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.     

Fundamental aspects of arm repair phase in two echinoderm models

Regeneration is a post-embryonic developmental process that ensures complete morphological and functional restoration of lost body parts. The repair phase is a key step for the effectiveness of the subsequent regenerative process: in vertebrates, efficient re-epithelialisation, rapid inflammatory/immune response and post-injury tissue remodelling are fundamental aspects for the success of this phase, their impairment leading to an inhibition or total prevention of regeneration. Among deuterostomes, echinoderms display a unique combination of striking regenerative abilities and diversity of useful experimental models, although still largely unexplored.Therefore, the brittle star Amphiura filiformis and the starfish Echinaster sepositus were here used to comparatively investigate the main repair phase events after injury as well as the presence and expression of immune system and extracellular matrix (i.e. collagen) molecules using both microscopy and molecular tools.Our results showed that emergency reaction and re-epithelialisation are similar in both echinoderm models, being faster and more effective than in mammals. Moreover, in comparison to the latter, both echinoderms showed delayed and less abundant collagen deposition at the wound site (absence of fibrosis). The gene expression patterns of molecules related to the immune response, such as Ese-fib-like (starfishes) and Afi-ficolin (brittle stars), were described for the first time during echinoderm regeneration providing promising starting points to investigate the immune system role in these regeneration models.Overall, the similarities in repair events and timing within the echinoderms and the differences with what has been reported in mammals suggest that effective repair processes in echinoderms play an important role for their subsequent ability to regenerate. Targeted molecular and functional analyses will shed light on the evolution of these abilities in the deuterostomian lineage.     

Evolutionary history of regeneration in crinoids (Echinodermata)

The fossil record indicates that crinoids have exhibited remarkable regenerative abilities since their origin in the Ordovician, abilities that they likely inherited from stem-group echinoderms. Regeneration in extant and fossil crinoids is recognized by abrupt differences in the size of abutting plates, aberrant branching patterns, and discontinuities in carbon isotopes. While recovery is common, not all lost body parts can be regenerated; filling plates and overgrowths are evidence of non-regenerative healing. Considering them as a whole, Paleozoic crinoids exhibit the same range of regenerative and non-regenerative healing as Recent crinoids. For example, Paleozoic and extant crinoids show evidence of crown regeneration and stalk regrowth, which can occur only if the entoneural nerve center (chambered organ) remains intact. One group of Paleozoic crinoids, the camerates, may be an exception in that they probably could not regenerate their complex calyx-plating arrangements, including arm facets, but their calyxes could be healed with reparative plates. With that exception, and despite evidence for increases in predation pressure, there is no compelling evidence that crinoids have changed though time in their ability to recover from wounds. Finally, although crinoid appendages may be lost as a consequence of severe abiotic stress and through ontogenetic development, spatiotemporal changes in the intensity and frequency of biotic interactions, especially direct attacks, are the most likely explanation for observed patterns of regeneration and autotomy in crinoids.     

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.     

Degeneration and Regeneration in Crustacean Neuromuscular Systems

Crayfish motor neurons seem to repair damage to peripheral axonsby selective fusion of outgrowing proximal stumps with severeddistal processes that can survive morphologically and physiologicallyintact for over 200 days. Survival of isolated motor and CNSgiant axons is associated with much hypertrophy of their glialsheath. The severed stumps of peripheral sensory neurons oftendegenerate within 21 days and their glial sheath does not hypertrophy.Denervation and immobilization produce relatively little changein the morphology and physiology of the opener muscle, whereastenotomy produces much atrophy within 30-60 days. Crayfish motor and CNS giant neurons show no capability forregenerating ablated cell bodies, whereas peripheral sensorysomata regenerate after limb autotomy. An entire opener musclecan be replaced after limb autotomy but the organism shows littleor no ability to redifferentiate an entire muscle in the absenceof body part regeneration. However, a few opener muscle fiberscan be regenerated if the bulk of the muscle mass remains intact.The significance of all these findings are interpreted withrespect to the developmental capabilities and environmentaladaptations of the crayfish together with the evolution of regenerativeabilities in anthropods and vertebrates.     

