THE JOURNAL OF COMPARATIVE NEUROLOGY 461:262275 (2003)

Increased Expression of Multiple Neurolament mRNAs during Regeneration of Vertebrate Central Nervous System Axons
CHRISTINE GERVASI, AMAR THYAGARAJAN, AND BEN G. SZARO* Department of Biological Sciences and the Center for Neuroscience Research, State University of New YorkUniversity at Albany, Albany, New York 12222

ABSTRACT Characteristic changes in the expression of neuronal intermediate laments (nIFs), an abundant cytoskeletal component of vertebrate axons, accompany successful axon regeneration. In mammalian regenerating PNS, expression of nIFs that are characteristic of mature neurons becomes suppressed throughout regeneration, whereas that of peripherin, which is abundant in developing axons, increases. Comparable changes are absent from mammalian injured CNS; but in goldsh and lamprey CNS, expression of several nIFs increases during axon regrowth. To obtain a broader view of the nIF response of successfully regenerating vertebrate CNS, in situ hybridization and video densitometry were used to track multiple nIF mRNAs during optic axon regeneration in Xenopus laevis. As in other successfully regenerating systems, peripherin expression increased rapidly after injury and expression of those nIFs characteristic of mature retinal ganglion cells decreased. Unlike the decrease in nIF mRNAs of regenerating PNS, that of Xenopus retinal ganglion cells was transient, with most nIF mRNAs increasing above normal during axon regrowth. At the peak of regeneration, increases in each nIF mRNA resulted in a doubling of the total amount of nIF mRNA, as well as a shift in the relative proportions contributed by each nIF. The relative proportions of peripherin and NF-M increased above normal, whereas proportions of xeltin and NF-L decreased and that of XNIF remained the same. The increases in peripherin and NF-M mRNAs were accompanied by increases in protein. These results are consistent with the hypothesis that successful axon regeneration involves changes in nIF subunit composition conducive to growth and argue that a successful injury response differs between CNS and PNS. J. Comp. Neurol. 461:262275, 2003. 2003 Wiley-Liss, Inc.
Indexing terms: peripherin; intermediate lament; Xenopus laevis; -internexin; optic nerve

Because the optic nerves of sh (Sperry, 1948) and frog (Sperry, 1944; Gaze, 1959) regenerate, they are excellent systems for studying genes involved in successful recovery from CNS injury. Neuronal intermediate lament (nIF) genes, whose proteins make neurolaments (NFs), are among the most abundantly expressed genes that are highly regulated during regeneration. Vertebrate nIF genes have been classied according to similarities in amino acid sequence and intron position as either type III or type IV intermediate laments (Steinert and Roop, 1988). In Xenopus, as in mammals, all nIFs are classied as type IV, with the exception of peripherin, which is type III. The stability of NFs and their great tensile strength have suggested that the changes in nIF gene expression at each phase of axon outgrowth may reect the growing
2003 WILEY-LISS, INC.

axons shifting demands for structural stability (Schwartz et al., 1990; Fliegner et al., 1994; Fuchs et al., 1994; Glasgow et al., 1994). For example, at early stages of outgrowth, NFs with immature compositions may facilitate axon elongation by helping to consolidate growth

Grant sponsor: National Institutes of Health; Grant number: NS30682. *Correspondence to: Ben G. Szaro, Department of Biological Sciences, SUNY University at Albany, 1400 Washington Avenue, Albany, NY 12222. E-mail: bgs86@cnsunix.albany.edu Received 14 November 2002; Revised 17 February 2003; Accepted 24 February 2003 DOI 10.1002/cne.10695 Published online the week of May 5, 2003 in Wiley InterScience (www. interscience.wiley.com).

