Brain regeneration in anuran amphibians

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1 Develop. Growth Differ. (2007) 49, doi: /j x x Blackwell Publishing Asia Review Brain regeneration in anuran amphibians Tetsuya Endo,* Jun Yoshino, Koji Kado and Shin Tochinai Department of Natural History Sciences, Faculty of Science, Hokkaido University, N10W8, Kita-ku, Sapporo , Japan 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. Key words: amphibian, anuran, brain, regeneration, Xenopus. Introduction Amphibians are known to be highly regenerative animals. They can regenerate a limb, tail including a spinal cord, jaw, liver and even brain. After a partial amputation, the remaining part of those organs reconstitutes the lost part completely. Mammals, including humans, also have regenerative ability to some extent, but in general this ability is considered to be very limited. Conversely, recent progress in regenerative medicine has demonstrated regenerative responses in the mammalian central nervous system (CNS), including the discovery of neural stem cells (NSCs) (Doetsch et al. 1999; Johansson et al. 1999). It has been shown that neurons are replaced even in the adult CNS using the NSC system (Gage 2002). This implies the presence of regenerative abilities in the adult mammalian CNS in response to an injury, although this *Author to whom all correspondence should be addressed. tendo@bio.sci.hokudai.ac.jp Received 30 November 2006; revised 15 December 2006; accepted 15 December Journal compilation 2007 Japanese Society of Developmental Biologists ability is largely confined to the regrowth of neural processes and the formation of new synapses (see Kirsche 1983). The type of massive regeneration after partial brain removal that is common among fish and amphibians is not observed in mammals. Therefore fish and amphibians are unique among vertebrates in having the ability to completely regenerate a brain. Whereas urodele amphibians (newts and salamanders) retain their regenerative ability in adult life, this is not the case in anuran amphibians (frogs) that have high regenerative abilities as larvae, but lose them as adults. The ontogenic decline of regenerative ability occurs not only in the brain but also the limb (Dent 1962). This phenomenon has fascinated researchers for a long time, because it enables us to study regeneration comparing a regenerative and non-regenerative phase in one organism. In recent years, molecular biological studies have revealed much about the mechanisms of larval and adult regeneration, particularly in the limb (reviewed by Gardiner et al. 2002; Suzuki et al. 2006). These recent studies have built on classical studies that were made before the 1980s by interpreting the old phenomenology in a more contemporary molecular language. Such studies will eventually lead us to understand the deeply hidden mechanisms of regeneration that researchers could never see in the past.

2 122 T. Endo et al. Although there are many classical studies of anuran brain regeneration, understanding of this phenomenon at the molecular level is still poor. The purpose of this review is to summarize the classical literature on brain regeneration in amphibians, particularly in anurans, in order to provide a foundation for the next generation of studies using molecular biological techniques. We will focus on the telencephalon and the optic tectum of the mesencephalon, because other encephalic regions are involved in maintaining the viability of the animal, such as respiratory and cardiovascular function. Therefore it is possible to resect a part of the telencephalon or optic tectum with low mortality, thus allowing for regeneration to occur. Also, they are large and swollen, so they are relatively easy to resect. Finally, because the telencephalon and optic tectum are the centers of olfaction and vision, respectively, functional recovery can be assessed behaviorally. Regeneration of the telencephalon Although successful brain regeneration occurs in adult urodeles, most studies of telencephalon regeneration have been carried out in larvae and juveniles (see Kirsche 1983). The paucity of studies on telencephalon regeneration in adult urodeles may be a consequence of the extremely long time it would take in adult-sized brains, and also because of a methodological problem, both of which would make analyses difficult. In many classic studies, for example, a whole telencephalon was extirpated to see if it could be regenerated from the stump on the diencephalon. However, because NSCs from different encephalic regions possess each regional character in mammals (Klein et al. 2005), it seems likely that each encephalic region can regenerate only itself, even in the highly regenerative amphibians. For example, removal of the optic tectum, a dorsal part of the mesencephalon, results in successful regeneration, as we will describe in the next section (Minelli & Del Grande 1974; Ohsawa et al. 2003). Therefore partial removal of telencephalon in adult urodeles should be tested to see if complete regeneration occurs from the cells of the remaining portion of the telencephalon. In