DEVELOPMENTAL DYNAMICS 226:295307, 2003

REVIEWS

A PEER REVIEWED FORUM

Urodele Spinal Cord Regeneration and Related Processes

Ellen A.G. Chernoff,1* David L. Stocum,1 Holly L.D. Nye,2 and Jo Ann Cameron2
Urodele amphibians, newts and salamanders, can regenerate lesioned spinal cord at any stage of the life cycle and are the only tetrapod vertebrates that regenerate spinal cord completely as adults. The ependymal cells play a key role in this process in both gap replacement and caudal regeneration. The ependymal response helps to produce a different response to neural injury compared with mammalian neural injury. The regenerating urodele cord produces new neurons as well as supporting axonal regrowth. It is not yet clear to what extent urodele spinal cord regeneration recapitulates embryonic anteroposterior and dorsoventral patterning gene expression to achieve functional reconstruction. The source of axial patterning signals in regeneration would be substantially different from those in developing tissue, perhaps with signals propagated from the stump tissue. Examination of the effects of broblast growth factor and epidermal growth factor on ependymal cells in vivo and in vitro suggest a connection with neural stem cell behavior as described in developing and mature mammalian central nervous system. This review coordinates the urodele regeneration literature with axial patterning, stem cell, and neural injury literature from other systems to describe our current understanding and assess the gaps in our knowledge about urodele spinal cord regeneration. Developmental Dynamics 226:295307, 2003. 2003 Wiley-Liss, Inc. Key words: axolotl; Ambystoma mexicanum; urodele; spinal cord development; regeneration; spinal cord regeneration; spinal cord; neurulation; ependymal cells; ependymoglia; radial glia; axial pattern formation; newt; salamander Received 21 October 2002; Accepted 5 November 2002

INTRODUCTION
Anuran amphibians, reptiles, birds, and mammals can only regenerate spinal cord as larvae or embryos. The anuran amphibians and higher vertebrates can regenerate limited regions of the spinal cord spontaneously or can regenerate after specic types of lesions (reviewed in Chernoff et al., 2002). Urodele amphibian spinal cord regeneration is a unique experimental system in that urodeles are the only tetrapod vertebrates that are strong regenera-

tors in adulthood. Retention of embryonic character is cited as a property that supports this regeneration process, but it is not clear to what extent embryonic processes involved in central nervous system (CNS) development must be retained or re-expressed. Regenerating urodele spinal cord clearly does not recapitulate the early events of neurulation. However, the example of limb regeneration suggests that some embryonic patterning and differentiative processes would be re-

quired to reconstruct spinal cord structure and restore function completely (Stocum, 1996; Brockes, 1997; Chistensen et al., 2002). Caudal regeneration studies have shown that urodele ependymal cells produce neuronal cells (Egar and Singer, 1972; Nordlander and Singer, 1978; Arsanto et al., 1992; Benraiss et al., 1996; Zhang et al., 2000). The modulation of urodele ependymal cell proliferation and differentiation by broblast growth factor-2 (FGF-2) and epidermal growth factor (EGF)

1 Indiana University-Purdue University Indianapolis, Department of Biology, and Indiana University Center for Regenerative Biology and Medicine, Indianapolis, Indiana 2 University of Illinois Department of Cell and Structural Biology and College of Medicine, B107 Chemical and Life Sciences Laboratory, Urbana, Illinois Grant sponsor: NSF; Grant number: PFI EHR-0093092; Grant sponsor: Indiana 21st Century Research and Technology Fund; Grant number: 031500-0064. *Correspondence to: Ellen Chernoff, Indiana University-Purdue University Indianapolis, Department of Biology, and Indiana University Center for Regenerative Biology and Medicine, 723 W. Michigan St., Indianapolis, IN 46202. E-mail: echernof@iupui.edu

DOI 10.1002/dvdy.10240

2003 Wiley-Liss, Inc.

296 CHERNOFF ET AL.

demonstrates properties reminiscent of mammalian neural stem cells (OHara and Chernoff, 1994; Zhang et al., 2000; Temple, 2001). It is possible that, instead of retention of embryonic properties per se, regeneration capacity reects expression of neural stem cell properties in specialized cell populations. The higher vertebrate neural injury literature has dened inhibitory pathways and cellular responses (Fawcett and Asher, 1999; Kwon and Tetzlaff, 2001; McDonald and Sadowsky, 2002). These processes include excitotoxicity due to excessive calcium transport and mobilization, glial scars formed by astrocytes or meningeal cells, and toxic myelin breakdown products. The corresponding processes and cell behavior in urodele systems will be discussed and compared. In this review, we will integrate the information available from several urodele species and several areas of research to provide a picture of the literature that forms the background for current urodele spinal cord regeneration research.

URODELE INJURY MODELS

Several parameters must be considered when using a urodele spinal cord injury and regeneration model system. Choices must be made regarding phase of life cycle, neotenic or metamorphosing species, region of the spinal cord lesioned, and type of lesion. The spinal cord regenerates in embryonic, larval, juvenile, and adult urodeles. The regeneration that occurs in embryonic or larval animals involves the formation of substantial numbers of new neurons and is qualitatively different from adult regeneration (Detwiler, 1947; Holtzer, 1951, 1952; Butler and Ward, 1965, 1967; Stensaas, 1983; Davis et al., 1989). The time course of regeneration is also more rapid in larval and young juvenile urodeles than in adults (Piatt, 1955). Different aspects of spinal cord regeneration can be examined at different points in the life cycle, emphasizing neurogenesis or axonal regrowth. The choice between metamorphosing and neotenic animals can be a compromise among factors

such as ease of husbandry, animal availability, and biological differences. It has been argued that the persistent regenerative capacity of the adult axolotl (Ambystoma mexicanum) derives from its neotenic state and continuous growth, leading to retention of embryonic/immature tissue properties (Holder and Clarke, 1988; Holder et al., 1991). Neoteny does not necessarily decrease the desirability of a spinal cord model system because metamorphosing amphibians undergo extensive neural changes that affect spinal cord regeneration and complicate studies of properties permissive to neural regeneration (Shi, 2000). Adult neotenic urodeles produce a strong regeneration response from a predominantly stable, fully differentiated, fully functional spinal cord. The use of axolotl specifically has the advantage of using a captive-bred, purpose-bred animal, relatively extensive spinal cord regeneration literature, and an emerging molecular biology database (Indiana University Axolotl Colony: http://www. indiana.edu/ axolotl/; S. Randal Voss, Salamander Genome Project: http://www.lamar.colostate.edu/ srvoss/SGP/ (Note: Web site will be moving to the University of Kentucky)). The progress of regeneration is easiest to follow in complete transections. In transection models the relationship of the regenerating axons with ependymal cell processes and the pia mater is very clear (Egar and Singer, 1981). In suction wounds, the meninges and pia mater can be spared, resulting in axonal regrowth along the inner surface of the pia (Stensaas, 1983). Although widely used in mammalian injury models, crush injuries are not widely used in urodele regeneration studies (Steward et al., 1999). Crushed cord undergoes a more extensive period of cell death, debris removal, and tissue remodeling than cleanly transected cord (personal observation). Recovery of locomotion has been demonstrated after transection of brachial, thoracic, lumbar, and tail spinal cord (Steffaneli, 1950; Holtzer, 1951, 1952; Piatt, 1955; Butler and Ward, 1967; Nordlander and Singer, 1978; Stensaas, 1983; Clarke et al.,

1988; Davis et al., 1989; OHara et al., 1992). Most of these studies use axonal regrowth as the measure of regeneration. Some axonal regrowth occurs in axolotls as early as 2 weeks after transection (reviewed in Stensaas, 1983). In a systematic study of the restoration of normal swimming behavior after thoracic transection lesions in adult Eastern Spotted Newts (Notaphthalmus viridescens), the earliest recovery of coordinated swimming was 4 weeks after lesioning, with 50% of the animals showing swimming with decits at 6 weeks and fully coordinated swimming at 8 weeks (Davis et al., 1990). Retrograde labeling studies show a strict correlation of coordinated swimming with regrowth of the descending supraspinal axons to the level of the lumbar enlargement (Davis et al., 1990). Experiments following regeneration for periods of 6 months or less routinely report that the spinal cord does not return to its original thickness and that some urodele spinal cord tracts do not regenerate (Piatt, 1955; Stensaas, 1983; Holder et al., 1989). However, the time required for complete replacement of cord structures can be very long, e.g., 23 months in adult axolotls (Clarke et al., 1988). Variation in injury type and other experimental parameters can make it difcult to compare the results of different laboratories. For example, experiments that describe failure of regeneration in adult urodeles may have produced gross lateral displacement of the cord due to severing of the vertebral column. This type of disruption prevents regenerating ends of the cord from meeting (Butler and Ward, 1967). The use of any one injury type may be limited to a single laboratory and performed at a time when no biochemical or molecular analysis was possible. This nding makes direct comparison of current experiments with some of the older literature difcult. Instead of cataloguing differences and aberrations in the literature, we will concentrate on processes and mechanisms that are signicant in the context of current knowledge.