Tissue distribution of monoamine neurotransmitters in normal and regenerating arms of the feather star Antedon mediterranea

Crinoid echinoderms can completely and rapidly regenerate arms lost following self-induced or traumatic amputation. Arm regeneration in these animals therefore provides a valuable experimental model for studying all aspects of regenerative processes, particularly with respect to the nervous system and its specific contribution to regenerative phenomena. Taking into account the primary role of the nervous system in regeneration in other invertebrates, we have investigated the specific involvement of neural factors, viz. the monoamine neurotransmitters dopamine and serotonin, in arm regeneration of Antedon mediterranea. In the present work, the presence of classical monoamines has been revealed by employing specific immunocytochemical and histofluorescence tests in association with biochemical detection by means of high pressure liquid chromatography. The distribution pattern of these neurohumoral molecules at standard regenerative stages has been compared with that of normal non-regenerating arms. Results indicate that both dopamine and serotonin dramatically change in both their distribution and concentration during the repair and regenerative processes. Their remarkably enhanced pattern during regeneration and widespread presence at the level of both nervous and non-nervous tissues indicates that they are important neural growth-promoting factors in crinoid arm regeneration. Received: 18 December 1995 / Accepted: 23 February 1996     

Dichotomy of the glial cell response to axonal injury and regeneration

Neurons in the mammalian central nervous system (CNS) have a poor capacity for regenerating their axons after injury. In contrast, neurons in the CNS of lower vertebrates and in the peripheral nervous system (PNS) of mammals are endowed with a high posttraumatic capacity to regenerate. The differences in regenerative capacity have been attributed to the different compositions of the respective cellular environments and to different responses to injury the nonneuronal cells display, which range from supportive and permissive to nonsupportive and hostile for regeneration. The same cell type may support or inhibit regeneration, depending on its state of maturity or differentiation. Astrocytes and oligodendrocytes are examples of cells in which such a dichotomy is manifested. In developing and in spontaneously regenerating nerves, these cells support (astrocytes) and permit (oligodendrocytes) growth. However, in nonregenerating adult mammalian nerves, astrocytes form the nonsupportive scar tissue; and the mature oligodendrocytes inhibit axonal growth. Maturation of these cells may be regulated differently during development than after injury. Among the putative regulators are factors derived from astrocytes, resident microglia; or cytokines produced by macrophages. During development, regulation leads to a temporal separation between axonal growth and maturation of the cellular environment, which might not occur spontaneously after injury in a nonregenerating CNS without intervention at the appropriate time. Data suggest that temporal intervention aimed at the glial cells might enhance the poor regenerative capacity of the mammalian CNS. Possible regulation of the nonneuronal cell response to injury via involvement of protooncogenes is proposed.     

Echinoderm regenerative response as a sensitive ecotoxicological test for the exposure to endocrine disrupters: effects of p,p′DDE and CPA on crinoid arm regeneration

Echinoderms are valuable test species in marine ecotoxicology and offer a wide range of biological processes appropriate for this approach. Regenerating echinoderms can be regarded as amenable experimental models for testing the effects of exposure to contaminants, particularly endocrine disrupter compounds (EDCs). As regeneration is a typical developmental process, physiologically regulated by humoral mechanisms, it is highly susceptible to the action of pseudo-hormonal contaminants which appear to be obvious candidates for exerting deleterious actions. In our laboratory experiments, selected EDCs suspected for their antiandrogenic action (p,p′-DDE and cyproterone acetate) were tested at low concentrations on regenerating specimens of the crinoid Antedon mediterranea. An integrated approach which combines exposure experiments and different morphological analyses was employed; the obtained results suggest an overall pattern of plausible endocrine disruption in the exposed samples, showing that processes such as regenerative growth, histogenesis, and differentiation are affected by the exposure to the selected compounds. These results confirm that (1) regenerative phenomena of echinoderms can be considered valuable alternative models to assess the effects of exposure to exogenous substances such as EDCs, and (2) these compounds significantly interfere with fundamental processes of developmental physiology (proliferation, differentiation, etc…) plausibly via endocrine alterations. In terms of future prospects, taking into account the increasing need to propose animal models different from vertebrates, echinoderms represent a group on which ecotoxicological studies should be encouraged and specifically addressed.     

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.     

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.     

