Considering the evolution of regeneration in the central nervous system

The ability of many animals to regenerate substantial parts of their body is a remarkable phenomenon of biology. A particularly fascinating aspect of regeneration is the reconstitution of a functional nervous system. Since Tremblay’s discovery in 1740 that Hydra could regenerate itself after bisection, this trait has been seen in most major branches of the animal kingdom. However, there are many animals, including ourselves, that have poor regenerative ability1. It has long been argued whether the ability to regenerate was present in the ancestral metazoan and was lost during evolution (an ancestral trait), or whether it was not present in the ancestor and arose multiple times in evolution (an adaptive trait). If this capacity is an adap-tive trait, is regeneration a truly advantageous trait that was selected for or does it represent an epiphenomenon?2. Although a definitive evolutionary account of regenera-tion and its natural selection (or non-selection) is not yet possible, it is timely to address this issue not only because of the commemoration of Darwin’s bicentennial birthday year, but owing to the increasing cellular and molecular understanding of development, tissue formation and regeneration. As the process of regeneration becomes understood in greater detail in different species, we may start to better compare how specific aspects of regenera-tion may have changed over evolutionary history. Several excellent reviews have recently addressed evolutionary aspects of regeneration3–5, allowing us to focus here more specifically on regeneration of the CNS.

Neurogenesis versus axonogenesisWhat precisely is meant by CNS regeneration? Broadly speaking, this means re-acquiring nervous system

function after injury or disruption, with or without the need to replicate the original structure or its full func-tionality. In this Review we refer to regeneration as an attempt to faithfully replace the CNS structures that were damaged or removed. ‘Perfect’ and ‘imperfect’ CNS regeneration occurs among vertebrates, and both examples are discussed here. On a cellular level, the topic can be subdivided into two distinct but related areas: first, axon regrowth from pre-existing, injured neu-rons through the injury site to re-establish connections (axonogenesis), and, second, the production of new neu-ronal cells from precursor populations that subsequently produce neurites to make connections with host cells (neurogenesis) (FIG. 1). If an animal is capable of sub-stantial neurogenesis, it is also capable of axonogenesis that can result in faithful or perfect regeneration, but there are a number of examples in which axonogenesis occurs without substantial neurogenesis, and this results in imperfect regeneration (BOX 1)6,7. Here we focus on neurogenesis, not only because it leads to more faithful replacement of the original tissue but also because recent data on adult neurogenesis in different species provides an interesting basis for the interspecies comparison.

CNS regeneration across speciesCaution must be taken when comparing regeneration between species based on the accumulated experiments from many researchers because regeneration, even when focusing on neurogenesis, is a multifaceted trait that can vary according to location and type of damage. Within one organism, different regeneration responses can be observed depending on whether the injury is made in

*Center for Regeneration Therapies, University of Technology, Dresden, c/o Max-Planck Institute for Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany. ‡Developmental Biology Unit, University College London Institute of Child Health, 30 Guilford Street, London WC1N 1EH, UK. e-mails: [email protected]; [email protected]:10.1038/nrn2707

MetazoanAny multicellular member of the animal kingdom.

EpiphenomenonA by-product of another essential metazoan trait associated with development, tissue organization or asexual reproduction.

Considering the evolution of regeneration in the central nervous systemElly M. Tanaka* and Patrizia Ferretti‡

Abstract | For many years the mammalian CNS has been seen as an organ that is unable to regenerate. However, it was also long known that lower vertebrate species are capable of impressive regeneration of CNS structures. How did this situation arise through evolution? Increasing cellular and molecular understanding

of regeneration in different animal species coupled with studies of adult neurogenesis in mammals is providing a basis for addressing this question. Here we compare CNS regeneration among vertebrates and speculate on how this ability may have emerged or been restricted.

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Larva and adult

Larva and adult

Larva and adult

Larva

Adult

Embryo

Embryo

Lamprey

Fish

Salamander

Frog

Lizard

Bird

Mammal

Larva? Adult?

Larva and adult

Larva and adult

Larva

Adult None

Embryo

Embryo?

Larva? Adult?

Larva and adult

Larva and adult

Larva

Embryo?

Embryo?

Nature Reviews | Neuroscience

Axonogenesis Ependymal tube healing and outgrowth New neurogenesis Sensoryganglia

Radial glia-like ependymoglial cells Neuron

EpendymaltubeSpinal cord

Blood–brain barrierA barrier between the blood and the CNS, which is established by specialized capillaries and astrocytes and allows selective entry of compounds from the blood into the CNS parenchyma.