contrast to urodeles, anuran amphibians can only regenerate the telencephalon as larvae and lose the ability as adults. Young Xenopus tadpoles, such as stage larvae, have quite high regenerative ability and complete regeneration is observed after ablation of a massive part of the telencephalon (Srebro 1957; Yoshino & Tochinai 2004). Within a month after tissue removal, an almost normal structure is reformed (Fig. 1b d). In mature Xenopus adults, no regeneration occurs and structural defects persist (Srebro 1965). The response to injury at the intermediate developmental stages is unclear, given the conflicting results reported for the regenerative response in froglets (young adults). One is that no regeneration occurs even in 10-day-old froglets, as observed in mature adults (Fig. 1e g) (Yoshino & Tochinai 2004); whereas the other is that regeneration takes 3 months in froglets that undergo cephalotomy immediately after metamorphosis (Jordan 1958). At this point, it is not possible to resolve these apparently contradictory findings because it is unclear if the experimental conditions were the same between them. At least, postlarval regeneration takes a long time and results in a deformed structure based on Jordan s experiment, and postmetamorphic Xenopus eventually lose the ability to regenerate the telencephalon. Regeneration of the telencephalon requires reconnection with the olfactory nerves in order to reform the olfactory bulb. Some classic studies, which were mainly based on histology, suggested that numerous cells migrate from the olfactory organ to the brain along the olfactory nerves and participate in brain regeneration as neuroblasts (Srebro 1957; Jordan 1958; Kosciuszko 1958; Kirsche & Kirsche 1964). In this way, reconnection between the telencephalon and olfactory nerves was thought to be a prerequisite for regeneration; however, this report regarding the source of cells in regeneration has not been confirmed using appropriate cell lineage markers. Conversely, considerable regeneration of the telencephalon is observed even before the reconnection (Yoshino & Tochinai 2004). This regenerated part is the anterior part of the cerebrum, which has differentiated gray matter and white matter; however, the olfactory bulb is missing from the regenerate (Yoshino & Tochinai 2006). The phenomenology of forebrain regeneration corresponds to what is seen in the development of the olfactory bulb. In Xenopus embryos and tadpoles, when the connection of the olfactory nerves to the forebrain is disturbed, the olfactory bulb fails to develop (Graziadei & Monti-Graziadei 1992). In contrast, transplantation of the olfactory placode near the diencephalon of the tadpole is enough to induce well-defined structures such as glomeruli that are a part of the olfactory bulb (Stout & Graziadei 1980; Magrassi & Graziadei 1985). In mammals, innervation of the olfactory nerves into the forebrain controls cell cycle kinetics of anterior telencephalic progenitor cells to induce formation of the olfactory bulb (Gong & Shipley 1995). When neurogenesis in the olfactory epithelium is inhibited by disturbing fibroblast growth

3 Brain regeneration in anuran amphibians 123 Fig. 1. Telencephalon (cerebrum and olfactory bulb) regeneration in Xenopus larvae and froglets. (a) Illustration of the brain in Xenopus. The anterior half of the telencephalon was removed. The broken line indicates the amputation site. cer; cerebrum, ob; olfactory bulb. (b g) Horizontal sections of the telencephalon in larvae (b d) and froglets (e g). (b, e) Intact; (c, f) immediately after amputation; (d, f) 30 days after amputation. Scale bars, 300 µm. factor (FGF) signaling, the olfactory bulb is not formed (Hebert et al. 2003; Kawauchi et al. 2005). In regeneration of the Xenopus telencephalon, innervation of the olfactory nerves into the regenerating telencephalon may play a crucial role in the regeneration of the olfactory bulb through the same mechanisms involved in the development of this organ. Regeneration of the optic tectum It is repeatedly demonstrated that regeneration of the optic tectum is possible in adult urodeles (Minelli & Del Grande 1974; Ohsawa et al. 2003). Ohsawa et al. removed 70% of the tissue of the left optic tectum from adult newts and observed optic nerve projections onto the regenerated structure by anterograde labeling with horseradish peroxidase from the right eye. The left optic tectum regenerated up to 80% of the volume of the contralateral intact tectum and regeneration was accompanied by de novo differentiation of nerve cells from ependymal cells (Ohsawa et al. 2003; M. Okamoto, pers. comm., 2006). Anuran amphibians have the ability to regenerate the optic tectum, as well as the telencephalon, at early larval stages and there is also a decline in regenerative ability in a stage-dependent manner. In Rana pipiens, embryos and early larvae until stage 20 (Shumway 1940), which is comparable to about stage 37/38 in Xenopus (Nieuwkoop & Faber 1956), show considerable regeneration after a short period following the removal of an optic lobe unilaterally. In contrast, none of the