URODELE SPINAL CORD REGENERATION 297

EMBRYONIC PROPERTIES OF THE URODELE SPINAL CORD

Urodele neural development can be divided into three steps: neuralization, the induction of the neural plate; neurulation, the formation and closure of the neural tube; and neural differentiation, the formation of neurons and glia (reviewed in Duprat, 1996). Not all of these processes are relevant to urodele spinal cord regeneration: the neural plate is not reconstructed, and the regenerated spinal cord does not develop by the folding of a neural plate, but there are mechanisms involved in developmental patterning and differentiation that may be relevant in regeneration competence and tissue reconstruction. Is there any recapitulation of neurulation events in urodele spinal cord regeneration? Traditionally neural tube formation in amphibian embryos has been described as a primary neurulation process (folding, not cavitation), and this is true for urodeles (Jacobson, 1981). In some anurans, a process of fusion and relumination occurs (Davidson and Keller, 1999). In Xenopus, prospective dorsal neural tube cells undergo medial cell movement and intercalation that integrates deep and supercial cells to form the roof of the neural tube. These rearrangements are followed by relumination of the neural tube, a process that shares some features with secondary neurulation processes in amniotes (Davidson and Keller, 1999). A relumination-like process could be part of regeneration in urodele cord. The reconstructed spinal cord innervates muscles and organs along the anteroposterior (A-P) axis. Ascending and descending axons must be targeted appropriately along the dorsoventral (D-V) axis, and the various types of spinal neurons must connect with each other. This nding suggests that some aspects of embryonic A-P and D-V patterning might be re-expressed. The aspects of spinal cord development that may help in understanding regenerative mechanisms are discussed here in conjunction with the corresponding regenerative process.

What are the residual embryonic qualities of the adult axolotl spinal cord that could facilitate regeneration? Postembryonic neurogenesis and the persistence of radial ependymal or radial glial bers are two often-cited properties. Labeling studies demonstrate that new neurons are produced fairly frequently in intact axolotls smaller than 7.5 cm (juveniles 6 7 months of age; Holder et al., 1991). In larger/older intact animals, new spinal cord neurons are formed infrequently, reecting the stability of the mature CNS tissue (Holder et al., 1991). The axolotl ependymal cell spans the spinal cord from lumen to basal lamina, as do the embryonic radial glia. The radial bers of the ependymal cells contain glial brillary acidic protein (GFAP) in the outer (basal) segment, a region that starts in the outer gray matter and spans the white matter, whereas the cytoplasm of the cell body contains intermediate laments composed of cytokeratins (Holder et al., 1990). However, the presence of transepithelial radial ependymal (radial glial) processes also occurs in the spinal cord of adult frogs, animals that do not regenerate their spinal cords; and frog ependymal cell processes also express GFAP in the outer segment of the process (Miller and Liuzzi, 1986). There is probably no correlation between intermediate lament content of the ependyma and regeneration capacity, as phylogenetic studies have shown no systematic differences that would correlate with success or failure of regeneration (Bodega et al., 1994; Clarke and Ferretti, 1998). So, transepithelial extension and regionalization of intermediate lament proteins are not sufcient indicators of spinal cord regeneration capacity.

SPINAL CORD STRUCTURE

The mature urodele spinal cord consists of an ependymal zone lining the central canal, gray matter, and white matter. The boundary between white and gray matter is not as distinct as in mammals. Within the gray matter, the ventral horns can be distinguished, but a dorsal horn region is not well-developed. As in all

amphibians, the ependymal cells maintain radial bers (ependymal processes) with pial endfeet that terminate on, and produce, the glia limitans (basal extracellular matrix surrounding the cord; Roots, 1986; Miller and Liuzzi, 1986; Holder et al., 1989). Thick layers of brillar collagen (piaarachnoid matrix) produced by the leptomeningeal cells (neural crest origin) surround the basal lamina. Numerous blood vessels terminate on the surface of the spinal cord. The ependymal processes and endfeet form the blood/spinal cord barrier. In pigmented animals, melanocytes are found on the meningeal collagen. The developing urodele spinal cord contains nine groups of spinal cord neurons: (1) dorsal RohanBeard primary sensory neurons, (2) giant dorsolateral commissural interneurons, (3) multipolar dorsolateral commissural interneurons, (4) dorsolateral ascending interneurons, (5) multipolar dorsolateral ascending interneurons, (6) unipolar mid-cord commissural neurons with glycinelike immunoreactivity, (7) bipolar or multipolar descending interneurons, (8) ventrolateral motor neurons, and (9) -aminobutyric acidlike KolmerAgduhr cerebrospinal uid-contacting neurons (Harper and Roberts, 1993; Roberts, 2000). These nine groups of neurons are identiable in embryos of the smooth newt, Triturus vulgaris, at a stage that can swim when removed from their egg membranes (stages 32 48, Harper and Roberts, 1993; Gallien and Bidaud, 1959). The giant dorsolateral commissural neurons are also present in axolotl embryos from stage 39 on and in other Ambystomatid salamanders (Bordzilovskaya et al., 1989; Harper and Roberts, 1993; Roberts, 2000). A signicant gap in urodele cord regeneration studies is analysis of the fate of each of these types of neurons.

SPINAL CORD REMODELING AFTER INJURY

Regeneration of the urodele spinal cord has been examined in tail amputation and lesions of the cervical, thoracic, and lumbar regions, although not, unfortunately, all in the

298 CHERNOFF ET AL.

Fig. 1. Spinal cord regeneration: gap replacement. A: Cartoon of gap replacement in the urodele spinal cord. (1) A complete transection results in retraction of the stumps. (2) Mesenchymal ependymal outgrowth from cranial and caudal stumps lls the lesion site. (3) The ependymal cells reepithelialize, and axons regrow through ependymal cell processes. B: Early in the gap replacement process in adult axolotl spinal cord, 2 weeks of regeneration, ependymal cells grow out as a solid mesenchymal mass. Shown in cross-section. C: Cranial to the lesion site, the ependymal cells remain in epithelial form. Mitotic cells are prominent. Voids are visible in the damaged tissue. D: At a later stage in the regeneration process, 4 weeks after lesioning, white and gray matter are reconstituted. Small voids are still present in the tissue. Images taken from material supplied by M.W. Egar.

same species (reviewed in Piatt, 1955; Davis et al., 1989). The most signicant differences are those between regeneration of the spinal cord in amputated tail (caudal regeneration) and in spinal cord transected more cranially (gap replacement). The type of tissue remodeling that occurs and the inductive effects of the spinal cord on surrounding tissue differ (Piatt, 1955; Egar and Singer, 1972; Stensaas, 1983). Figure 1 shows a diagrammatic representation of the gap replacement process and representative sections through regenerating adult axolotl cord. Tail regeneration is much more

complicated than body spinal cord regeneration in the sense that complex non-neural structures must also be replaced. Contact between the regenerating spinal cord and the tail stump wound epithelium results in induction of an epimorphic regeneration process similar to limb regeneration (Godlewski, 1928; Efrimov, 1951; Polazhaev, 1972). The wound epithelium is not necessary to cord regeneration of the tail spinal cord but plays a role in the regeneration of muscle and connective tissue. Tail regeneration is a bit like regenerating a limb with a spinal column in it instead of long bones and autopodia. The response of each of the

principal spinal cord cell types in the regeneration process will be considered.

Ependymoglia
One feature common to the animals that regenerate their spinal cord (sh, urodele amphibians, young anuran tadpoles, lizard tails, fetal birds, and mammals) is an ependymal response (described below) after lesioning (reviewed in Chernoff, 1996; Clarke and Ferretti, 1998). The mechanism that initiates this response is unknown, and many of the contributions of these ependymal