Using zebrafish as the model organism to understand organ regeneration

The limited regenerative capacity of several organs, such as central nervous system(CNS), heart and limb in mammals makes related major diseases quite difficult to recover. Therefore, dissection of the cellular and molecular mechanisms underlying organ regeneration is of great scientific and clinical interests. Tremendous progression has already been made after extensive investigations using several model organisms for decades. Unfortunately, distance to the final achievement of the goal still remains. Recently, zebrafish became a popular model organism for the deep understanding of regeneration based on its powerful regenerative capacity, in particular the organs that are limitedly regenerated in mammals. Additionally, zebrafish are endowed with other advantages good for the study of organ regeneration. This review summarizes the recent progress in the study of zebrafish organ regeneration, in particular regeneration of fin, heart, CNS, and liver as the representatives. We also discuss reasons of the reduced regenerative capacity in higher vertebrate, the roles of inflammation during regeneration, and the difference between organogenesis and regeneration.     

Spinal cord regeneration in a tail autotomizing urodele

Adult urodele amphibians possess extensive regenerative abilities, including lens, jaws, limbs, and tails. In this study, we examined the cellular events and time course of spinal cord regeneration in a species, Plethodon cinereus, that has the ability to autotomize its tail as an antipredator strategy. We propose that this species may have enhanced regenerative abilities as further coadaptations with this antipredator strategy. We examined the expression of nestin, vimentin, and glial fibrillary acidic protein (GFAP) after autotomy as markers of neural precursor cells and astroglia; we also traced the appearance of new neurons using 5‐bromo‐2′‐deoxyuridine/neuronal nuclei (BrdU/NeuN) double labeling. As expected, the regenerating ependymal tube was a major source of new neurons; however, the spinal cord cranial to the plane of autotomy showed significant mitotic activity, more extensive than what is reported for other urodeles that cannot autotomize their tails. In addition, this species shows upregulation of nestin, vimentin, and GFAP within days after tail autotomy; further, this expression is upregulated within the spinal cord cranial to the plane of autotomy, not just within the extending ependymal tube, as reported in other urodeles. We suggest that enhanced survival of the spinal cord cranial to autotomy allows this portion to participate in the enhanced recovery and regeneration of the spinal cord. J. Morphol. 2011. © 2011 Wiley Periodicals, Inc.     

Echinoderm regeneration: an in vitro approach using the crinoid Antedon mediterranea

Among echinoderms, crinoids are well known for their remarkable regenerative potential. Regeneration depends mainly on progenitor cells (undifferentiated or differentiated), which migrate and proliferate in the lesion site. The crucial role of the “progenitor” elements involved in the regenerative processes, in terms of cell recruitment, sources, and fate, is a central problem in view of its topical interest and biological implications. The spectacular regenerative potential of crinoids is used to replace lost internal and external organs. In particular, the process of arm regeneration in the feather star Antedon mediterranea is the regeneration model most extensively explored to date. We have addressed the morphological and functional characterization of the cell phenotypes responsible for the arm regenerative processes by using an in vitro approach. This represents the first successful attempt to culture cells involved in crinoid regeneration. A comparison of these results with others from previous in vivo investigations confirms the diverse cell types contributing to regeneration and underscores their involvement in migration, proliferation, and dedifferentiation processes.     

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.     

Deciphering proteins and their functions in the regenerating retina

Neurons of the mammalian CNS, including retinal ganglion cells, lack, in contrast to the PNS, the ability to regenerate axons spontaneously after injury. Regeneration of the CNS is extremely complex and involves various molecular factors and cells. Therewith the regenerative process remains an enormous scientific and clinical challenge. This article provides an overview of proteins that play a crucial role in axon regeneration of retinal ganglion cells and their underlying signaling pathways. In this context, we elucidate the role of 2D gel electrophoresis and highlight some additional proteins, altered upon regeneration by using this highly sensitive method.     

Deciphering proteins and their functions in the regenerating retina

Neurons of the mammalian CNS, including retinal ganglion cells, lack, in contrast to the PNS, the ability to regenerate axons spontaneously after injury. Regeneration of the CNS is extremely complex and involves various molecular factors and cells. Therewith the regenerative process remains an enormous scientific and clinical challenge. This article provides an overview of proteins that play a crucial role in axon regeneration of retinal ganglion cells and their underlying signaling pathways. In this context, we elucidate the role of 2D gel electrophoresis and highlight some additional proteins, altered upon regeneration by using this highly sensitive method.     