the brain, the retina or the spinal cord8. Furthermore, dif-ferent injury paradigms have been used either to selec-tively remove specific neuronal populations (through the injection of toxins) or to non-selectively remove many cell layers (mechanical injury)8–10. Considering the overall structure of neural tissue and the location of the stem cells that will replace the missing cells in injured tissue, this distinction has important mechanistic implications. It is now widely accepted that stem cells and/or progeni-tor cells that are functionally important for neurogenesis line the internal lumen of the nervous system, the ven-tricular zone and the adjacent layer, the subventricular zone, and that neuronal cells ultimately migrate to their final destination (FIG. 2)11,12. many neurotoxins selectively remove neuronal subtypes and not the ventricular or sub-ventricular layer, thereby leaving the stem cells and/or progenitor cell source intact. By contrast, physical dam-age often disrupts both the neuronal and ventricular lay-ers and therefore requires that the tissue elicits additional responses to ultimately achieve regeneration (FIG. 3). These include: first, a functional wound-healing response to reconstitute the ventricular layers; second, control of the potentially increased number of non-neuronal cells that will have infiltrated because of breaches in the blood–brain barrier; and third, regeneration of multiple cell types. Therefore, the ability to regenerate after physical damage may be the most demanding, and most variable, criterion for regeneration. Contradictory results have even been reported for the same animal species after brain, retina or spinal cord lesion, these inconsistencies are probably due to the variability in the injury protocol that was used in each study (for example, see BOX 2).

A classic paradigm to induce spinal cord regeneration in lower vertebrates has been tail or caudal amputation

and, as this may be the most reproducible and com-prehensive procedure, we have used spinal cord regen-eration in the context of tail or caudal regeneration to compare regeneration across species (when possible) and have summarized the results in FIG. 4 and TABLE 1. As tail regeneration studies are not available in birds and mammals, internal lesions to the spinal cord have been used instead. Although not ideal for comparison, the general profile that chick and mammals can regenerate to a certain extent in the embryo but not after birth holds true not only for the spinal cord, but also for other neural structures, such as the retina, and non-neural structures, such as the limb, which suggests that the comparison is valid. Retinal regeneration is the area where perhaps the most comprehensive interspecies comparison is avail-able (see BOX 2). The overall species profile of retinal regeneration resembles that depicted in TABLE 1 and observations made in the retina reinforce many points made in this Review.

Taking a general look across the vertebrates in FIG. 4 and TABLE 1, spinal cord regeneration after tail amputation is observed in the aquatic anamniotes sur-veyed so far and in reptiles, but becomes more restricted in birds and mammals, which could be consistent with the notion that regeneration is an ancestral trait that was diminished, lost or blocked over evolution13–18. Although, regeneration can be scored as positive or negative based on the capability to regenerate an innervated tail, in fact, the faithfulness and perdurance of regeneration varies from species to species (FIG. 1; TABLE 1). These differ-ences probably represent a mixture of processes that have changed sporadically in individual animal lineages and those that represent major transitions in evolution-ary history. Salamanders (which belong to the order

Figure 1 | Different cellular modes of regenerating spinal cord structures after amputation. Injury of the spinal cord can lead to outgrowth of axons (axonogenesis) from surviving, differentiated neurons that can make connections to targets, but that do not necessarily regenerate the missing cells at the injury site. In addition, in some animals, the cells that line the central canal of the spinal cord extend a tube of cells through proliferation. In fish, frog tadpoles and salamanders, this ependymal tube of cells generates new neurons, including sensory ganglia neurons in salamanders, that extend axons to reconstitute neuronal architecture and connectivity. The limited data in lamprey suggests that it undergoes ependymal tube growth and neurogenesis but this is not as extensively described as in other animals.

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Caudata) have been best studied for their comprehensive regeneration of the spinal cord and although the descrip-tions of caudal spinal cord regeneration in cyclostomes and fish suggest that these animals can extensively regen-erate the spinal cord, this still needs to be confirmed. Brain regeneration in fish, however, has been extensively documented8,15–21. larval frogs (anurans) and reptiles are capable of functional but imperfect regeneration, with spinal cord regeneration during lizard tail regeneration being rather limited (described later)7,18,20,22,23. By con-trast, lizards can robustly replace neurons in different brain areas after neurotoxin damage, which highlights that the variable regeneration response is dependant on anatomical location and/or injury paradigm24 (FIG. 3).

Another important dimension of regeneration is its life-stage dependence. Frogs, birds and mammals display partial spinal cord regeneration at larval and embryonic stages, respectively, but all lose regeneration capacity over the course of maturation not only in the tail ampu-tation paradigm but in various regeneration contexts such as spinal cord lesion, brain lesion and regeneration of non-CNS structures such as the limbs25–29. Is this loss of regeneration over development (and possibly evolu-tion) due to a change in a single trait? Although a precise molecular understanding of what restricts regeneration over development and evolution is not yet clear, we would suggest that differences in regeneration ability

are due to alterations in several key attributes, including changes in wound-healing responses in neurogenic and gliogenic cell populations (as well as in surrounding cell types, such as immune cells) and changes in the genetic and epigenetic circuitry of stem cell and/or progeni-tor cell pools that maintain or restrict access to certain embryonic transcriptional programmes.

Cellular traits underlying regenerative abilityHow can the interspecies differences described in the previous section be understood mechanistically? On the cellular level, regeneration of new CNS neurons requires that functioning CNS tissue responds to injury or cell loss by resealing and reconstructing the ventricu-lar layer. This wound-healing process requires stem cells to migrate, re-establish cell contacts, self-renew and undergo specification and spatial patterning to form the required diversity of neurons and glia that will inte-grate into host tissue to replace the damaged structure. As discussed later, the steps of progenitor-cell patterning and controlled neurogenesis that are used for successful regeneration in animals such as the salamander largely recapitulate the steps carried out during early embryonic development to establish the CNS in the first place. This means that in organisms with a capacity to regenerate, cells can successfully resolve a lesion by reconstructing the tissue architecture necessary to support stem cells that will then recapitulate the developmental steps of cell type specification and neurogenesis.