URODELE SPINAL CORD REGENERATION 299

cells to the regeneration process are still under investigation. The tissue reorganization involved in the ependymal response has been described in detail. During caudal regrowth in the regenerating tail, ependymal cells form a hollow terminal vesicle continuous with the central canal of the stump cord. The ependymal outgrowth occurs in the form of a tubular extension that does not lose its apical/basal polarity. The epithelial intercellular junctional complexes and basal lamina are maintained (Egar and Singer, 1972, 1981; Clarke and Ferretti, 1998). Cell proliferation is associated with the ependymal response (see section on Mitotic Capacity In Vivo). An ependymal tube extends as the cells continue to proliferate. The remodeling of lesioned body spinal cord (above the cauda equina) also starts with an ependymal response. After nontail spinal cord is transected, the ependymal cells rearrange to seal the cut ends of the spinal cord, forming an ependymal bulb. The ependymal cells migrate into the lesion site from both cranial and caudal stumps during the process of gap replacement (Butler and Ward, 1967; Singer et al., 1979; Stensaas, 1983). Early in the regeneration process, the cell population within the lesion site consists of ependymal cells and inltrating macrophages. Cells within the ependymal outgrowth proliferate, migrate, and remove existing extracellular matrix material and debris from dead cells (Simpson, 1968; Egar and Singer, 1972; Singer et al., 1979; Stensaas, 1983; Anderson et al., 1986; Chernoff et al., 1990, 2000). In gap replacement, ependymal outgrowth involves reorganization of the epithelial ependymal cells into a mesenchyme with characteristic changes in cell cell adhesive junctions, intermediate laments, and extracellular matrix components (Butler and Ward, 1967; Egar and Singer, 1972; OHara et al., 1992). This outgrowth is in the form of a solid mass lacking apical/basal polarity. In adult axolotls ( 13 cm), there is extensive outgrowth from cranial and caudal stumps at 2 weeks after lesioning. The mesenchymal ependymal outgrowths meet and join at

approximately 3 4 weeks to form an ependymal bridge. The ependymal bridge reorganizes from a solid cord of cells into an epithelial tube at 4 5 weeks (OHara et al., 1992; Fig. 1). There are differences in individuals based in part on the extent to which the spinal cord retracts when cut, and the amount of trauma to the cut ends. Neuropil and newly myelinated axons can be seen in the ependymal bridges of some animals as early as 4 weeks of regeneration (OHara et al., 1992). The axons and dendrites grow in the spaces between ependymal endfeet or between ependymal processes in close contact with the basal lamina (Egar and Singer, 1981). Studies that rigorously examine restoration of coordinated movement are relatively rare. There are two reasons for this. Either lesions are made caudally to avoid paralyzing the bladder and thus minimizing motor decits or axonal regrowth is monitored in lieu of scoring coordinated movement. The characterization of the disorganization of ependymal cells in gap replacement/body cord as an epithelial/mesenchymal transformation is based on characteristics found in other epithelialmesenchymal transformations in vitro and in embryonic development (Hay and Zuk, 1995). E-cadherin is lost, matrix metalloproteinases are secreted, and basal lamina is removed (OHara et al., 1992; Chernoff et al., 2000; Chernoff, unpublished observations). The apical intercellular junctional complex is removed with concomitant loss of the subapical microlament bands and loss of desmosome-associated tonolaments (OHara et al., 1992; Chernoff et al., 1998).

et al., 1990; OHara et al., 1992). During mesenchymal outgrowth in the earlier stages of gap replacement regeneration, the ependymal cells lose their GFAP and epithelial cytokeratins and produce vimentin (OHara et al., 1992). This change in intermediate lament content with tissue reorganization is seen in culture as well as in vivo (OHara et al., 1992).

Extracellular matrix and adhesion molecules.

Ependymal cells in intact axolotl spinal cord produce laminin. During mesenchymal outgrowth, the laminin is lost and bronectin is produced (OHara et al., 1992). In vitro, a bronectin-coated substratum and the presence of EGF in the culture medium maintain injury-reactive ependymal cells as mesenchyme (Chernoff et al., 1998). Tenascin is observed around ependymal cells in urodele spinal cord and in the axonal tracts in developing animals and may play a role in channeling axonal regeneration. In the intact adult, the tenascin level remains high in the axonal tracts but is low in the ependymal cells. The tenascin distribution in regenerating animals is like that in developing animals (Caubit et al., 1994). The neural cell adhesion molecule (N-CAM) shows an interesting distribution in the urodele spinal cord. During development, the polysialylated form of N-CAM (PSA-N-CAM) is present around the ependymal cells and axonal tracts. N-CAM is also present in the adult, but PSA-N-CAM is only detected in the ependymal zone. PSAN-CAM is up-regulated in the regenerating spinal cord (Caubit et al., 1993).

Intermediate lament content.

The intermediate lament composition in ependymal cells of intact adult or juvenile axolotl spinal cord shows the regional localization characteristic of all amphibian ependymoglia. The outer (basal) segment of the ependymal process contains GFAP. The cytoplasm of the juxtaluminal cell body contains epithelial cytokeratins 8 and 18, simple epithelial cytokeratins, and also reacts with antibodies to tonolaments (Holder

Matrix-degrading enzymes.
During spinal cord regeneration in the axolotl, matrix remodeling includes removal of the basal lamina (glia limitans) early in ependymal outgrowth, and remodeling of a substantial amount of brillar collagen in the meninges. Matrix remodeling involves the action of matrixdegrading enzymes such as the matrix metalloproteinases (MMPs). MMPs are characterized by a zinc-

300 CHERNOFF ET AL.

binding region required for protease activity and calcium-binding required for substrate binding (Birkedal-Hansen et al., 1993; Nagase and Woessner, 1999). They are found in zymogen (granule storage) form within cells and are proteolytically processed and secreted. MMPs and related enzymes are active in a variety of normal and abnormal processes in the nervous system (Yong et al., 2001). Intact adult axolotl spinal cord does not produce levels of MMPs detectable by zymography (Chernoff et al., 2000). Injury-reactive ependymal cells, however, contribute to the remodeling of lesioned urodele spinal cord through MMP production. Ependymal outgrowth in vivo and mesenchymal ependymal cells in vitro produce MMP-1 (interstitial collagenase), MMP-2, and MMP-9 (gelatinase a and b). The amount of MMP activity is high at 2 and 4 weeks after lesioning and declines by 8 weeks of regeneration. The endogenous MMP inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), is detected in the intact spinal cord but is absent during mesenchymal ependymal outgrowth (when cells have deepithelialized and are dividing and migrating; Chernoff et al., 2000). The action of other matrix-degrading enzymes has not been analyzed in the urodele spinal cord.

modeoxyuridine (BrdU) labeling, Benraiss et al. (1999) reported a high level of mitosis in ependymal cells inside of and rostral to the terminal bulb. Within the regenerating ependymal tube, there does not appear to be a dened growth zone; mitotic gures are distributed along the tubes length, and new neurons differentiate (Clarke and Ferretti, 1998; Benraiss et al., 1999). Directly comparable studies in other urodele species and in nontail spinal cord regeneration have not been published.

are ependymal cells that have retracted their endfeet from the basal surface. Although the status of urodele astrocytes may be ambiguous, it is clear that the lesioned urodele cord does not produce astrocytic scars formed by GFAP-positive cells, or broblastic scars formed by invading meningeal cells that inhibit axonal regrowth in mammals (Fawcett and Asher, 1999; Steward et al., 1999).

Oligodendrocytes
In anuran amphibians, as in other vertebrates, the notochord is required to induce formation of oligodendrocytes in the ventral spinal cord during dorsoventral patterning of the spinal cord (reviewed in Orentas and Miller, 1996; Maier and Miller, 1997). The alteration of white matter that results from early surgical ablation of the notochord in salamanders suggests that urodele oligodendrocyte formation is similar (Triturus, formerly Triton, Lehmann, 1928; axolotl, Kitchin, 1949). Oligodendrocytes have been described in the periependymal stratum of the mature urodele spinal cord and also more basally (P. waltl, Zamora, 1978). Oligodendrocytes can affect the regeneration process positively by remyelinating new axons or negatively through the production of toxic or inhibitory myelin products after injury. In mammals, myelin components such as myelin-associated glycoprotein (MAG) and Nogo (NI35/250) are known to inhibit regeneration after injury (reviewed in Ng and Tang, 2002). Other CNS components known to inhibit axonal outgrowth in mammals include chondroitin sulfate proteoglycan and tenascin-R (Becker et al., 1999; Ng and Tang, 2002). Nogo (NI35/ 250) has not been found in regeneration-competent lower vertebrate, such as sh and larval Xenopus, but the other components are present (Wanner et al., 1995; Lang et al., 1995; Becker et al., 1999). It has been suggested that they are rapidly removed after injury. In anuran amphibian (Xenopus laevis), spinal cord myelin becomes nonpermissive to axonal outgrowth with metamorphosis and reacts with IN-1, the

Astrocytes
The identication of GFAP as a brous astrocyte marker in vertebrate produces some confusion in interpretation of glial differentiation in amphibians (Bignami et al., 1972; Holder et al., 1990; Soula et al., 1990; Cochard et al., 1995). In the developing amphibian spinal cord, the radial glia and, later, the ependymal cells span the neural epithelium. As described earlier, these cells contain GFAP in the basal (outer) portion of the cell processes, with cytokeratin comprising the intermediate laments within the cell body (Miller and Liuzzi, 1986; Holder et al., 1990). The radial glia/ependymoglia in amphibians have been identied as astrocytes in several studies (e.g., Cochard et al., 1995). Astrocyte differentiation has been described as precocious in amphibians because of the presence of GFAP in radial glia (Pleurodeles waltl: Soula et al., 1990; Xenopus: Szaro and Gainer, 1988). There is disagreement on the existence of true astrocytes in adult amphibian CNS. Some laboratories have identied cells, present in low numbers, that express only GFAP in their intermediate laments, but it is clear that most of the GFAP in the intact axolotl spinal cord is in the outer segments of radial ependymal bers (Zamora and Mutin, 1988; Holder et al., 1990; OHara et al., 1992). Electron microscopic examination has suggested that there are astrocytes that lack radial processes (Schonbach, 1969; Egar and Singer, 1972). It remains to be determined whether these cells are truly equivalent to brous astrocytes, or

Mitotic capacity in vivo.