Neural regeneration and cell replacement: a view from the eye

Neuronal degenerations in the retina are leading causes of blindness. Like most other areas of the CNS, the neurons of the mammalian retina are not replaced following degeneration. However, in nonmammalian vertebrates, endogenous repair processes restore neurons very efficiently, even after complete loss of the retina. We describe the phenomenon of retinal regeneration in nonmammalian vertebrates and attempts made in recent years to stimulate similar regenerative processes in the mammalian retina. In addition, we review the various strategies employed to replace lost neurons in the retina and the recent use of stem cell technologies to address problems of retinal repair.     

3‐acetylpyridine‐induced degeneration in the adult ascidian neural complex: Reactive and regenerative changes in glia and blood cells

Ascidians are interesting neurobiological models because of their evolutionary position as a sister‐group of vertebrates and the high regenerative capacity of their central nervous system (CNS). We investigated the degeneration and regeneration of the cerebral ganglion complex of the ascidian Styela plicata following injection of the niacinamide antagonist 3‐acetylpyridine (3AP), described as targeting the CNS of several vertebrates. For the analysis and establishment of a new model in ascidians, the ganglion complex was dissected and prepared for transmission electron microscopy (TEM), routine light microscopy (LM), immunohistochemistry and Western blotting, 1 or 10 days after injection of 3AP. The siphon stimulation test (SST) was used to quantify the functional response. One day after the injection of 3AP, CNS degeneration and recruitment of a non‐neural cell type to the site of injury was observed by both TEM and LM. Furthermore, weaker immunohistochemical reactions for astrocytic glial fibrillary acidic protein (GFAP) and neuronal βIII‐tubulin were observed. In contrast, the expression of caspase‐3, a protein involved in the apoptotic pathway, and the glycoprotein CD34, a marker for hematopoietic stem cells, increased. Ten days after the injection of 3AP, the expression of markers tended toward the original condition. The SST revealed attenuation and subsequent recovery of the reflexes from 1 to 10 days after 3AP. Therefore, we have developed a new method to study ascidian neural degeneration and regeneration, and identified the decreased expression of GFAP and recruitment of blood stem cells to the damaged ganglion as reasons for the success of neuroregeneration in ascidians. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 877–893, 2015     

Comparative immunological analysis of echinoderm cellular and humoral defense factors

Coelomocyte are found in the fluid filling coelomic cavity of echinoderms and depending on species can be a mixture of several morphologically different types. There are among them: granular and agranular amoebocytes, morula cells, vibratile and lymphocyte-like cells. All these cells take part in cellular response to immune challenges through phagocytosis, clotting, encapsulation of foreign particles, cytotoxicity, and the production of antimicrobial agents, such as reactive oxygen and nitric oxide. The data are given on a variety of humoral factors found in the coelomic fluid, including different types of lectines, agglutinins, hemolysins, acute phase proteins and antimicrobial factors. The discussion on cooperation between cellular and humoral arms of defense reactions during inflammation reveals the crucial role of coelomocytes in immune response. It is suggested that the sea urchin complement system (that is homologous to the alternative pathway in vertebrates) is appeared initially in echinoderms as a protein cascade that points to opsonization of foreign cells and particles, augmenting their phagocytosis and subsequent destruction by coelomocytes. So the identification of a simple complement system as a part of the echinoderm immune response shows that these animals as well as all invertebrate deuterostomes share innate immune system homologies with vertebrates. Studying the simpler immune response demonstrated by echinoderms is important for understanding the ancestral deuterostome defense system and reconstructing the evolution of immune system in higher vertebrates.     

3.6 kb genomic sequence from Takifugu capable of promoting axon growth-associated gene expression in developing and regenerating zebrafish neurons

Unlike mammals, fish have the capacity for functional adult CNS regeneration, which is due, in part, to their ability to express axon growth-related genes in response to nerve injury. One such axon growth-associated gene is gap43, which is expressed during periods of developmental and regenerative axon growth, but is not expressed in CNS neurons that do not regenerate in adult mammals. We previously demonstrated that cis-regulatory elements of gap43 that are sufficient for developmental expression are not sufficient for regenerative expression in the zebrafish. Here we have identified a 3.6kb genomic sequence from Fugu rubripes that can promote reporter gene expression in the nervous system during both development and regeneration in zebrafish. This compact sequence is advantageous for functional dissection of regions important for axon growth-associated gene expression during development and/or regeneration. In addition, this sequence will also be useful for targeting gene expression to neurons during periods of growth and plasticity.