cases operated between stages 21 (Shumway s normal stage, 1940) and XV (Taylor & Kollros s normal table 1946), which are comparable to Xenopus stages 40 57, show a complete regenerative response (Terry 1956). Similar results were obtained in Xenopus, where a gradual loss of regenerative ability was observed (Fig. 2) (Filoni & Gibertini 1969). Tadpoles before stage 50 can repair the lesion in the operated ventricle with undifferentiated cells quickly and can reconstitute the optic lobe completely with regards to the number of layers of the tectum and its thickness after removal. After stage 51, irregularities in the thickness and alignment of the layers increase progressively. The stratification disappears from the regenerates that are operated at stage 55. At stage 59, the ventricle never completely heals even 90 days after operation and has no stratified organization (Filoni & Gibertini 1969). After metamorphosis, the wound surface is never closed and its histological appearance has not changed around the surface even a month after the operation (Fig. 2f,g). However an increase in the BrdU labeling index is observed 9 days after the tectotomy, suggesting

4 124 T. Endo et al. Fig. 2. Optic tectum regeneration in Xenopus larvae and froglets. (a) Illustration of the brain in Xenopus. mes; mesencephalon. (b) A 3-D image of the anterior half of the mesencephalon as seen from the posterior. The right optic tectum was removed from the whole mesencephalon. (c g) Crosssections of the mesencephalon in larvae (c e) and froglets (f, g). Immediately (c, f), 4 days (d) and 30 days (e, g) after tissue removal. Scale bar, 300 µm. that tectum cells might be able to respond to injury even in froglets (Kado & Tochinai; unpubl. data, 2006). Complete stratification of the tectum during regeneration seems to require neural connection into the regenerating tectum in a manner that is comparable to the requirement of the olfactory nerve connection in telencephalon regeneration. As described above, the highly stratified structure is a distinctive feature in the optic tectum and is a good measurement of how much of the tectum has been regenerated. After the operated ventricle is closed with undifferentiated cells, laminar formation begins as a consequence of the migration of the cells from the inner layers toward the outer layers (Filoni & Gibertini 1969). There are nine layers in the Xenopus optic tectum (Lazar 1973; Lazar 1984). The outer layers nine, eight and seven are always the first ones to appear in regeneration (Filoni & Gibertini 1969), which is comparable to the sequence of formation during tectum development (Kollros 1953). If the tectum is permanently denervated during development by removing a contralateral eye, the total number of synapses decreases rapidly to about 40% of the original number, and the thickness of the superficial layers eight and nine is reduced to 80% of the control tectum (Ostberg & Norden 1979). It is likely that innervation of the optic nerves is also of importance in the regenerating tectum. When a single eye is removed concomitantly with ablation of a contralateral tectum in order to eliminate the effect of the optic nerves during tectum regeneration, a hypoplastic tectum is regenerated, suggesting that the final migration of tectal cells to the definitive position in the regenerate is dependent on the invasion of the optic nerves into the regenerating tectum (see Levine 1984). Functional assay for brain regeneration Typically, studies on amphibian tissue/organ regeneration have focused only on morphology and rarely on restoration of function. The assessment of function in tissues/organs that are moved by the organism, such as a limb or tail, is less challenging than the assessment of brain function. In the former case, it wound be obvious if regenerates are functional, if operated animals actually recovered to walk or swim using the regenerate. For brain regeneration, however, the answer is not as simple, because the brain is an information-processing organ that consists of complex neural networks. Morphological evaluation is not enough to determine if the neural networks are functionally recovered and therefore a functional assay

5 Brain regeneration in anuran amphibians 125 is required. For this reason, evaluation tests have been developed for studies on mammalian spinal cord regeneration, including the Basso-Beattie-Bresnahan (BBB) method (Basso et al. 1995). In this test, functional recovery from a spinal cord injury can be scored by observing behavioral outcomes. It is possible to assess functional regeneration of the telencephalon by examining if frogs can sense odors, because the telencephalon in frogs principally processes olfactory information (Scalia 1976). Watersoluble odorants are primarily detected by olfactory receptor neurons in the nasal cavity. Olfactory receptor neurons innervate the olfactory bulb through the olfactory nerve layer and make primary synapses onto mitral and tufted cells in the glomerular layer, where they are the only output neurons in the olfactory bulb (Wilson & Mainen 2006), and project axons onto the main