Glial cells and their wound-healing responsesBecause the wound-healing response may be considered to be a regeneration-specific step that is distinct from the developmental steps required to build the CNS in the first place, it is likely to be an important deter-minant of regenerative ability. How does this step differ among vertebrates? Glial cells respond to injury within the CNS, and organisms that robustly regenerate the spi-nal cord after amputation or lesion, such as salamanders and larval frogs, show a distinct glial cell response at the wound site compared with mammals.

In salamanders and frogs, the spinal cord contains two types of glia, myelinating oligodendrocytes and radial glia, the cell bodies of which reside next to the central canal or the grey matter and extend processes radially to the outer pial surface of the spinal cord. Radial glia are identifiable as expressing the glial-cell marker glial acidic fibrillary protein (GFAP) (FIG. 2). These radial glia also have proliferative and neurogenic capacity. After injury, they transiently lose GFAP, re-express vimentin and nes-tin, which are normally expressed by embryonic radial glia, and undergo an epithelial to mesenchymal transi-tion that allows them to migrate into surrounding tissue before reforming a single-cell layered neuroepithelial tube30–33. This cellular tube undergoes elongation through cell division and ultimately undergoes neurogenesis to reconstruct the spinal cord, providing a permissive envi-ronment for axonal growth through the lesion.

The glial cell populations in the mammalian spinal cord have a number of distinct properties, and respond differently to injury, ultimately generating a wound

Box 1 | Axonogenesis

Severed CNS axons display remarkable regenerative ability in anamniotes and developing amniotes. Although adult lower vertebrates can regenerate their axons much more efficiently than birds and mammals, great interspecies variability is observed, as exemplified by studies of optic nerve regeneration in reptiles. These animals, unlike fish, do not spontaneously recover vision even when axonal regrowth to the tectum occurs, unless they undergo visual conditioning83–85. Thus, extensive axonal regrowth is not always tightly coupled with establishment of appropriate connectivity to the target. The difference between lizards and fish in their ability to re-establish target innervation might be associated with increased brain complexity86.

Several factors are known to impair regrowth of injured axons within the adult mammalian CNS, including presence of inhibitory molecules, decrease in growth-promoting molecules, glial scar formation and inflammation. The relative importance attributed to these factors has changed over the years. The emerging view is that most extrinsic factors investigated can have both beneficial and deleterious effects on preserving injured neurons and nerve regeneration depending on extent and timing. Additional regulation of axon regeneration may come from intrinsic differences within neuronal populations and possibly from mechanisms that govern axon degeneration.

The changes in the CNS environment that may occur with development, such as myelination, and formation of a glial scar following injury, undoubtedly have an important role in the loss of regenerative ability. Intrinsic changes in the ability to regrow axons, however, may also be at play87. Indeed, some adult vertebrates, such as lampreys (jawless fish), other fish and lizards regenerate CNS axons through a glial scar, which is inhibitory for the regeneration of rodent axons88–90. It should be noted that even within the same animal, different subsets of neurons show different regenerative capability. For example, in the lamprey spinal cord, only about 50% of the axons regenerate following spinal cord resection, yet they are sufficient to re-establish functionality91,92. These studies emphasize that reconnection of surviving neurons can restore useful CNS function even if a faithful replacement of all missing cells is not achieved.

Evidence exists for an intrinsic control of axonal degeneration, as exemplified by the significant protection from nerve degeneration following insult in the WldS (slow Wallerian degeneration) mutant mouse93–94. Therefore, it is conceivable that differences in the extent and/or mode of axon degeneration among species following nerve lesion may also have a role in their subsequent regenerative capability.

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Central canal

Mantel zone

Grey matter

Marginal zone

White matter

Pia mater

Ependymalzone

Radial glia indeveloping amniotes

Ependymal cells in adult amniotes

Ependymoglial cellsin adult salamandersfish and reptiles

Pia mater


Central canal

White matterGrey matter

Glial scarA barrier composed mainly of reactive astrocytes and proteoglycans that forms after CNS injury to separate healthy from damaged tissues.

response that does not reconstitute a neuroepithelial tube. many of the GFAP positive cells that reside in the mammalian spinal cord lose their radial character as well as their contact with the central lumen to form astrocytes34. upon injury, a subset of these cells upregu-late GFAP, infiltrate the wound site and deposit extra-cellular matrix and proteoglycans to form a glial scar, which impedes axonal extension through the injury site, rather than reconstructing the spinal cord tube35–37. A separate descendent of the radial glia, the ependymal cells, which are mostly composed of cuboidal epithelial cells that line the central canal, seem to act as astrocyte and oligodendrocyte progenitor cells following spinal cord injury, but these cells home to the glial scar and do not form neurons after tissue damage38. Therefore, the composition of the glial cell population and its response to injury is a major difference among regenerative and non-regenerative vertebrates: regenerative species have retained the radial glial cells that were present at the developmental stages and which serve both a neuro-genic stem cell function and glial cell function, whereas non-regenerative vertebrates have subfunctionalized or diversified glial cell function by developing distinct cell populations, such as the cuboidal ependymal cells to line the lumen and astrocytes that reside solely in the white matter39–41.

