about the molecular controls of the process. This review provides an overview of forelimb regeneration in the newt, addressing what we know about cellular and molecular aspects. Particular focus is placed on the dedifferentiation process, which yields a population of embryonic-like pluripotent cells that will eventually reform the lost structure. This cellular plasticity seems to be the key to regenerative ability. We discuss the dedifferentiation process in newt forelimb regeneration and outline the various studies that have revealed that mammalian cells also have the ability to dedifferentiate if given the appropriate triggers.     

Brain regeneration in anuran amphibians

Urodele amphibians are highly regenerative animals. After partial removal of the brain in urodeles, ependymal cells around the wound surface proliferate, differentiate into neurons and glias and finally regenerate the lost tissue. In contrast to urodeles, this type of brain regeneration is restricted only to the larval stages in anuran amphibians (frogs). In adult frogs, whereas ependymal cells proliferate in response to brain injury, they cannot migrate and close the wound surface, resulting in the failure of regeneration. Therefore frogs, in particular Xenopus, provide us with at least two modes to study brain regeneration. One is to study normal regeneration by using regenerative larvae. In this type of study, the requirement of reconnection between a regenerating brain and sensory neurons was demonstrated. Functional restoration of a regenerated telencephalon was also easily evaluated because Xenopus shows simple responses to the stimulus of a food odor. The other mode is to compare regenerative larvae and non-regenerative adults. By using this mode, it is suggested that there are regeneration-competent cells even in the non-regenerative adult brain, and that immobility of those cells might cause the failure of regeneration. Here we review studies that have led to these conclusions.     

Planarian stem cells: a simple paradigm for regeneration

Planarians are capable of profound regenerative feats dependent upon a population of self-renewing adult stem cells called neoblasts. The key features of neoblasts are their capacity for indefinite self-renewal, their totipotency and the ability of their progeny to interpret differentiation and polarity signals and correctly replace lost structures after tissue damage. Regeneration in planarians offers a paradigm for understanding the molecular and cellular control of the repair and regeneration of animal tissues, and could provide valuable insights for the safe use of stem cells to repair damaged, diseased and ageing human tissues with little or no regenerative capacities. Here, I review recent progress in understanding neoblasts in regeneration and the growing potential this research has to be broadly informative for human biology.     

Improving cardiac regeneration after injury: Are we a step closer?

During regeneration, lost functional tissue can, in general, be replaced by different mechanisms, including proliferation of terminally differentiated cells or through differentiation of resident stem cells. It is a well-accepted dogma that the mammalian heart cannot efficiently regenerate upon injury as a consequence of insufficient oxygen supply. This is in sharp contrast to the hearts of adult zebrafish or newts that are able to replace lost ventricular tissue. Novel data indicate that the young murine heart also has the ability to regenerate within the first week after birth using mechanisms apparently quite similar to those observed in fish. This now provides us with a good starting point to identify the molecular mechanisms that led to the loss of the regenerative capacity of the adult mammalian heart. These future studies will also indicate whether it will be possible to reawaken the regenerative capability of cardiomyocytes in the human heart by treatment with selected pharmaceuticals.     

The influence of fundamental traits on mechanisms controlling appendage regeneration

One of the most compelling questions in evolutionary biology is why some animals can regenerate injured structures while others cannot. Appendage regeneration appears to be common when viewed across the metazoan phylogeny, yet this ability has been lost in many taxa to varying degrees. Within species, the capacity for regeneration also can vary ontogenetically among individuals. Here we argue that appendage regeneration along the secondary body axis may be constrained by fundamental traits such as body size, aging, life stage, and growth pattern. Studies of the molecular mechanisms affecting regeneration have been conducted primarily with small organisms at early life stages. Such investigations disregard the dramatic shifts in morphology and physiology that organisms undergo as they age, grow, and mature. To help explain interspecific and intraspecific constraints on regeneration, we link particular fundamental traits to specific molecular mechanisms that control regeneration. We present a new synthesis for how these fundamental traits may affect the molecular mechanisms of regeneration at the tissue, cellular, and genomic levels of biological organization. Future studies that explore regeneration in organisms across a broad phylogenetic scale, and within an ontogenetic framework, will help elucidate the proximate mechanisms that modulate regeneration and may reveal new biomedical applications for use in regenerative medicine.     

General characterization of regeneration in Aeolosoma viride (Annelida,Aeolosomatidae)

Regeneration has long been the focus of scientific interest for its potential to restore lost, damaged, or aged tissues and organs. A wide range of regenerative studies have been conducted on different vertebrate and invertebrate model organisms. Annelids are known for their regenerative capacities, and because of their relatively complex organ systems, they are an ideal organism for regeneration study. Our present work focused on the freshwater annelid Aeolosoma viride, an asexually reproducing annelid capable of regenerating both anteriorly and posteriorly. Even though regenerative ability has been documented in this animal in previous studies, detailed characterization of the process is still unavailable. The objective of this study was to evaluate the regenerative ability of A. viride. We described the sequential morphological events during the process of regeneration, such as wound healing and the formation of blastema, mouth, and pygidium. In order to clarify the capacity and type of regeneration, we conducted a series of observations and experiments using a cell proliferation assay. Massive proliferation and the absence of cell migration indicated that the animal regenerates primarily through epimorphosis. Our study of the epimorphic regenerative process of A. viride provides a clearer picture of the evolutionary origin of regeneration in annelids.     

Neurotrophic regulation of fibroblast dedifferentiation during limb skeletal regeneration in the axolotl (Ambystoma mexicanum)

The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a “critical-size defect” (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment.     

Unraveling tissue regeneration pathways using chemical genetics

Identifying the molecular pathways that are required for regeneration remains one of the great challenges of regenerative medicine. Although genetic mutations have been useful for identifying some molecular pathways, small molecule probes of regenerative pathways might offer some advantages, including the ability to disrupt pathway function with precise temporal control. However, a vertebrate regeneration model amenable to rapid throughput small molecule screening is not currently available. We report here the development of a zebrafish early life stage fin regeneration model and its use in screening for small molecules that modulate tissue regeneration. By screening 2000 biologically active small molecules, we identified 17 that specifically inhibited regeneration. These compounds include a cluster of glucocorticoids, and we demonstrate that transient activation of the glucocorticoid receptor is sufficient to block regeneration, but only if activation occurs during wound healing/blastema formation. In addition, knockdown of the glucocorticoid receptor restores regenerative capability to nonregenerative, glucocorticoid-exposed zebrafish. To test whether the classical anti-inflammatory action of glucocorticoids is responsible for blocking regeneration, we prevented acute inflammation following amputation by antisense repression of the Pu.1 gene. Although loss of Pu.1 prevents the inflammatory response, regeneration is not affected. Collectively, these results indicate that signaling from exogenous glucocorticoids impairs blastema formation and limits regenerative capacity through an acute inflammation-independent mechanism. These studies also demonstrate the feasibility of exploiting chemical genetics to define the pathways that govern vertebrate regeneration.