olfactory cortex in the cerebrum through the lateral olfactory tract (Schwob & Price 1984). Consequently, if the anterior half of the telencephalon is removed and it is not functionally regenerated, frogs cannot recognize a smell at all. The forelimb sweeping behavior in response to food odors provides the opportunity to evaluate functional regeneration of the telencephalon. The detection of food by olfaction is thought to initiate the appetitive stage of the normal feeding behavior in Xenopus (Hutchison 1964). During this stage, the animal becomes active and begins to swim randomly, flicking its forelimbs to its snout. Once they touch a food object by the forelimbs or the snout during this stage, the animal attempts to ingest the food (Hutchison 1964). Therefore, flicking forelimbs, also referred to as a sweeping action by Avila and Frye (1977), is a behavioral response by which to evaluate olfactory function of a regenerated telencephalon. The response behavior to food odors has been used as a functional assay to demonstrate that a regenerated telencephalon that was injured at stage 53 recovered the ability to process olfactory information (Yoshino & Tochinai 2006). Trout food pellets were dissolved in distilled water, filtered and centrifuged to obtain a colorless supernatant. The supernatant, as an olfactory stimulus, was applied to intact (positive control), olfactory nerve-cut (negative control) and telencephalon-regenerated frogs (experiment) to test if it triggered a feeding response as evidenced by the sweeping action. As expected, none of the frogs with non-regenerated, severed olfactory nerves showed the sweeping action, while 100% of intact frogs showed the sweeping action within a minute. The experimental animals had the anterior half of the telencephalon removed at stage 53 and had been allowed to undergo regeneration for a period of time that would be sufficient to restore the missing structures. Among these animals, half responded to the odor stimulus within 2 min, flicking their forelimbs to the mouth. When the animals that did not show the sweeping action were dissected, it turned out that all of them had regenerated only the cerebrum but not the olfactory bulb, which resulted from the failure of olfactory reinnervation. Thus, all animals at this stage can regenerate the telencephalon and if the olfactory nerves reconnect to the brain, olfactory function is also restored. In contrast to studies of regeneration of the telencephalon, behavioral experiments have not yet been reported for regeneration of the optic tectum. There are some behavioral studies about vision in Xenopus (see Elepfandt 1996) that might be useful for evaluating functional recovery of the regenerated optic tectum. However, it must be noted that Xenopus young tadpoles have the pineal eye as a third photoreceptor organ (Roberts 1978; Jamieson & Roberts 1999; Jamieson & Roberts 2000), and pineal-dependent responses during swimming are observed up to stage 44 (Jamieson & Roberts 2000). Thus researchers need to pay attention to the role of this visual organ when designing experiments using such young tadpoles. Origin of the cells In regeneration of both the telencephalon and optic tectum in amphibians, it is presumed that the cells participating in the regenerative response originated from the periventricular zone (the ventricular zone (VZ) and subventrucular zone (SVZ) at the embryonic/larval stages and the ependymal layer (EL) and subependymal layer (SEL) in the adult. The terminology for these layers is based on The Boulder Committee 1970). The VZ is located adjacent to the ventricular lumen and differentiates perinatally into a cuboidal epithelial cell layer, the EL (Takahashi et al. 1996). The SVZ originates from the VZ embryonically and is considered to be the SEL in the adult that contains several layers of tightly packed SVZ-like cells between the EL and the mature nervous tissue (Takahashi et al. 1996; see Peretto et al. 1999). The term SVZ seems to be preferred recently to SEL even for the adult tissue. In adult mammals, it is suggested that the SEL retains multipotent neural stem cells (Doetsch et al. 1999; see also Alvarez-Buylla et al. 2002). Although ependymal cells in mammalians have also been suggested to function as neural stem cells for the SEL (Johansson et al. 1999), several other studies do not support this interpretation (Chiasson et al. 1999; Doetsch et al. 1999; see also Alvarez-Buylla