The close functional association of radial glia with stem cells able to produce new neurons in the adult is supported by the existence of germinal niches that undergo continued neurogenesis in the brains of adult lizards, birds and mammals. In these niches, radial glial-like cells that have direct contact with the lumen are retained mosaically into adulthood41,42. This strong correlation between lumen-contacting radial glia and

regeneration or adult neurogenesis suggests that certain properties of radial glia are important for their extensive neurogenic capacity. The increasing appreciation of the role of cilia in receiving and transmitting crucial mor-phogenetic cues suggests that the distinctive cilial archi-tecture of radial glia compared with ependymal cells may represent a key functional determinant43,44.

Although the properties of radial glia are strongly associated with regeneration and adult neurogenesis, their presence alone is not sufficient for regeneration. After post-metamorphic maturation in frogs, the spinal cord GFAP–expressing glia retain their radial organi-zation, yet the ability of these cells to reform a tube is apparently blocked and regeneration does not occur. The reasons for this change in the regeneration capac-ity of frog radial glia has not been studied in the spinal cord but it has been examined in the forebrain, which regenerates in tadpoles but not in adult Xenopus laevis. In the adult, the disrupted forebrain ventricular layer is not properly resealed after injury. Partial forebrain regeneration can however be restored if the injured area is augmented by simply transplanting more brain cells that help to close the wound with consequent brain lay-ering45 (FIG. 3). The authors of this study speculated that in the adult X. laevis, brain radial glial cell migration is not efficient enough to heal the area and start regenera-tion, but if regrowth of epithelial tissue is promoted by the additional cells, then the brain area can be regen-erated. This interpretation implies that changes in the initial response to physical injury might be crucial for permitting or restricting regeneration ability.

Similarly in birds, although the presence of radial glia is strongly associated with adult neurogenic brain niches, the ability to regenerate following spinal cord injury is lost early in development, well before the complete loss of radial glia, which suggests that either changes in the ability of radial glia to respond to injury, their neuro-genic capacity or other environmental signals restrict their regeneration response46,47,48. Significant regenera-tion is still observed at embryonic day 11 when, unlike at later non-regenerative stages, radial glia markers are upregulated in response to spinal cord injury, and ongo-ing neurogenesis probably maintains the spinal cord in a state compatible with regeneration49,50.

A key question concerning why radial glia versus astrocytes or ependymal cells can respond to injury by undergoing wound healing and neurogenesis rather than gliosis is whether there is an intrinsic difference in response to the same cues, or differences in the envi-ronment. Several studies indicate that certain mamma-lian astrocyte and ependymal cell subsets have intrinsic self-renewal and neurogenic potential, suggesting that differences in the environment underlie radial glia regeneration capacity. However, many of these experi-ments rely on selecting rare cells in culture for analysis, and these might represent a small, spatially restricted population of cells48,51,52. In future it will be important to compare how the different, major astrocytic populations in mammals respond to defined extracellular stimuli at injury sites with cognate fish and amphibian cells that undergo regeneration.

Figure 2 | Neural progenitors in spinal cord development and regeneration. The early structure of the neural tube of developing amniotes is very similar to the ependymal tube that gives rise to the regenerated spinal cord following tail amputation in salamanders. Cells that line the central canal and which have radial processes that contact the pial surface are crucial for neurogenesis early in mouse development (inset). In amniotes these cells disappear with the progression of neural differentiation, but in the CNS of adult salamanders, fish and reptiles, which display significant regenerative ability, radial glia-like cells (ependymoglia) are retained.

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a Neurogenesis in response to neurotoxins (e.g. as performed in lizard telencephalon)

b Regeneration in response to forebrain injury (e.g. as performed in fish, salamander and frog)

Neurotoxine.g. 3-acetylpyridine

Ventricle

Ventricle

Parenchymal cell

Neuron

New neurogenesis

Ventricular layerradial glia

Mechanical injury

Reconstitution of ventricular zone and neurogenesis

Damaged neuron

Damaged radial glia

Cellular automataA method to simulate physical phenomena in space and time by using an array of units (cells) that have certain properties that change according to a given set of rules.

Changes in extracellular wound environment and the immune system. Differences in the environment of injured tissue especially with regards to the immune system have long been speculated to affect regeneration ability. The salamander seems to have an inefficient adaptive immune system, whereas in frogs the adaptive immune system develops rapidly around metamorpho-sis, coinciding with loss of regeneration capacity. Based on these and other observations it has been speculated that this is one feature that allows salamanders to keep regenerating into mature animal stages (for a review see REF. 53). Recently Fukazawa et al. using X. laevis tail regeneration as a model, showed that changes in genes controlling T regulatory cells and other immune func-tions change significantly during a stage-specific decline in tail regeneration and that treatment of animals with immunosuppressants or depletion of immune cells par-tially rescued the decline in regeneration54. Therefore, the surveillance of damaged tissue, especially in situa-tions in which the blood–brain barrier is broken may have a large influence on regeneration.