PERIPHERIN IN REGENERATING FROG OPTIC NERVE without inhibiting it (Walker et al., 2001), whereas after synaptogenesis NFs of mature composition may further enhance axon strength and caliber. Consistent with this view, two nIF subunits characteristic of immature developing axons [peripherin (Parysek and Goldman, 1988; Troy et al., 1990b) and -internexin (Fliegner et al., 1994)] increase in expression during mammalian PNS regeneration (Oblinger et al., 1989; Troy et al., 1990a; McGraw et al., 2002), whereas three subunits most abundant in mature axons [low (NF-L), medium (NF-M), and high (NF-H) molecular mass NF triplet proteins] decrease (Wong and Oblinger, 1990). Peripherinand -internexin-like nIFs also increase during goldsh optic nerve regeneration (Glasgow et al., 1992; Fuchs et al., 1994), but comparable changes in nIF expression fail to occur in injured mammalian CNS (Mikucki and Oblinger, 1991), arguing that they are linked to successful recovery from injury. Moreover, the relative magnitudes of changes in the expression of individual subunits must be highly regulated, since substantially altering nIF stoichiometries can cause NFs to aggregate and axons to degenerate (Beaulieu et al., 1999, 2000). The regenerating optic nerve of juvenile Xenopus laevis frogs is a good system for studying nIF expression during CNS regeneration, since the time-course of regeneration, as well as changes in the expression of several nIF proteins, have already been characterized for it (Szaro et al., 1985; Zhao and Szaro, 1994, 1995, 1997b): Newly regenerating axons rst cross the lesion between 5 and 6 days after an orbital nerve crush, and approach the chiasm at 9 days. At 15 days the rst axons reach the optic tectum and by 18 days they cover it. For several weeks afterward, innervation of the tectum increases, eventually restoring a retinotopic map. For the rst week after injury, expression of NF-M and two -internexin-like nIF proteins, xeltin and XNIF, is suppressed and then subsequently increases, but only in those axons that successfully enter the optic tract. Our current study had two principal objectives. The rst was to determine whether peripherin expression increases during Xenopus optic nerve regeneration, as it does after injury in the goldsh optic nerve and mammalian PNS. In developing Xenopus retinal ganglion cells (RGCs), peripherin is the most abundant nIF and it disappears as RGCs mature (Gervasi et al., 2000). Thus, demonstrating that its expression returns during regeneration would further strengthen the argument that peripherin-like, type III nIFs are important for axonal growth. The second objective was to estimate the relative magnitudes of changes in multiple nIFs during regeneration. This would better indicate which ones may promote successful regeneration and would help identify possible differences between regenerating CNS and PNS. Such differences might be expected, since mature, uninjured CNS neurons normally have much lower NF contents than do PNS neurons. We accomplished these objectives using in situ hybridization and video densitometry. In addition, we immunostained regenerating axons with antibodies to peripherin and NF-M to determine whether increases in nIF mRNAs produced increases in axonally transported protein and NF aggregation. As in mammalian regenerating PNS, we found that during early phases of regeneration, levels of peripherin mRNA increased and those of other nIFs decreased. Unlike mammalian PNS, however, levels of several nIF mRNAs, including NF-M, XNIF, and xeltin, also

263 increased above normal well before regeneration was nished. Increased levels of axonally transported peripherin and NF-M did not cause NF aggregates, arguing that changes in nIF subunit stoichiometries were balanced to avoid disruption of NFs. At each phase of regeneration, both the level of all nIF mRNAs combined and their relative proportions changed, arguing that successful CNS axon regeneration involves both a change in the total amount of nIFs expressed and a unique mix of nIFs conducive to growth.

MATERIALS AND METHODS Optic nerve surgery

Periodic albino Xenopus laevis (Hoperskaya, 1975; Tompkins, 1977) were bred in our laboratory and used within 3 months after metamorphosis. Frogs were anesthetized by immersion in 0.1% 3-aminobenzoic acid ethylester (Sigma, St. Louis, MO), according to protocols approved by our institutional Animal Care and Use Committee and conforming to NIH guidelines, and their left optic nerve was crushed at the orbit as described previously (Zhao and Szaro, 1994). Our present study used juvenile frogs from the same stock, reared under similar conditions, and operated upon in the same manner as those used previously (Zhao and Szaro, 1994). In addition, the reappearance of NF-M immunoreactivity within the regenerating optic nerves of our animals (data not shown) followed the same time-course as in these earlier studies. Consequently, we were condent that the time-course of regeneration in our study was similar to what had been previously established.