6 126 T. Endo et al. Fig. 3. Schematic illustration of regenerative differences between larval and adult brain tissues in Xenopus. (b) After brain injury, periventricular cells in the vicinity of the wound migrate and close the wound in larvae, but not in adults. (c) The rate of cell proliferation increases around the regeneration site and the regenerating tissue thickens in larvae. A temporal increase in cell proliferation is also observed in adult brains. (d) Regeneration of a functional brain is completed in about a month in larvae. Conversely, the wound site is never closed and no regeneration occurs in adults. et al. 2002). In amphibians, cells from both the EL (or VZ) and SEL (or SVZ) proliferate after CNS injury and appear to participate in CNS regeneration (Srebro 1957; Jordan 1958; Srebro 1959; Filoni & Gibertini 1969; Filoni & Margotta 1971; Minelli et al. 1987; Minelli et al. 1990; Zhang et al. 2000; Yoshino & Tochinai 2004; for a review see Kirsche 1983; Chernoff et al. 2003). The relationship between the response of periventricular cells and the ontogenetic decline of regenerative ability has been studied in Xenopus. In the case of the regeneration of the telencephalon in larvae (regeneration-competent), an early response to tissue removal is closure of the wound surface, which occurs after several days (Yoshino & Tochinai 2004) (cf. Fig. 2d). Presumably the cells that close the wound are derived from the VZ. The level of cell proliferation is high during regeneration; however, because the animal is still developing at this stage, the labeling index of an uninjured, control brain is also high. Eight days after ablation, the proliferation activity in the VZ is at its maximum, which corresponds to the stage in regeneration when the reforming brain tissues thicken. The proliferating cells are positive for the NSC marker, Musashi-1, suggesting that they are NSCs. Although postmetamorphic froglets do not regenerate the telencephalon, the frequency of proliferating cells is increased in response to injury. However, wound closure does not occur in froglets (Fig. 1g) (Yoshino & Tochinai 2004) and thus it is obvious that the proliferating cells do not participate in regeneration. Wound closure seems to be critical for successful regeneration because it is one of the earliest events that are followed by a sequence of regeneration processes. We consider that the failure of wound closure is one of the causal events that make froglets unable to regenerate the telencephalon (Fig. 3). It may also be the case that regenerative failure of the optic tectum is a consequence of a failure to close wounds after metamorphosis (Fig. 2g). In young regenerative tadpoles, high mitotic activity is observed particularly in the VZ of the non-operated optic lobe and the area of transition between the removed optic lobe and the ipsilateral semicircular torus. It is the cells from these two areas that are presumed to migrate to cover the cut surface. In later stages, as regenerative ability declines, the presumptive source of cells for regeneration becomes confined eventually to the non-operated optic lobe (Filoni & Gibertini 1969). There is a temporal increase in cell proliferation in froglets in response to tectotomy; however, BrdU is incorporated only in ependymal cells in the vicinity of the wound edge. In contrast, during regeneration at larval stages, BrdU-positive cells are widely distributed, including within the SVZ of the intact side of the mesencephaolon (Kado & Tochinai; unpubl. data, 2006). This difference might be potentially of interest, as we describe below.

7 Brain regeneration in anuran amphibians 127 Cell proliferation in response to the ablation of brain tissues might be mediated by a diffusible factor(s). The increase in the cell proliferation rate is observed not only in the vicinity of the removed portion but also in other parts of the brain (Del Grande et al. 1990; Minelli et al. 1990; Franceschini et al. 1992; Filoni et al. 1995). These findings are suggestive of the existence of a diffusible factor(s) that is released into the cerebrospinal fluid and/or blood where it is mitogenic for its target tissues (Yoshino & Tochinai 2004). It has been demonstrated that the fluid secreted into the wounded brain cavity in rats has neurotrophic activity (Nieto-Sampedro et al. 1982). In mammals, there are cytokines that are upregulated around the wounded area following brain injury, such as FGF2 and transforming growth factor (TGF)-β (see Ghirnikar et al. 1998). Although we have limited information on the molecular mechanism of CNS regeneration in amphibians, it has been shown that FGF2 induces proliferation of neural progenitor cells in urodele tail regeneration (Zhang et al. 2000) and that epidermal growth factor (EGF) enhances cell migration and proliferation from ependymal explants in vitro (O Hara & Chernoff 1994). Therefore these factors might be released into the ventricles and induce NSCs to proliferate during brain regeneration in amphibians. Attempts to induce brain regeneration Because anuran amphibians progressively lose regenerative abilities in various tissues and organs at later developmental stages, researchers have attempted to rescue the regenerative ability in older tadpoles or metamorphosed frogs. As regenerative decline appears to be intrinsic to the cells that participate in regeneration (Sessions & Bryant 1988), it is likely that regenerative abilities could be restored by treatments that cause these cells to revert to a more pluripotent state, or by grafting tissue or cells from young regenerative animals into adult non-regenerative hosts. Srebro (1965) tried to implant the brains from Xenopus tadpoles at various stages in place of the removed telencephalon; however, in most cases the implants did not persist