The evolution of wound-healing ability. If wound heal-ing is an important determinant of regeneration capacity, then how might these cellular responses have evolved to

promote a successful outcome? The successful regrowth of epithelial tissue that is required for regeneration may not have been selected for but rather may have been a by-product of the requirement to maintain or re-access embryonic conditions to regenerate. A computational study that models the evolution of a developmental sys-tem using cellular automata, showed that wound healing and regeneration were non-essential, unselected traits that developed during the evolution of a successful developmental system. This theoretical study shows that wound healing that permits regeneration may not neces-sarily be an adaptive trait, but a natural consequence of assembling a dynamic system that undergoes cell–cell rearrangements, proliferation and death55. This would suggest that loss of regeneration is related to cells taking on non-developmental traits required for adult func-tion that are incompatible with this natural regeneration trait. The emergence of diversified ependymal and astro-cyte populations with different cellular functions and responses, as well as strong immune surveillance, may represent such changes.

Access to developmental programmesHow regenerative organisms build mature CNS struc-tures yet maintain or re-access embryonic programmes

Figure 3 | regeneration in the forebrain after neurotoxin damage versus injury. a | Neurotoxin damage causes selective removal of neurons without extensive damage to the ventricular layer. Ventricular layer radial glia can increase proliferation and neurogenesis to replace missing cells. b | Physical damage can disrupt ventricular zone cells that then must reseal and renew in addition to replacing missing neurons. The mammalian forebrain shows continuous adult neurogenesis and the organization of this neurogenic zone can be seen in REF. 101.

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Retinalprogenitors

Ciliary margin

RetinaRPE

Optic nerve

Lens

Peripheralretina

Axonexit

Rod progenitors in fish Ganglion cell

Amacrine cella Rod ConeMueller glia Bipolar cell

RPEHorizontal cell

Growthregion

Salamander:retina ablation

RPE Retina

Fish:neurotoxicdamage

RPE

b RPE delaminates and transdifferentiates

Stem cell recruitment from Mueller glia

Damagedcells


to undertake the complex process of rebuilding the diverse cell types that have been damaged in an injured CNS, and how this access may have become limited, is important for understanding the mechanism and evolution of regeneration. In the salamander, a species with powerful regeneration abilities, maintaining and re-accessing developmental programmes of progenitor cell control is crucial for regeneration. Based on grafting experiments, the regenerating spinal cord of salamanders has long been known to have potent patterning effects on

surrounding mesodermal tail tissues, indicating that the regenerating spinal cord is organized into distinct spatial domains along the dorsoventral axis and that it secretes growth-promoting and patterning cues56. Recent molec-ular analysis of the regenerating spinal cord in axolotl showed that ventricular radial glial cells residing in the uninjured spinal cord, which represent the progeni-tors for spinal cord regeneration, maintain expression of the combinatorial transcription-factor code arrayed along the dorsoventral axis that is normally used during

Box 2 | Regeneration in the retina

The eye has been a fertile area for comparing regeneration between species because different strategies for regenerating the retina have arisen over evolution95–98. Teleost fish recruit cells from within the retinal layer — mainly Mueller glia which are analogous to radial glia (see part a of the figure) — to regenerate the retina. Newts and larval frogs display a different mode of regeneration in which cells from the apposing retinal pigment epithelium (RPE) divide, delaminate and convert into a new retinal neuroepithelium that recapitulates developmental steps to form the retina (see part b of the figure). Adult frogs were long thought incapable of retina regeneration, but it was recently demonstrated that damaging the retina without removing the vascular bed beneath it allowed RPE-mediated retina regeneration81, which suggests that changes in the environment might be important variables in the evolution of retina regeneration. It will be important to perform an analogous surgery in cyclostomes and fish to determine if the mechanism is ancestral. Avian and mammalian embryos display some retina regeneration potential for a very limited window, when the RPE can be forced to form retina by adding ectopic fibroblast growth factor 2 (FGF2). However, this ‘induced’ retina has the wrong apical–basolateral polarity and occurs by directly converting the RPE layer to retina without cellular delamination and RPE self-renewal99. The induced RPE to retina conversion in chicken embryos also provides insight into how both extrinsic versus intrinsic factors constrain regeneration. FGF2 alone is normally capable of forcing this conversion up to stage 25. However, this period can be prolonged to stage 31 by blocking activin signalling, which normally reinforces RPE formation. After this period, reducing activin signalling cannot stimulate RPE to retina conversion, and a yet-to-be-defined cell intrinsic mechanism apparently operates to prevent RPE to retina conversion100. An interesting future direction is to determine whether regenerative animals such as newts ever assemble this cell intrinsic inhibitory step, whether they have a powerful mechanism to dismantle the inhibitory machinery or whether they implement a partial or alternative block of RPE to retina that is more easily reverted.