In tail cord regeneration, it is generally agreed that ependymal cells possess a high level of mitosis. Based on distribution of mitotic gures, the ependymal cells are highly proliferative just cranial to the amputation site, more so than in the ependymal bulb (Egar and Singer, 1972; Nordlander and Singer, 1978). Proliferating cell nuclear antigen (PCNA) labeling in Pleurodeles waltl (the Spanish ribbed newt) at early postamputation stages shows that most of the ependyma cells near the amputation plane are labeled. Lower numbers are labeled within the terminal vesicle (Zhang et al., 2000). Later, new neurons are detected by using a beta-3 tubulin probe, and ependymal cells are still proliferating at this time. Few PCNA-labeled cells exist in intact P. waltl spinal cord (Zhang et al., 2000). By using bro-

URODELE SPINAL CORD REGENERATION 301

Nogo-neutralizing antibody (Lang et al., 1995). The recent literature on urodele oligodendroglia is notably sparse. In salamander optic nerve, inhibitory components such as MAG and tenascin-R are present but are rapidly removed after injury, providing an environment more supportive to axonal outgrowth (Pleurodeles waltl; Becker et al., 1999). Specic information on urodele spinal cord myelin is absent.

mitotic as their ependymal parents (Benraiss et al., 1999). When BrdUlabeled regenerating cord cells were cultured, they gave rise to labeled neurons, showing directly that new neurogenesis was indeed occurring.

Axonal regrowth.
Studies of urodele spinal cord regeneration have reported a range in the extent of functional recovery. The number of axons that regenerate, the number of functional synapses that occur, and the extent to which coordinated locomotion is recovered varies (Davis et al., 1989, 1990). In the newt Notophthalmus viridescens (then Triturus the Eastern redspotted newt), retrograde labeling shows that all of the regions of the brainstem that project to the lumbar spinal cord can regenerate (Davis et al., 1989). Regeneration can occur across a gap of 10 mm. It is widely observed that the regenerated urodele spinal cord is thinner than the intact cord, there are fewer axons, and not all of the connections made by regenerating neurons are correct after regeneration over a period of weeks or a few months (Stensaas, 1983; Davis et al., 1989). Long-term regeneration studies in the axolotl (up to 23 months) show that with enough time, the number of neurons in the nuclei of the medulla, midbrain, and diencephalon with axons that reach regenerated lumbar spinal cord attains control levels (Clarke et al., 1988).

Neurons
In urodele tail regeneration, morphologic studies and markers of neuronal differentiation show that new peripheral nervous system (PNS) and CNS neurons are produced from the ependymal tube (Notophthalmus viridescens and Pleurodeles waltl; Egar and Singer, 1972; Nordlander and Singer, 1978; Arsanto et al., 1992; Benraiss et al., 1999; Zhang et al., 2000). Neural crest cells are no longer present, so the regenerated peripheral nervous system is derived from either the ventrolateral part of the regenerated ependymal tube or dorsal aspect of the terminal vesicle (Arsanto et al., 1992; Nicolas et al., 1996; Benraiss et al., 1996). Labeled, tail regenerate-derived ependymal cells form cell types normally of neural crest origin in culture as well: melanocytes and Schwann cells (Benraiss et al., 1996). Pulse-chase experiments using BrdU show that new CNS neurons are produced in Pleurodeles waltl tail cord, in situ. In these studies, GFAP was used as a glial marker, and neuron-specic enolase (NSE) and neurolament antibodies were used as neuronal markers. Threeweek regenerates show the proliferative ependyma giving rise to new neurons (Benraiss et al., 1999). Double labeling of long-term labeled cells shows that NSE- and GFAP-positive cells both label with BrdU at the rostral and median levels of the regenerating cord, indicating the formation of new neurons and glia. Confocal microscopy resolved individual labeled cells, showing that both glia and neurons are generated from the ependyma. The stronger labeling of the glial and neuronal progeny showed they are not as

decrease, and intracellular calcium levels increase (Happel et al., 1981; Stokes et al., 1983). Calcium inuxes occur in association with glutamate excitotoxicity that can kill neurons and oligodendrocytes (Panter et al., 1990). Examination of calcium activity in injured rat spinal cord suggests that cells within the dorsal gray matter are strongly involved and the effects are then propagated into the white matter (Moriya et al., 1994; McDonald and Sadowsky, 2002). By using an in vitro system, it was shown that stimulation of increases in cytosolic calcium in individual astrocytes is propagated to neighboring astrocytes and to adhering neurons, providing an additional path for calcium-mediated toxic events after CNS injury (Nedergaard, 1994). This process has been shown to have consequences in vivo in mammalian CNS (Araque et al., 2001). The meningeal cells can also participate in transfer of calcium (Grafstein et al., 2000).

Calcium and the urodele CNS.

One major difference in the urodele and mammalian responses to spinal cord injury may lie in the ability of injury-reactive ependymal cells to act as a buffer, possibly through calcium uptake and sequestration that protects neurons and oligodendrocytes from the processes that would trigger secondary cell death. Although some cell death would occur, the propagation of toxic effects would be limited. This putative calcium uptake process could also serve as the trigger for the onset of the ependymal response itself through second messenger signaling pathways. This mechanism remains theoretical, the subject of future investigations using cell culture model systems. What is known about calcium and the urodele CNS is that modulation of intracellular calcium levels is important throughout amphibian neural development. Calcium inux plays a specic role in amphibian neuralization (induction of the neural plate; Moreau et al., 1994; Duprat, 1996). There are species differences among amphibian embryos: axolotl embryos exhibit autoneuralization in that their cells have much

MODULATORS OF REGENERATION Calcium

The intrinsic properties and environmental cues that trigger the onset of the ependymal response after injury have yet to be determined. The involvement of calcium early in postinjury events in mammalian cord suggests that it should be considered in initiation of the regeneration process. Excitotoxicity is among the damaging events and toxic reactions that occur in the mammalian spinal cord after serious injury (reviewed in McDonald and Sadowsky, 2002). Extracellular calcium levels

302 CHERNOFF ET AL.

higher resting internal Ca levels than P. waltl and X. laevis, and there is no incremental effect with calcium channel agonists (Duprat, 1996). During neurulation, the lifting of the neural folds is calcium-dependent. This nding was shown in studies with Ambystomatid embryos (axolotls and spotted salamander Ambystoma maculatum; Moran, 1976; Moran and Rice, 1976). The developmental role of calcium that is most relevant to regeneration may be in neuronal differentiation. There is a threshold level of calcium required for neuronal differentiation that can be achieved by different mechanisms in different species. Ca is known to be necessary for differentiation of spinal neurons in Xenopus laevis, and the Ca transients associated with this process occur spontaneously (Holliday and Spitzer, 1990). One consequence of the activation of L-type calcium channels in neuronal differentiation is the activation of immediate early gene proteins such as c-fos or Jun-B, demonstrated in tissue culture cell lines, such as PC12 cells (Sheng and Greenberg, 1990). During neurite outgrowth in cultured Xenopus neurons, calcium is required in the culture medium, but the optimum calcium concentration for axonal outgrowth is low compared with levels for neuralization (Bixby and Spitzer, 1984). In general, calcium homeostasis changes during development with progressively lower levels of intracellular calcium from neuralization to axonal outgrowth (Duprat, 1996).

tein is present in the ependymal cell (radial glial) bodies surrounding the central canal in spinal cord and hindbrain as well as in the glial processes of the ventral oor plate. Cytoplasmic retinoic acid binding protein shows a reciprocal antibody binding pattern within the axons of the spinal cord white matter (Hunter et al., 1991). Endogenous retinol and retinoic acid are detected in the spinal cord. Combined information from the axolotl studies and embryonic chick and mouse studies show that ependymal cells can take up retinol, convert it to retinoic acid, and secrete it. Neurons can then take up retinoic acid, which promotes neurite outgrowth. The retinoic acid source elicits directional axonal outgrowth in vitro, suggesting that the retinoic acidproducing ependymal cells could exert a chemoattractant effect on axons, but an in vivo effect in spinal cord regeneration has not yet been identied (Hunter et al., 1991).

keeps cells dividing and undifferentiated (Svendsen and Caldwell, 2000; Temple, 2001).