RNase protection assay

Total RNA was isolated from eyes using a Masterpure RNA Purication kit (Epicenter Technologies, Madison, WI) and pooled as follows: 1) 12 unoperated eyes from six animals; 2) 12 eyes whose optic nerve had been crushed 9 days before; and 3) 12 unoperated, contralateral control eyes from these same animals. RNase protection assays were performed using an RPAIII kit (Ambion, Austin, TX) and the following probes, which were synthesized and labeled by in vitro transcription with 32P: Xenopus peripherin (spanning nt #14352012; Sharpe et al., 1989); Xenopus NF-M2 (spanning nt #456 822; Gervasi and Szaro, 1997); Xenopus Elongation Factor 1- (EF1-; spanning nt #30753225 from exon 5; Johnson and Krieg, 1995; GenBank M25697). Aliquots of 10 g of total RNA from each sample were hybridized simultaneously with 7 104 cpm each of the Xenopus peripherin and the NF-M2 probes; 3 g were hybridized separately with 4 104 cpm of EF1- probe. Hybridizations were done overnight at 53C. The single-stranded, unprotected RNA was then digested with RNase T1 (100 U/ml) for 1 hour at room temperature. The protected RNA fragments were separated on a 5% acrylamide/8 M urea denaturing gel, which was dried and exposed (15 hours) rst to X-ray lm (XAR-1 lm, Kodak, Rochester, NY) with an intensifying screen at 70C, and then to a phosphoimaging screen (18 and 27 hours; Amersham-Molecular Dynamics, Piscataway, NJ). The exposures on the phosphoimaging screen were then read and quantitated using a Storm 860 Image Scanner (Amersham-Molecular Dynamics) running Image Quant (v. 5.0) software. Background was corrected in each lane

264 by the histogram peak method. The ratios for each probe between the operated and contralateral control eye samples were equal for the 18 and 27 hours exposures, which conrmed that exposures fell within the linear range of the assay.

C. GERVASI ET AL. the conditions described below raised background without increasing the number or the intensity of labeled RGCs. Slides were hybridized with 0.25 g/ml of cRNA probe at 55C overnight. High stringency washes were performed in 0.1 standard saline citrate at 60 64C. The digoxigenin signal was visualized by immunostaining slides with anti-digoxigenin Fab fragments coupled to alkaline phosphatase and then developing them in 4-nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate (Genius 3 kit; Roche Molecular Biochemicals). For slides to be compared, the conditions of immunostaining and development were standardized. Sections were mounted under glass coverslips in aqueous mounting medium (Shandon Lipshaw, Pittsburgh, PA).

Preparation of frozen histological sections

At various intervals after crush, the frogs were anesthetized as before and xed by intracardial perfusion with 4% paraformaldehyde/10% sucrose in amphibian Ringers solution (113 mM NaCl, 2 mM KCl, 1.1 mM CaCl2, 1.2 mM NaHCO3). Sections were cut transversely at 12 m as described in Gervasi et al. (2000) and thaw-mounted onto commercially available glass slides treated with aminoalkylsilane (Polysciences, Warrington, PA). Adjacent sections through the eye were collected successively on separate slides to permit a fair comparison of multiple probes on each animal. Sample slides containing sections of optic nerve from each animal were stained with cresyl violet to conrm that the optic nerve had been crushed successfully, i.e., depending on the time of sacrice, that the optic nerve either showed signs of degeneration (3 and 6 days postcrush) or contained the halo of new axons that is characteristic of regenerating optic nerves (9 days and later; Zhao and Szaro, 1994). Any animal not meeting these criteria was excluded from further analysis.