The retina also provides a model to compare adult neurogenesis and regeneration. Robust adult neurogenesis occurs in fish and amphibians, in which the retina grows throughout life from a cell source called the ciliary margin, which is positioned at the junction between the neural retina and RPE (see part a of the figure). But, this germinal zone is not the major source of cells for retinal regeneration in newts as cells from the RPE in the central retina are also used (see part b of the figure). The distinction between adult neurogenesis and regeneration is also evident in the post-metamorphic frog, in which after severe retinal injury the retina heals while growth from the ciliary margin continues as normal without making up the lost tissue and this results in the formation of a smaller, functional eye96. These observations indicate that the cell source of adult neurogenesis is not necessarily mobilized to elicit regeneration in response to injury.

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Cyclostomes(lamprey)

Fish Caudata(salamanders)

Amphibians

Anurans(frogs)

Reptiles Birds Mammals

Larvae and possibly adults regenerate

Larvae and adults regenerate

Embryos or larvae regenerate

Neurogenesis after injury

Limited neurogenesis after injury

Neurogenesis probably at least in embryos and larvae

( )

MelanophoresPigment cells of neural crest origin that, during development, migrate from the neural tube to the skin and other pigmented tissues.

neural tube development to direct the formation of dif-ferent neuronal subtypes57. Furthermore, the salamander maintains into adulthood a crucial signalling centre for neural tube development found in all vertebrates called the floorplate that secretes the well-known extracellu-lar morphogen, sonic hedgehog58. During spinal cord regeneration, this source of sonic hedgehog is involved in determining the size of the dorsoventral neuronal progenitor cell domain, spinal cord growth, as well as the patterning and growth of tissues surrounding the regenerating spinal cord, including the cartilage precur-sors that will eventually form the vertebrae.

By contrast, sonic hedgehog expression and recep-tivity changes relatively early during neural tube development in chickens and mammals, which may underlie some of the loss of regeneration ability. During the course of embryonic development in chickens, the dorsoventral transcription factor domains become refractory to sonic hedgehog control despite its con-tinued expression, and before birth these transcription factors and the floorplate sonic hedgehog expression are extinguished, which presumably contributes to reduced regeneration capacity59. When the adult mouse spinal cord is injured, scattered cells within the spinal cord parenchyma, far from the lumen, express PAX7, one of the ‘pattern associated’-transcription factors, but there is no evidence of productive neurogenesis; it is likely that these cells are too scattered or only partially competent to promote regeneration60. Therefore, losing access to embryonic conditions in the mammalian spinal cord is likely to be a major factor that limits the regenera-tive response. How the salamander retains expression of these factors in its stem cells when it reaches the adult state and how this system is repressed in mammals is an important question.

A promising direction in molecularly understand-ing how regeneration becomes restricted comes from comparing species that are capable of full versus par-tial regeneration. Although tail amputation leads to

regeneration in salamanders, frog tadpoles and lizards, the true extent of CNS regeneration varies between these animals (FIG. 1). For example, in X. laevis, tail regenera-tion involves neurogenesis of motor and sensory neurons but bonafide formation of dorsal root ganglia, which originate from the neural crest, does not occur22. During X. laevis tail regeneration, melanophores have been found to arise from melanophore precursors in the unampu-tated part of the skin rather than the regenerating spi-nal cord, which indicates that regeneration might occur without producing neural crest cells within the spinal cord that migrate out to the periphery, as is observed during embryogenesis. This is in contrast to the sala-mander in which immunohistochemical and cell tracing experiments indicate that within the regenerating spinal cord a neural crest cell population forms that migrates peripherally to regenerate bonafide dorsal root ganglia61,62 (v. mazurov, l. mchedlishvili and e.m.T., unpublished results). Within the regenerating salamander spinal cord the molecular factors that direct neural crest formation are expressed in the same spatial domains that are found in early development58. In X. laevis, the profile of molec-ular factors controlling neural crest formation has not been examined in detail so it is not yet known if the lack of a neural crest represents a cell-intrinsic limitation in the neural precursors, or a lack of signal. On the ventral side of the spinal cord, however, some molecular factors have been analysed. Interestingly, sonic hedgehog is not expressed in the X. laevis floorplate (but it is expressed in the notochord), which suggests that the environmental signals to diversify cells into different neural cell types are lacking63. Therefore, species-specific differences in the expression of crucial morphogens may modu-late the extent of the regeneration response. In frogs, loss of sonic hedgehog expression also correlates with a decreased capacity to regenerate limbs, and analyses of the DNA methylation status of a limb-specific sonic hedgehog enhancer revealed increased methylation in adult X. laevis compared with axolotl and young frogs, implicating species- and stage-specific repression of gene expression as a means by which capacity to regenerate can be lost64. An interesting future question is whether such changes in DNA methylation status occur on a gene by gene basis or whether they reflect genome-wide changes in the DNA methylation between species with multiple functional outcomes.

Spinal cord regeneration is even more limited in liz-ards than in frogs, forming only an ependymal tube with apparently no neurogenesis, but there is little molecular information to allow a comparison with other verte-brates (FIG. 1)20. The molecular make-up of this regen-erating ependymal tube is not known, so it is unclear which components of the neurogenesis pathway might be missing in the lizard, and whether the ependymal cells have the potential to carry out a full programme of regeneration. Certainly the molecular profiling of the liz-ard ependymal tube could help determine whether there are molecular commonalities with X. laevis and chickens that help to account for regenerative loss — an impor-tant issue to establish the phylogenic profile of regen-erative ability. Of further benefit would be to perform

Figure 4 | Phylogenetic overview of cNs regeneration ability. Squares represent a stage-dependent regeneration ability. Circles represent neurogenesis in the spinal cord during regeneration.