Epidermal Growth Factor

Injury-reactive adult axolotl ependymal cells show a differential response to a variety of growth factors in vitro. The most important observation is that exogenous epidermal growth factor (EGF) is essential for stimulating axolotl ependymal proliferation from background levels (10%) to high levels (50 70%) in vitro (OHara and Chernoff et al., 1994). EGF is also required to maintain the migration of the axolotl ependymal cells in culture (OHara and Chernoff, 1994). The source of EGF (or its potential substitute, transforming growth factor- ) in vivo remains to be determined. Cultured axolotl ependymal cells did not respond to exogenous FGF as did the P. waltl tail cord cell in situ (OHara and Chernoff, 1994; Zhang et al., 2000). Perhaps axolotl ependymal cells make sufcient endogenous FGF. EGF receptor expression appears during development of the rat cerebral cortex in response to stimulation by FGF-2 (Lillien and Raphael, 2000). EGF is important in the behavior of adult mammalian neural stem cells as well as in development (Weiss et al., 1996).

Fibroblast Growth Factor

In the intact urodele tail cord, FGF-2 is produced in ventrolateral neurons, not ependymal cells. FGF-2 mRNA is produced in increasingly higher levels in the blastema of the regenerating tail. In this case, production of FGF-2 has been shown to be associated with the ependymal cells. The FGF-2 levels are high as the ependymal tube is extending and being rebuilt (Zhang et al., 2000). Two-week regenerates are actively expressing FGF-2 and making new neurons. FGF2 is also expressed in the regenerating spinal sensory ganglia. Exogenous FGF-2 increases the number of proliferating cells in vivo in P. waltl (Zhang et al., 2000). In other systems, FGF-2 plays a role in regulation of neural differentiation (Temple, 2001). In adult mammalian CNS, FGF-2 is expressed by brous astrocytes and subsets of neurons. It is up-regulated after injury to adult mammalian CNS, where it and the FGF-2 receptor probably play a role in the astrocyte proliferation that generates glial scars and in oligodendrocyte proliferation (Fawcett and Asher, 1999). In neural stem cell studies in vitro, exogenous FGF-2

NEURAL STEM CELL PROPERTIES

The growth factor requirements and endogenous growth factor production of regenerating urodele cord ependymal cells t the emerging picture of vertebrate neural stem cells. During development, FGF-2 maintains the progenitor cell properties of early embryonic mammalian neural stem cells and maintains their proliferation (Temple, 2001). EGF is involved in supporting proliferation of mammalian neural stem cells derived from later embryos and adult tissue (Weiss et al., 1996; Burrows et al., 1997; Lillien and Raphael, 2000; Temple, 2001). There are regional differences in the mammalian CNS. Adult murine spinal cord stem cells respond to EGF plus FGF, whereas brain lateral ventricle stem cells proliferate in the presence of EGF alone

Retinoic Acid
To examine the effects of retinoic acid on neurite outgrowth, explants of axolotl spinal cord were grown in collagen gels with a source of alltrans RA (Hunter et al., 1991). These studies used juvenile animals between 4 and 8 cm in length, which are still growing and adding new CNS neurons (Holder et al., 1991). Neurite outgrowth occurs in the presence of RA with no other growth factors present, although the outgrowth is somewhat sparse. In vivo immunohistochemical studies show that cytoplasmic retinol binding pro-

URODELE SPINAL CORD REGENERATION 303

(Weiss et al., 1996). The stem cells that respond to FGF and/or EGF maintain pluripotency. One important marker of stem cell properties is activity of the Notch-1 signaling pathway. Notch-1 signaling maintains neural stem cell properties, but does not create them (Hitoshi et al., 2002). Musashi-1 is an RNA-binding protein rst identied in Drosophila for its involvement in the maintenance of Notch-1 signaling (Nakamura et al., 1994). MouseMusashi-1 is highly enriched in the CNS stem cells and increases Notch-1 signal activity by posttranscriptional down-regulation of Numb (Sakakibara et al., 1996; Imai et al., 2001). Downstream of Notch-1 receptor activity is activation of E(spl)C/Hes genes (enhancer of splithairy). Hes gene activity represses expression of proneural genes such as Mash and neurogenin, maintaining stem cell or progenitor cell properties (Artavanis-Tsakonas et al., 1999; Ohtsuka et al., 1999; Johansson et al., 1999). Expression of these genes is being evaluated in amphibian spinal cord. One difference between amphibian spinal cord and most mammalian neural stem cell source tissues is that the amphibian spinal cord stem cells are ventricular rather than subventricular (Momma et al., 2000; Morshead and van der Kooy, 2001; Alvarez-Buylla et al., 2002). In amphibians, spinal cord cells with stem cell properties have been identied from regeneration-competent Xenopus tadpole and adult axolotl spinal cord (Chernoff et al., 2001; unpublished results of the Chernoff Laboratory). These cells express nrp-1, the Xenopus homolog of Musashi-1 (Richter et al., 1990). Nrp-1 expression is down-regulated as regenerative capacity is lost in Xenopus and is up-regulated during adult axolotl spinal cord regeneration (unpublished results of the Chernoff Laboratory).

cord, we must look primarily to nonamphibian systems for molecular mechanisms of axial patterning of the developing cord, as there is limited information available from urodeles or other amphibians. For spinal cord regeneration to completely reproduce missing structures and produce functional axonal targeting, it may be necessary to express or re-express A-P and D-V axis cues.

A-P Patterning
Detailed studies of A-P and D-V patterning of the developing spinal cord have been performed primarily in amniote embryos (reviewed in Altmann and Brivanlou, 2001). A-P patterning of the embryonic spinal cord starts with additive expression of Hox-a and Hox-b cluster genes and progresses to establishing the identities of subgroups of spinal motoneurons through expression of genes such as engrailed (McGinnis and Krumlauf, 1992; Pfaff and Kintner, 1998). Cross-inhibitory regulation between the bHLH transcription factors neurogenin, Math1 and Mash1 helps to determine progenitor cell domains and neuronal fate in the spinal cord of higher vertebrates (Shirasaki and Pfaff, 2002). Although A-P patterning of the spinal cord occurs early in CNS development, there are later A-P cues, such as those that pattern subgroups of motor neurons (Pfaff and Kintner, 1998). The persistent expression of patterning genes in adult tissue has been seen in other urodele systems. HoxA11 and distal-less family genes are expressed in intact adult newt limb and in limb regeneration (Beauchemin and Savard, 1992; Beauchemin et al., 1994). There are similar patterning gene examples in spinal cord regeneration. In Pleurodeles waltl, two Nkx3-related genes (PwNkx3.3 and PwNkx3.2 ) exhibit graded expression in normal adult spinal cord along the A-P axis (Nicolas et al., 1999). The gradient is more pronounced for PwNkx3.3, which is stronger posteriorly. Reverse transcriptase-polymerase chain reaction of mRNA from 11 regions (anterior to posterior along the body) shows increased expression of both genes at the level of the lumbar en-

largement. These genes are up-regulated in many tissues of the regenerating tail and may be particularly important in skeletal regeneration. Wnt growth factor genes provide a second example of adult expression of pattern regulating genes. In Pleurodeles waltl, Pwnt-10a and Pwnt-7a are expressed in normal adult tissue and up-regulated during tail regeneration (Caubit et al., 1997a,b). Several of the Wnt genes have also been identied with A-P axis-associated expression during regeneration. Pwnt-5a, -5b, and -10a are expressed in an A-P gradient. No identication has been made of Wnt gene involvement in D-V axis regeneration comparable to that found in dorsal neural tube differentiation (Muroyama et al., 2002). Patterning genes are associated specically with regeneration of missing structures in tail regeneration. Dlx3 (PwDlx3), a distal-less gene, appears in the tail cord ependymal cells that re-form the dorsal root ganglia (Nicolas et al., 1996). This is a gene that is not expressed in normal adult tissue and may not be associated with the embryonic origin of dorsal root ganglia (Beauchemin and Savard, 1992; Nicolas et al., 1996).

D-V Patterning
D-V patterning of the embryonic spinal cord is also a complex process involving many different pathways and transcription factors. At the top of the patterning cascade Sonic Hedgehog (SHH), from the notochord and neural tube oor plate, is key in the ventralizing process, whereas the bone morphogenetic proteins, from the ectoderm and neural tube roof plate, are important for dorsalization (Altmann and Brivanlou, 2001; Eggenschwiler et al., 2001). Specic information on urodele patterning responses is sparse, and the details of patterning in development or regeneration may have species-specic variations. The Gli genes Gli1 and Gli2 are expressed in response to SHH in amniote embryos. One of the known targets of Gli is the forkhead family winged-helix transcription factor HNF3- , which

PATTERNING OF THE REGENERATING CORD

In amphibian limb regeneration, it is clear that the patterning process recapitulates a substantial portion of limb development. In the spinal

304 CHERNOFF ET AL.

can activate oor plate marker production (Briscoe and Ericson, 2001). In Xenopus the winged-helix forkhead transcription factor XFKH1 (Pintallavis, XFD-1) is expressed during gastrulation in the dorsal lip of the blastopore and, subsequently, in the notochord and the oor plate of the neural tube (Dirksen and Jamrich, 1992; Ruiz i Altaba and Jessel, 1992; Knochel et al., 1992). The apparent axolotl homolog AxFKH1 is expressed only in the oor plate (Whitely et al., 1997). It has been suggested that the relationship between the notochord and oor plate is different in Xenopus and axolotls. Alternatively, it is suggested that another fork head gene, such as Ax FKH2 is actually functionally homologous to XFKH1 (Whitely et al., 1997). There is currently no information about expression and regulation of the urodele homologues of the amniote embryo homeodomain genes involved in production of the dorsal and ventral spinal cord interneurons and motoneurons (Briscoe and Ericson, 1999, 2001; Lee and Jessell, 1999; Briscoe et al., 2000; Patten and Placzek, 2000; Altmann and Brivanlou, 2001; Shirasaki and Pfaff, 2002). This involvement of these genes, Pax, Nkx, Dbx, Irx, and LIM-homeodomain genes, in regeneration is an area of active interest at this time. In regeneration, D-V signaling might be required to establish the identity and differentiation of new neurons and for the targeting of regrowing axons. Possibly, stump tissues provide signals to adjacent cells that induce the patterning of the regenerate. Also unknown is the relationship between D-V signaling and the A-P patterning of the regenerate. There is a strong indication that the patterning process exists and is generated differently than it is during development: in tail regeneration, PNS ganglia are produced from the ependymal tube rather than from neural crest cells (see the axonal regrowth section above). Studies on the expression of patterning genes in urodele spinal cord regeneration are ongoing in several laboratories.