Morphological analysis and intensity measurements

Slides processed for in situ hybridization were viewed on a Leitz Laborlux compound microscope with a 25 objective (Leitz PL Fluotar, 0.6 NA). Images of retinal sections were captured with a Dage CCD300T-RC video camera. To set the limits of the range of the intensity measurements, the lightest and darkest regions of the sections of the most intensely stained slides were used for setting the gain and black levels on the camera in order to minimize the number of saturated pixels while maximizing the range over which the intensities could be collected. This was done while also adjusting the level of illumination and the analog contrast on the frame grabber, so that the morphological details captured by the computer through the camera faithfully reproduced those seen by eye through the microscope. These settings were then kept constant for all images to be compared. This ensured that the light absorbed by the color precipitant present on the slides fell within the linear range of intensities captured in the computer image and that separate images could be reliably compared. With the morphometric analysis functions of Metamorph (v. 4.0, Universal Imaging, West Chester, PA), the average labeling intensity per pixel was then measured within the RGC layer and corrected for background (average labeling intensity over the neighboring, unstained inner nuclear layer). For each retina, 12 such backgroundcorrected measurements were averaged. These measurements were then averaged among animals to obtain the mean value (SE) for each time point. Measurements were transferred to an Excel spreadsheet, which was used for all further statistical analyses. To allow for fair comparisons among separate animals and probes in this analysis, consecutive slides from each animal were hybridized with equivalent amounts of probes to each of the ve nIF mRNAs and processed in parallel. To ensure the consistency of the comparisons, measurements were taken from only the central portion of the retina. In all, two animals each at 3, 6, 9, 12, 18, and 35 days postcrush and three animals at 21 days postcrush were used. Several additional animals (one each at 3, 18, 21, 28, 35 days postcrush hybridized with probes to all ve nIFs; and one each at 6, 9, 12, and 15 days postcrush hybridized with probes to just peripherin, xeltin, and XNIF) showed equivalent staining intensities, but were not included in the quantitative analyses because they were not processed in parallel.

Preparation of cRNA probes for in situ hybridization

Digoxigenin-labeled cRNA probes were transcribed (Gervasi et al., 2000) using clones generated in our laboratory and a Genius 4 Kit (Roche Molecular Biochemicals, Indianapolis, IN). Plasmids containing full-length Xenopus peripherin, NF-L, XNIF, and xeltin (Zhao and Szaro, 1997a; Gervasi et al., 2000) and a partial length Xenopus NF-M (clone 6B-1a of NF-M1, Gervasi and Szaro, 1997) were linearized for in vitro transcription to generate digoxigenin-labeled antisense cRNA probes of comparable length and activity (2.1 kb for XNIF, xeltin and NF-L, 2.0 kb for peripherin, 1.7 kb for NF-M). For a negative control, a sense cRNA to peripherin was transcribed. The amount of probe synthesized was determined by measuring uorescence in a TD-700 spectrouorometer (Turner Designs) using a RiboGreen Quantitation kit (Molecular Probes, Eugene, OR). The quality and size of the probes were veried on formaldehyde/agarose gels (Davis et al., 1994). To verify that probes were labeled, a known mass was spotted onto nitrocellulose and then assayed with a horseradish peroxidase labeled anti-digoxigenin antibody (Gervasi et al., 2000). To improve penetration into the tissue, probes were chemically fragmented into lengths of approximately 300 nucleotides (Angerer et al., 1987).

In situ hybridization
In situ hybridization on sectioned tissue was performed essentially as described previously (Zhao and Szaro, 1997a). Pilot experiments were used to nd conditions that reliably produced a signal that minimized saturation of the labeling (intense black staining) and background while maximizing the sensitivity of the assay (the number of RGCs that were labeled at least lightly). In general, increasing developing time and probe concentration above

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Quantitative shifts in the distribution of nIF mRNAs among RGCs

For determining the percentage of cells within the RGC layer expressing a given nIF mRNA, sections were viewed on a Leitz Laborlux compound microscope with a 25 objective. Within a eld of view, all cells within the RGC layer were scored as either unlabeled (i.e., labeled below background), weakly (labeled just above background), moderately (visible distinctly and medium blue in color), or strongly labeled (dark blue and labeled over a signicant area of the cell body; Jacobs et al., 1997). These numbers were normalized against the total number of cells within the RGC layer for each eld of view. For comparisons between animals, cells were scored within comparable regions of the retina and in the same number of experimental animals as used for the analyses described above.