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TaxonThe unit used in taxonomy (classification of plants and animals) to group related plants or animals together at any level of the hierarchy.

PaedomorphosisA term describing the situation in which embryonic or juvenile characteristics of the ancestor are evident in an adult organism irrespective of the mechanism by which this state came about (encompasses neoteny).

Selfish junk DNADNA elements in the genome that were thought to have no functional role but to self-replicate and multiply within the host genome. Recent work suggests that this non-protein coding DNA contains important regulatory sequences.

NeotenicA term describing a state whereby the development of a species’ somatic body structures has slowed down or is absent, resulting in juvenile traits being present in an adult stage

cross-species and heterochronic transplantation of cells from regeneration restricted donors to regeneration permissive hosts and analyse cell potential in the dif-ferent environments. Such cross-species transplantation studies have previously been performed with X. laevis and axolotl; transplantation of frog limb skin from non-regenerative stages to axolotl limbs supported the early stages of regeneration65,66.

Paedomorphosis, metamorphosis and regenerationmany amniotes lose the ability to regenerate during the course of development, and some potential reasons have been discussed in the previous section. Frogs display robust regeneration as larvae but lose this capacity after metamorphosis. Considering that frogs are the sister taxon to the salamander in the amphibian lineage, how this situation arose — whether salamanders never devel-oped this stage-dependent regeneration loss, whether they lost it or whether it arose in frogs independently of the warm-blooded vertebrates — is not known. Salamanders are well-known for their paedomorphic features and it is interesting to consider regeneration and paedomorphosis in light of inter-species studies correlat-ing anatomical brain complexity with genome and cell size67–69. Salamanders have an unusually large genome, which has been speculated to have arisen multiple times in metazoan evolutionary history. When compar-ing brain anatomy between different anuran (frogs and toads) and caudate (salamander) species, it was noted that the larger genome (which correlates with larger cell size) in salamanders correlates with a simplified brain organization and with signs of retaining a juvenile character, even though these animals display complex prey-catching behaviour that seems to be due to com-pensatory development of specific brain regions. It was speculated that this simple brain architecture represents a secondary simplification of brain architecture from a more complex, ancestral state seen in frogs, which have smaller cell size. Theoretical work on the evolution of cell differentiation has led to the hypothesis that the acquisition of large genome size by incorporating selfish junk DNA is actually the condition that allows evolution of salamander nervous system paedomorphosis70.

Does paedomorphosis also extend to re-accessing a regeneration-competent environment and maintaining

juvenile progenitor cell pools? These large-scale genome changes may not only affect the evolution of cell differ-entiation, as has been proposed, but may alter the epi-genetic mechanisms by which important developmental genes are repressed and activated, making developmen-tal genes more dynamically accessible to extracellular injury cues, rather than being stably repressed in adult cells. The secondary simplification hypothesis described above would imply that regeneration was an ancestral trait, as seen in fish, that then became stage-dependent in amphibians. The extended, lifelong regeneration capabilities in the salamander would then be a feature that was re-gained through paedomorphosis, after the emergence of the amphibian lineage. Such a scenario would suggest that the stage-dependent regeneration ability of frogs, chickens and mammals could derive from a common trait present in an ancestral amphibian. The concept of paedomorphosis also provides an inter-esting prospect for regenerative medicine in mammals. If paedomorphosis that leads to regeneration could arise during evolution, what are the cellular and molecular differences that gave rise to this state, and could some of these traits be reproduced at the cellular level acutely in mammalian neural cells?

The juvenile characteristics found in salaman-ders, combined with the decline of regeneration after metamorphosis in frogs may lead many to wonder if metamorphosis or lack thereof is the key attribute that explains lifelong regeneration in the salamander compared with frog and other vertebrates, but this is unlikely. Although many salamanders are considered neotenic, there are many salamander species, such as Notophthalmus viridescens, that undergo metamorpho-sis yet retain the ability to regenerate their tail, CNS structures and limbs throughout their lifespan. This implies that the paedomorphic character of salamander CNS is not necessarily related to metamorphosis. How metamorphosis has evolved among vertebrates and its relationship to tissue maturation is a complex issue (for a review see REF. 71). It is, however, clear that thyroid hormone signalling as occurs during frog metamorpho-sis or that peaks just after birth in mammals (long after spinal cord regeneration capacity is lost) has profound effects on the CNS72,73 (for a review see REF. 74). Thyroid hormone signalling has documented effects on glial and

Table 1 | Profile of spinal cord regeneration across animal families*

Animal embryo or larva regeneration

Adult regeneration

ependymal tube formation

successful neurogenesis

Dorsal root ganglia

Axonogenesis

Lamprey + + (+)/? (+)/? (+)/? +

Fish + + + + + +

Salamander + + + + + +

Frog + – +/– +/– – +

Lizard ? + + – – +

Bird + – +/– +/– ?/ – +/–

Mammal + – +/– ?/– ?/– +/–

*Tail/caudal spinal cord regeneration is profiled across different vertebrate species. In birds and mammals, information from spinal cord lesion was used in lieu of the caudal spinal cord regeneration. In the last three columns, x/x denotes the response at larval/adult stage. ? denotes no known information.