PERSPECTIVES
Urodele spinal cord regeneration does not appear to be a recapitulation of spinal cord development. There are fundamental, structural reasons for the differences. The predominant difference is that the progenitor tissues no longer exist and are not reconstructed during regeneration. The neural plate is not reconstructed; therefore, regeneration does not repeat the events of neurulation. The notochord is buried in the centrum of the vertebrae and is no longer a source of ventralizing signals. Presumptive skin ectoderm no longer exists, but the skin ectoderm does produce a wound epithelium that can act as a source of signals to the spinal cord in caudal regrowth (Godlewski, 1928; Polezhaev, 1972). In caudal replacement, the PNS ganglia must regenerate from the CNS, as the neural crest no longer exists (Egar and Singer, 1972; Nordlander and Singer, 1978; Arsanto et al., 1992; Benraiss et al., 1999; Zhang et al., 2000). The structural differences suggest that at least some of the expression of axial patterning genes will also differ from that in embryonic neurulation. This has been shown in A-P patterning of caudal regrowth (Beauchemin and Savard, 1992; Nicolas et al., 1996; Caubit et al., 1997a,b). Studies of D-V patterning during regeneration are just beginning. Without the notochord and skin epidermis as signaling centers, it is possible that the D-V patterning cues are propagated in a planar manner from the stump tissue. The identity of these signals and their spatial and temporal patterns of expression are areas of current research interest. The ependymal outgrowth and sprouting axons would serve as the target for A-P and D-V patterning signals in reconstruction of the regenerating spinal cord. These processes could be coordinated by signals induced or up-regulated in stump tissue. Differentiation of the ependymal outgrowth could be coordinated with rearrangement of cells in the remodeling stump tissues and axonal regrowth from stump tissue neurons. Experimental observations of FGF

and EGF responsiveness, along with studies of neurogenesis in urodeles, link the ependymal cell response in cord regeneration with studies of mammalian neural stem cells (Holder et al., 1991; OHara and Chernoff, 1994; Benraiss et al., 1999; Zhang et al., 2000). The expression of the neural stem cell marker Musashi-1 and other elements of the Notch-1 signaling pathway will allow us to examine the differentiated state of ependymal cells throughout the process of spinal cord growth and regeneration. Understanding the regulation of stem cell properties of urodele ependymoglia will be important in understanding the urodele spinal cord regeneration process.

ACKNOWLEDGMENTS
E.A.G.C. received funding from the NSF, and D.L.S. was funded by an Indiana 21st Century Research and Technology Fund.

REFERENCES
Altmann CR, Brivanlou AH. 2001. Neural patterning in the vertebrate embryo. Int Rev Cytol 203:447 482. Alvarez-Buylla A, Bettina S, Doetsch F. 2002. Identication of neural stem cells in the adult vertebrate brain. Brain Res Bull 57:751758. Anderson MJ, Choy CY, Waxman SG. 1986. Self organization of ependyma in regenerating teleost spinal cord: evidence from serial section reconstructions. J Embryol Exp Morphol 96:118. Araque A, Carmignoto G, Haydon PG. 2001. Dynamic signaling between astrocytes and neurons. Annu Rev Physiol 63:795 813. Arsanto J-P, Komorowski TE, Dupin F, Caubit X, Diano M, Geraudie J, Carlson BM, Thouveny Y. 1992. Formation of the peripheral nervous system during tail regeneration in urodele amphibians: ultrastructure and immunohistochemical studies of the origin of the cells. J Exp Zool 264:273292. Artavanis-Tsakonas S, Rand MD, Lake RJ. 1999. Notch signaling: cell fate control and signal integration in development. Science 284:70 76. Beauchemin M, Savard P. 1992. Two distal-less related homeobox-containing genes expressed in regeneration blastemas of the newt. Dev Biol 154:55 65. Beauchemin M, Noiseus N, Trembley M, Savard P. 1994. Expression of Hox A11 in the limb and the regenerating blastema of adult newt. Int J Dev Biol 38: 641 649.

URODELE SPINAL CORD REGENERATION 305

Becker CG, Becker T, Meyer RL, Schachner M. 1999. Tenascin-R inhibits the growth of optic bers in vitro but is rapidly eliminated during nerve regeneration in the salamander Pleurodeles waltl. J Neurosci 19:813 827. Benraiss A, Caubit X, Coulon J, Nicolas S, Le Parco Y, Thouveny Y. 1996. Clonal cell cultures from adult spinal cord of the amphibian urodele Pleurodeles waltl to study the identity and potentialities of cells during tail regeneration. Dev Dyn 205:135149. Benraiss A, Arsanto JP, Coulon J, Thouveny Y. 1999. Neurogenesis during spinal cord regeneration in adult newts. Dev Genes Evol 209:363369. Bignami A, Eng LF, Dahl D, Uyeda CT. 1972. Localization of the glial brillary acidic protein in astrocytes by immunouorescence. Brain Res 43:429 435. Birkedal-Hansen H, Moore WGI, Bodden MK, Windsor LJ, Birkedal-Hansen B, DeCarlo A, Engler JA. 1993. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med 4:197250. Bixby JL, Spitzer NC. 1984. Early differentiation of vertebrate spinal neurons in the absence of voltage-dependent Ca2 and Na inux. Dev Biol 106:89 96. Bodega G, Suarez I, Rubio M, Fernandez B. 1994. Ependyma: phylogenetic evolution of glial brillary acidic protein (GFAP) and vimentin expression in vertebrate spinal cord. Histochemistry 102: 113122. Bordzilovskaya NP, Dettlaff TA, Duhon ST, Malacinski GM. 1989. Developmentalstage series of axolotl embryos. In: Armstrong JB, Malacinski GM, editors. Developmental biology of the axolotl. New York: Oxford University Press. p 201219. Briscoe J, Ericson J. 1999. The specication of neuronal identity by graded sonic hedgehog signalling. Semin Cell Dev Biol 10:353362. Briscoe J, Ericson J. 2001. Specication of neuronal fates in the ventral neural tube. Curr Opin Neurobiol 11:43 49. Briscoe J, Pierani A, Jessell TM, Ericson J. 2000. A homeodomain protein code species progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101:435 445. Brockes JP. 1997. Amphibian limb regeneration: rebuilding a complex structure. Science 276:81 87. Burrows RC, Wancio D, Levitt R, Lillien L. 1997. Response diversity and the timing of progenitor cell maturation are regulated by developmental changes in EGFR expression in the cortex. Neuron 19:251267. Butler EG, Ward MB. 1965. Reconstitution of the spinal cord following ablation in urodele larvae. J Exp Zool 160:47 66. Butler EG, Ward MB. 1967. Reconstitution of the spinal cord following ablation in adult Triturus. Dev Biol 15:464 486. Caubit X, Arsanto J-P, Figarella-Branger D, Thouveny Y. 1993. Expression of polysialylated neural cell adhesion mole-