at the host site, suggesting that they were rejected because of the immune response. Yoshino and Tochinai (2004) used the Xenopus J strain (JJ) in their experiments to eliminate immunological rejection. No immunological rejection occurs in JJ against JB (hybrids between J females and X. borealis males) and they can be distinguished from each other by differential quinacrin staining (Thiebaud 1983; Koibuchi & Tochinai 1999). In this experiment, tissue removed from regions of the telencephalon was taken out from larvae or froglets, dissociated into single cells and grafted as a dense cell suspension into the cavity that had been made in froglets after partial brain removal. Surprisingly, grafted cells closed the wound surface of the telencephalon and formed a brain-like structure. Although it was deformed, it connected with the olfactory nerves and roughly separated into three layers, including an EL lined smoothly from the host ependymal cells. These results suggest that even froglet brain cells, that might be NSCs, have the ability to not only proliferate, but can participate in the regeneration of well-patterned brain structures. Although a functional analysis has yet to be performed for brain-reconstituted animals, these results provide a clue as to the relationship between the decline of the regenerative ability and metamorphosis. In contrast to the classic and simple idea that the number of undifferentiated and regeneration-competent cells in the brain decreases during metamorphosis, it appears that regeneration-competent cells persist in the adult brain. Our current view is that regenerationcompetent brain cells exist in both the larval and adult brains, but that with progressive development and metamorphosis, environmental conditions eventually make the cells unable to initiate brain regeneration. Filoni et al. (1979) examined the effect of inhibition of metamorphosis by 4 (6)-Propyl-2-thiouracil (PTU) treatment on regeneration of the optic tectum and suggested the main cause of the decreasing regenerative ability is the progressive differentiation of the optic tectum by the time of surgery. While a certain concentration of thyroid hormone (TH) treatment on early stage Xenopus larvae can increase the mitotic activity in the regenerating optic lobe, it also accelerates histological differentiation (Filoni et al. 1974). During TH-induced metamorphosis, the brain cells differentiate and constitute complex structures together with the extracellular matrix (ECM). Regeneration-competent cells in late-stage tadpoles and adults might be unable to participate in brain regeneration in this mature environment. Conclusions Given the information about the difference between Xenopus larvae and adults during brain regeneration, we consider it noteworthy that the ability of ependymal cells to become mobilized in response to injury is correlated with regenerative ability (summarized in Fig. 3). Because there are proliferative and NSC marker-positive cells in the EL of the adult brain, immobility of regeneration-competent cells, rather than the lack of such cells, might cause failure of

8 128 T. Endo et al. the wound surface to close. Compared with the thick periventricular zone in larvae, the adult periventricular zone is very thin. This morphological difference is a consequence of the differentiation of progenitor cells into nerve cells to form the neural network in the adult brain, leaving a small population of undifferentiated cells localized only in the EL. Because of this complex neural network, regeneration-competent cells in the EL might be physically constrained to this region and thus unable to migrate to the wound surface. In urodele amphibians, ependymal cells produce matrix metalloproteinases (MMPs) during spinal cord regeneration (Chernoff et al. 2000) that would liberate cells from the matrix and thus allow the ependymal cells to migrate. It will be important to examine MMP activity during brain regeneration in Xenopus larvae and adults and to test if regeneration can be initiated by application of MMP into the non-regenerative adult brain in Xenopus. Acknowledgements We are grateful to Dr David Gardiner for his critical reading of the manuscript and for Dr Mitsumasa Okamoto s helpful information. We also thank the members of the Tochinai laboratory for their helpful advice and discussions. Tetsuya Endo and Shin Tochinai were supported by the 21st Century Center of Excellence (COE) Program on Neo-Science of Natural History (Program Leader Hisatake Okada) at Hokkaido University, financed by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. Tetsuya Endo was also supported by a Grant-in-Aid for Young Scientists (B) from MEXT. References Alvarez-Buylla, A., Seri, B. & Doetsch, F Identification of neural stem cells in the adult vertebrate brain. Brain Res. Bull. 57, Avila, V. L. & Frye, P. G Feeding behavior in the African clawed frog Xenopus laevis (Daudin). Herpetologica 33, Basso, D. M., Beattie, M. S. & Bresnahan, J. C A sensitive and reliable locomotor rating scale for open field testing in rats. J. 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