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neuronal cell differentiation and neural progenitor pro-liferation75,76. Therefore, although metamorphosis may not be the sole explanation of stage-dependent regenera-tion among vertebrates, how thyroid hormone signalling during key developmental transitions affects the ability to regenerate is likely to be a fruitful avenue of molecular studies in future.

Another hypothesis to explain extensive adult neu-rogenesis and regeneration in fish and salamanders is the mechanism of tissue growth in these animals. In contrast to mammals, in which muscle tissue growth post-birth occurs by increasing the size of cells but not their number, in fish the number of fibres continues to increase throughout their entire life cycle. This type of growth may require a matching increase in neuronal input and therefore exerts selective pressure to maintain neurogenesis77.

A self-renewing, neuronal progenitor cellSpinal cord regeneration in salamanders continues in the face of ageing or repeated amputation, which implies that regenerative stem cells have extensive self-renewal properties78,79. In regenerating animals, the stem cell pool reconstructs the tissue by self-renewing and by gener-ating the appropriate number and cohort of differenti-ated cell types in response to injury. Do the neural stem cells that are maintained in limited locations in birds and mammals have a similar self-renewal potential? Some studies suggest that self-renewal might be lim-ited. Although an in vivo experiment that is equivalent to repeated spinal cord amputation and regeneration has not been performed in mammals, adult neural stem cell self-renewal has been studied by propagating isolated stem cells from the lateral ventricle of the forebrain in culture and examining their ability to clonally re-estab-lish cell clusters that form astrocytes, oligodendrocytes and neurons, known as neurospheres. Comparison of the self-renewal capacity of forebrain neural stem cells in young versus old mice, suggested that neural stem cells in old mice have a lower self-renewal capacity compared with embryonic, fetal and early postnatal equivalents80. molecular profiling of stem cells in young versus old animals identified high mobility group A2 protein (HmGA2) — an abundant nuclear protein that is involved in both chromatin structure and the recruit-ment of transcription factors — as being a promoter of self-renewal in young mice but that was switched off in old mice, correlating with a lower renewal capacity. An interesting question is whether the HmGA2 pathway is maintained in regenerative species. If so, how does it

contribute to their lifelong regeneration capacities? How might alterations in this pathway have arisen in different species?

Timing, contingency and environmentIt should be noted that loss of regenerative ability may not only be due to dramatic losses in cell potential or active blocks to regeneration. It could be that signals or conditions that are required for regeneration simply lose synchronization with the cells that have to receive those signals to proceed, and thus they are unable to initiate the regeneration processes. An interesting example is that of retina regeneration in post-metamorphic X. laevis, which was long considered not to occur. Simply chang-ing the means of removing the retina, thereby main-taining a crucial supportive cell population, the retinal vascular membrane revealed that retinal regeneration from the retinal pigment epithelium (RPe) can indeed occur (see BOX 2). These findings suggest that in the regenerative salamanders the signals required to regen-erate retina from the RPe might derive from a different cell population81.

PerspectiveThe evolution of CNS regeneration is a thought-pro-voking topic for which many experiments at the cellular and molecular level can now be performed in different species to dissect which genetic pathways have evolved to promote or restrict regeneration. As discussed here, it is likely that changes in several attributes underlie the interspecies differences. In future it will be impor-tant to establish truly comparable molecular and cell based assays among representatives of different tax-ons to disentangle how differences in wound-healing responses, stem cell state, access to embryonic neu-rogenesis programmes and other cell attributes have changed. In asking why differences in regenerative abil-ity have arisen, it is often speculated that regeneration must have been lost owing to the tumorigenic poten-tial of actively proliferating stem cells in adult tissue. However, regenerating tissue controls tumorigenic cell growth82. We speculate that the selective pressure to resolve wounds in a way that promotes survival and reproductive fitness was an important variable that affected regeneration. Furthermore the evolution of more complex CNS structures in amniotes probably occurred by re-using embryonic signalling pathways in new cellular, and epigenetic contexts at the cost of maintaining access to embryonic gene programmes required for regeneration.

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AcknowledgementsWe thank Y. Malashichev, V. Soukup and R. Voss for discus-sions on evolution. E.M.T. was supported by grants from the Deutsche Forschungsgemeinschaft: SFB655, SPP1356 and TA 274/3-1, and the Bundesministerium für Bildung und Forschung Biofutures. P.F. was suppported by grants from the Biotechnology and Biological Sciences Research Council and the Child Research Appeal Trust.

FURTHER INFORMATIONElly M. tanaka’s homepage: http://www.crt-dresden.de/index.php?id=46 Patrizia ferretti’s homepage: http://www.ich.ucl.ac.uk/ich/academicunits/Developmental_biology/Research

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Considering the evolution of regeneration in the central nervous system

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