cule (PSA-N-CAM) in developing, adult and regenerating caudal spinal cord of the urodele amphibians. Int J Dev Biol 37:327336. Caubit X, Riou JF, Coulon J, Arsanto JP, Benraiss A, Boucaut JC, Thouveny Y. 1994. Tenascin expression in developing, adult and regenerating caudal spinal chord in the urodele amphibians. Int J Dev Biol 38:661 672. Caubit X, Nicolas S, Shi DL, Le Parco Y. 1997a. Reactivation and graded axial expression pattern of Wnt-10a gene during early regeneration stages of adult tail in amphibian urodele Pleurodeles waltl. Dev Dyn 208:139 148. Caubit X, Nicolas S, Le Parco Y. 1979b. Possible roles for Wnt genes in growth and axial patterning during regeneration of the tail in urodele amphibians. Dev Dyn 210:110. Chernoff EAG. 1996. Spinal cord regeneration: a phenomenon unique to urodeles? Int J Dev Biol 40:823 832. Chernoff EAG, Henry LC, Spotts T. 1998. An ependymal cell culture system for the study of spinal cord regeneration. Wound Repair Regen 6:435 444. Chernoff EAG, Munck CM, Egar MW, Mendelsohn LG. 1990. Primary cultures of axolotl spinal cord ependymal cells. Tissue Cell 5:601 613. Chernoff EAG, OHara CM, Bauerle D, Bowling M. 2000. Matrix metalloproteinase production in regenerating axolotl spinal cord. Wound Repair Regen 8:282291. Chernoff EAG, Sato K, Smith RC. 2001. Nrp-1 (Musashi) and Shh expression in ependymal cells of regenerating and non-regenerating Xenopus spinal cord. Mol Biol Cell 12:366a. Chernoff EAG, Sato K, Corn A, Karcavich RE. 2002. Spinal cord regeneration: intrinsic properties and emerging mechanisms. Semin Cell Dev Biol 13:361368. Christensen RN, Weinstein M, Tassava RA. 2002. Expression of broblast growth factors 4, 8, and 10 in limbs, anks, and blastemas of Ambystoma. Dev Dyn 223:193203. Clarke JDW, Ferretti P. 1998. CNS regeneration in lower vertebrates. In: Ferretti P, Geraudie J. editors. Cellular and molecular basis of regeneration. New York: Wiley and Sons. p 255269. Clarke JDW, Alexander R, Holder N. 1988. Regeneration of descending axons in the spinal cord of the axolotl. Neurosci Lett 89:1 6. Cochard P, Soula C, Giess MC, Trousse F, Foulquier F, Duprat AM. 1995. Determination of glial lineages during early central nervous system development. In: Zagris N, Duprat AM, Durston A, editors. Organization of the early vertebrate embryo. New York: Plenum Press. p 227239. Davidson LA, Keller RE. 1999. Neural tube closure in Xenopus laevis involves medial migration, directed protrusive activity, cell intercalation and convergent extension. Development 126: 4547 4556.

Davis BM, Duffy MT, Simpson SB Jr. 1989. Bulbospinal and intraspinal connection in normal and regenerated salamander spinal cord. Exp Neurol 103:41 51. Davis BM, Ayers JL, Koran L, Carlson J, Anderson MC, Simpson SB. 1990. Time course of salamander spinal cord regeneration and recovery of swimming: HRP retrograde pathway tracing and kinematic analysis. Exp Neurol 108:198 213. Detwiler SR. 1947. Restitution of the brachial region of the cord following unilateral excision in the embryo. J Exp Zool 104:53 68. Dirksen ML, Jamrich M. 1992. A novel, activin-inducible, blastopore lip-specic gene of Xenopus laevis contains a fork head DNA-binding domain. Genes Dev 6:599 608. Duprat AM. 1996. What mechanisms drive neural induction and neural determination in urodeles? Int J Dev Biol 40:745754. Efrimov MI. 1951. O skhodstve vzaimootnoshenii tsentralnoi nervnoi sistemy s okruzhayushchimi tkanyami v ontogeneze I pri ee transplantatsii u aksolotlya (The coincidence of the interrelationship of the central nervous system with the circumuent tissue in ontogeny, also in transplantation in Axolotl). Doklady AN SSSR 76:149 152. Egar M, Singer M. 1972. The role of ependyma in spinal cord regeneration in the urodele, Triturus. Exp Neurol 37: 422 430. Egar M, Singer M. 1981. The role of ependyma in spinal cord regrowth. In: Becker RO, editor. Mechanisms of growth control. Springeld IL: Charles Thomas Publisher. p 93106. Eggenschwiler JT, Espinoza E, Anderson KV. 2001. Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway. Nature 412:194 198. Fawcett JW, Asher RA. 1999. The glial scar and central nervous system repair. Brain Res Bull 49:377391. Gallien L, Bidaud O. 1959. Table chronologique du developpement chez Triturus helveticus Razoumowsky. Bull Soc Zool Fr 84:2232. Godlewski E. 1928. Untersuchungen uber Auslosung und Hemmung der Regeneration beim Axolotl. Wilhelm Roux Arch Entwicklungsmech 114:108 143. Grafstein B, Liu S, Cotrina ML, Goldman SA, Nedergaard M. 2000. Meningeal cells can communicate with astrocytes by calcium signaling. Ann Neurol 47: 18 25. Happel RD, Smith K, Banik NL, Powers JA, Hogan EL, Balentine JD. 1981. Ca2 accumulation in experimental spinal cord trauma. Brain Res 211:476 479. Harper CE, Roberts A. 1993. Spinal cord neuron classes in embryos of the smooth newt Triturus vulgaris: a horseradish peroxidase and immunocytochemistry study. Philos Trans R Soc Lond B 340:141160.

306 CHERNOFF ET AL.

Hay ED, Zuk A. 1995. Transformations between epithelium and mesenchyme: normal, pathological and experimentally induced. Am J Kidney Dis 26:678 690. Hitoshi S, Alexson T, Tropepe V, Donoviel D, Elia AJ, Nye JS, Conlon RA, Mak TW, Bernstein A, van der Kooy D. 2002. Notch pathway molecules are essential for the maintenance, but not the generation, of mammalian neural stem cells. Genes Dev 16:846 858. Holder N, Clarke JDW. 1988. Is there a correlation between continuous neurogenesis and directed axon regeneration in the vertebrate nervous system? Trends Neurosci 11:94 99. Holder N, Clarke JDW, Wilson S, Hunter K, Tonge DA. 1989. Mechanisms controlling directed axon regeneration in the peripheral and central nervous systems of amphibians. In: Kiorstsis V, Koussoulakos S, Wallace H, editor. Advanced research workshop on recent trends in regeneration research. NATO ASI Series. New York: Plenum Press. p 179 190. Holder N, Clarke JDW, Kamalati T, Lane EB. 1990. Heterogeneity in spinal radial glia demonstrated by intermediate lament expression and HRP labelling. J Neurocytol 19:915928. Holder N, Clarke JDW, Stephens N, Wilson SW, Orsi C, Bloomer T, Tonge DA. 1991. Continuous growth of the motor system in the axolotl. J Comp Neurol 303:534 550. Holliday J, Spitzer NC. 1990. Spontaneous calcium inux and its roles in differentiation of spinal neurons in culture. Dev Biol 141:1323. Holtzer H. 1951. Reconstitution of the urodele spinal cord following unilateral ablation. J Exp Zool 117:523558. Holtzer H. 1952. Reconstitution of the urodele spinal cord following unilateral ablation. J Exp Zool 119:263301. Hunter K, Maden M, Summerbell D, Eriksson U, Holder N. 1991. Retinoic acid stimulates neurite outgrowth in the amphibian spinal cord. Proc Natl Acad Sci U S A 88:3666 3670. Imai T, Tokunaga A, Yoshida T, Hashimoto M, Mikoshiba K, Weinmaster G, Nakafuku M, Okano H. 2001. The neural RNAbinding protein Musashi1 translationally regulates mammalian numb gene expression by interacting with its mRNA. Mol Cell Biol 21:3888 3900. Jacobson AG. 1981. Morphogenesis of the neural plate and tube. In: Connelly TG, Brinkely LL, Carlson BM, editors. Morphogenesis and pattern formation. New York: Raven Press. p 233263. Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J. 1999. Identication of a neural stem cell in the adult mammalian central nervous system. Cell 96:2534. Kitchin IC. 1949. The effects of notochordectomy in Ambystoma mexicanum. J Exp Zool 112:393 415. Knochel S, Lef J, Clement J, Klocke B, Hille S, Koster M, Knochel W. 1992. Activin A

induced expression of a fork head related gene in posterior chordamesoderm (notochord) of Xenopus laevis embryos. Mech Dev 38:157165. Kwon BK, Tetzlaff W. 2001. Spinal cord regeneration. Spine 26:S13S22. Lang DM, Rubin BP, Schwab ME, Stuermer CA. 1995. CNS myelin and oligodendrocytes of the Xenopus spinal cord but not optic nerveare nonpermissive for axon growth. J Neurosci 15:99 109. Lee KJ, Jessell TM. 1999. The specication of dorsal cell fates in the vertebrate central nervous system. Annu Rev Neurosci 22:261294. Lehmann FC. 1928. Die Bedeutung der Unterlagerung fur die Entwicklung der Medularplatte von Triton. Wilhelm Roux Arch Entwicklungsmech 113:123171. Lillien L, Raphael H. 2000. BMP and FGF regulate the development of EGF-responsive neural progenitor cells. Development 127:49935005. Maier CE, Miller RH. 1997. Notochord is essential for oligodendrocyte development in Xenopus spinal cord. J Neurosci Res 47:361371. McDonald JW, Sadowsky C. 2002. Spinal cord injury. Lancet 359:417 425. McGinnis W, Krumlauf R. 1992. Homeobox genes and axial patterning. Cell 68:283302. Miller RH, Liuzzi FJ. 1986. Regional specialization of the radial glial cells of the adult frog spinal cord. J Neurocytol 15: 187196. Momma S, Johansson CB, Frisen J. 2000. Get to know your stem cells. Curr Opin Neurobiol 10:45 49. Moran DJ. 1976. A scanning electron microscopic and ame spectrometry study on the role of Ca2 in amphibian neurulation using papaverine inhibition and ionophore induction of morphogenetic movement. J Exp Zool 198: 409 416. Moran D, Rice RW. 1976. Action of papaverine and ionophore A23187 on neurulation. Nature 261:497 499. Moreau M, Leclerc C, Gualandris-Parisot L, Duprat AM. 1994. Increased internal Ca2 mediates neural induction in the amphibian embryo. Proc Natl Acad Sci U S A 91:12639 12643. Moriya T, Hassan AZ, Young W, Chesler M. 1994. Dynamics of extracellular calcium activity following contusion of the rat spinal cord. J Neurotrauma 11:255 263. Morshead C, van der Kooy D. 2001. A new spin on neural stem cells? Curr Opin Neurobiol 11:59 65. Muroyama Y, Fujihar M, Ikeya M, Kondoh H, Takada S. 2002. Wnt signaling plays an essential role in neuronal specication of the dorsal spinal cord. Genes Dev 16:548 553. Nagase H, Woessner JF Jr. 1999. Matrix metalloproteinases. J Biol Chem 274: 2149121494. Nakamura M, Okano H, Blendy JA, Montell C. 1994. Musashi, a neural RNAbinding protein required for Drosophila

adult external sensory organ development. Neuron 13:67 81. Nedergaard M. 1994. Direct signaling from astrocytes to neurons in cultures of mammalian brain cells. Science 263: 1768 1171. Ng CE, Tang BL. 2002. Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration. J Neurosci Res 67:559 565. Nicolas S, Caubit X, Massacrier A, Cau P, Le Parco Y. 1999. Two Nkx-3-related genes are expressed in the adult and regenerating central nervous system of the urodele Pleurodeles waltl. Dev Genet 24:319 328. Nicolas S, Massacrier A, Caubit X, Cau P, Le Parco Y. 1996. A distal-less-like gene is induced in the regenerating central nervous system of the urodele Pleurodeles waltl. Mech Dev 56:209 220. Nordlander R, Singer M. 1978. The role of ependyma in regeneration of the spinal cord in the urodele amphibian tail. J Comp Neurol 180:349 374. OHara CM, Egar MW, Chernoff EAG. 1992. Reorganization of the ependyma during axolotl spinal cord regeneration: changes in intermediate lament and bronectin expression. Dev Dyn 193:103115. OHara CM, Chernoff EAG. 1994. Growth factor modulation of injury-reactive ependymal cell proliferation and migration. Tissue Cell 26:599 611. Ohtsuka T, Ishibashi M, Gradwohl G, Nakanishi S, Guillemot F, Kageyama R. 1999. Hes1 and Hes5 as notch effectors in mammalian neuronal differentiation. EMBO J 18:2196 2207. Orentas DM, Miller RJ. 1996. The origin of spinal cord oligodendrocytes is dependent on local inuences from the notochord. Dev Biol 177:4353. Panter SS, Yum S, Faden AI. 1990. Alteration in extracellular amino acids after traumatic spinal cord injury. Ann Neurol 27:96 99. Patten I, Placzek M. 2000. The role of sonic hedgehog in neural tube patterning. Cell Mol Life Sci 57:16951708. Pfaff S, Kintner C. 1998. Neuronal diversication: development of motorneuron subtypes. Curr Opin Neurobiol 8:2736. Piatt J. 1955. Regeneration in the central nervous system of amphibia. In: Windle WF, editor. Regeneration in the central nervous system. Springeld, IL: C. Thomas Publishing. p 20 46. Polezhaev LV. 1972. Loss and restoration of regenerative capacity in tissues and organs of animals. Cambridge MA: Harvard University Press. p 75 82. Richter K, Good PJ, Dawid IB. 1990. A developmentally regulated, nervous system-specic gene in Xenopus encodes a putative RNA-binding protein. New Biol 2:556 565. Roberts A. 2000. Early functional organization of spinal neurons in developing lower vertebrates. Brain Res Bull 53:585 593. Roots BI. 1986. Phylogenetic development of astrocytes. In: Fedoroff S, Ver-

URODELE SPINAL CORD REGENERATION 307

nadakis A, editors. Astrocytes (development, morphology and regional specialization of astrocytes). New York: Academic Press. p 134. Ruiz i Altaba A, Jessel TM. 1992. Pintallavis, a gene expressed in the organizer and midline cells of frog embryos: involvement in the development of the neural axis. Dev 116:8193. Sakakibara S, Imai T, Hamaguchi K, Okabe M, Aruga J, Nakajima K, Yasutomi D, Nagata T, Kurihara Y, Uesugi S, Miyata T, Ogawa M, Mikoshiba K, Okano H. 1996. Mouse-Musashi-1, a neural RNA-binding protein highly enriched in the mammalian CNS stem cell. Dev Biol 176:230 242. Schonbach C. 1969. The neuroglia in the spinal cord of the newt, Triturus viridescens. J Comp Neurol 135:93120. Sheng M, Greenberg ME. 1990. The regulation and function of c-fos and other immediate early genes in the nervous system. Neuron 4:477 485. Shirasaki R, Pfaff SL. 2002. Transcriptional codes and the control of neuronal identity. Annu Rev Neurosci 25:251 281. Shi Y-B. 2000. Amphibian metamorphosis. From morphology to molecular biology. New York: John Wiley and Sons. p 6 19, 135153. Simpson SB Jr. 1968. Morphology of the regenerated spinal cord in the lizard, Anolis carolinensis. J Comp Neurol 134: 193210. Singer M, Nordlander RH, Egar P. 1979. Axonal guidance during embryogenesis and regeneration in the spinal cord of newt: the blueprint hypothesis of

neuronal pathway patterning. J Comp Neurol 185:122. Soula C, Sagot Y, Coshard P, Duprat AM. 1990. Astroglial differentiation from neural epithelial precursor cells of amphibian embryos: an in vivo and in vitro analysis. Int J Dev Biol 34:351364. Steffaneli A. 1950. Some comments on regeneration in the central nervous system. In: Weiss P, editor. Genetic neurology. Chicago, IL: University of Chicago Press. p 210 211. Stensaas LJ. 1983. Regeneration in the spinal cord of the newt Notophthalmus (Triturus) pyrrhogaster. In: Kao CC, Bunge RP, Reier, PJ, editors. Spinal cord reconstruction. New York: Raven Press. p 121149. Steward O, Schauwecker PE, Guth L, Zhang Z, Fujika M, Inman D, Wrathall J, Kemperman G, Gage F, Saatman KE, Raghupathi R, McIntosh T. 1999. Genetic approaches to neurotrauma research: opportunities and potential pitfalls of murine models. Exp Neurol 157: 19 42. Stocum DL. 1996. A conceptual framework for analyzing axial patterning in regenerating urodele limbs. Int J Dev Biol 40:773783. Stokes BT, Fox P, Hollinden G. 1983. Extracellular calcium activity in the injured spinal cord. Exp Neurol 80:561572. Svendsen CN, Caldwell MA. 2000. Neural stem cells in the developing central nervous system: implications for cell therapy through transplantation. Prog Brain Res 127:1334. Szaro BG, Gainer H. 1988. Immunocytochemical identication of non-neuro-

nal intermediate lament proteins in the developing Xenopus laevis nervous system. Dev Brain Res 43:207224. Temple S. 2001. The development of neural stem cells. Nature 414:112117. Wanner M, Lang DM, Bandtlow CE, Schwab ME, Bastmeyer M, Stuermer CA. 1995. Reevaluation of the growthpermissive substrate properties of goldsh optic nerve myelin and myelin proteins. J Neurosci 15:7500 7508. Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson AC, Reynolds BA. 1996. Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16:7599 7609. Whitely M, Mathers PH, Jamrich M. 1997. Expression pattern of an axolotl oor plate-specic fork head genes reects early developmental differences between frogs and salamanders. Dev Genet 20:145151. Yong VW, Power C, Forsyth P, Edwards DR. 2001. Metalloproteinases in biology and pathology of the nervous system. Nat Rev Neurosci 2:502511. Zamora AJ. 1978. The ependymal and glial conguration in the spinal cord of urodeles. Anat Embryol (Berl) 154:67 82. Zamora AJ, Mutin M. 1988. Vimentin and glial brillary acidic protein in radial glia of the adult urodele spinal cord. Neuroscience 27:279 288. Zhang F, Clarke JDW, Ferretti P. 2000. FGF-2 up-regulation and proliferation of neural progenitors in the regenerating amphibian spinal cord in vivo. Dev Biol 225:381391.