Immunocytochemistry
Three separate polyclonal rabbit antisera were used to detect Xenopus peripherin. One of these, made against a synthetic peptide (QVVTESRKEQSSEGE) derived from the C-terminus of Xenopus peripherin, had been characterized previously in developing Xenopus laevis (Gervasi et al., 2000). The other two were new antisera made in separate rabbits to a synthetic peptide (RHFGSPSPGPSSR) derived from the N-terminal head domain of Xenopus peripherin. The peptides were synthesized, conjugated at their C-terminus to keyhole limpet hemocyanin, and injected into New Zealand rabbits at a commercial facility (Covance, Richmond, CA). Three additional, previously characterized monoclonal antibodies to Xenopus NF-M were also used: RMO270, which recognizes a C-terminal, phosphorylation-independent epitope (Szaro et al., 1989; Wetzel et al., 1989), and S6 and S8, which recognize phosphorylated and dephosphorylated epitopes, respectively (Szaro and Gainer, 1988). Western blots were performed as described in Szaro and Gainer (1988) on total homogenates of Xenopus spinal cord. The secondary antibody was horseradish peroxidase conjugated, goat antirabbit (Kirkegaard and Perry, Gaithersburg, MD) and the chromogen was 4-chloro-1-napthol. The sections used for immunocytochemistry were from the same animals as those used for in situ hybridization, which permitted a better comparison between the distributions of protein and mRNA. Immunocytochemistry was performed essentially as described in Szaro and Gainer (1988), using a peroxidase-conjugated secondary antibody (2 g/ml, Kirkegaard and Perry), a glucose oxidase reaction as a source of peroxide, and diaminobenzidine and nickel chloride as chromogens. To test whether either epitope masking or phosphorylation interfered with immunocytochemical staining with the peripherin antisera, some slides were treated with either trypsin or phosphatase, respectively. Trypsin or phosphatase was applied after blocking nonspecic binding sites in 10% fetal calf serum. For the trypsin treatment, slides were incubated in 1 mg/ml of porcine trypsin type II (Sigma) at 37C for 5 minutes. For the phosphatase treatment, slides were incubated at 37C for 3 hours in E. coli alkaline phosphatase type III (Sigma) at a concentration of 4 U/ml of buffer (1 mM ZnSO4, 100 mM NaCl, 50 mM Tris HCl, pH 8.0). Slides were then incubated in 10% fetal calf serum, followed by a second incubation in 5%

Fig. 1. Increases in NF-M and peripherin mRNAs at 9 days postcrush demonstrated by RNase protection. Ten g of total RNA pooled from 12 eyes were hybridized simultaneously with 32P-labeled RNA probes against Xenopus NF-M and peripherin in one tube, and 3 g of RNA were hybridized with a probe to Elongation Factor 1- (EF1-) in a separate tube (B,C,D). A: Probe hybridized against yeast RNA, demonstrating that the RNase digestion went to completion. B: RNA from naive, unoperated eyes. C: RNA from the contralateral control eye, 9 days after optic nerve crush. D: RNA from the operated eye, 9 days postcrush. After correcting for a slight increase (10%) in the expression of EF1- mRNA, the ratios of RNA expression between the operated and contralateral control eyes (D,C) were 1.3 for NF-M and 2.3 for peripherin.

bovine serum albumin and 1% normal goat serum, and then processed further for immunocytochemistry.

RESULTS RNase protection demonstrated increases in peripherin and NF-M mRNAs during regeneration
To determine whether peripherin mRNA expression increases during regeneration, we performed an RNase protection assay (Fig. 1) and compared changes in the levels of peripherin mRNA with those of two other mRNAs: Xenopus NF-M, another nIF protein expressed during axon elongation, and Xenopus Elongation Factor 1- (EF1), a constitutively expressed gene whose expression can be used to monitor overall levels of transcription in Xenopus. Total RNA was isolated from operated (Fig. 1D) and contralateral control eyes (Fig. 1C) at 9 days after nerve crush (which coincides with the arrival of axons at the chiasm), as well as from unoperated animals (Fig. 1B). The ratio (OE/UE) of peripherin and NF-M mRNA levels between the operated eyes (OE) and the contralateral control eyes (UE) demonstrated increases of 2.5- and 1.4fold, respectively. These increases were greater than the slight (1.1-fold) increase that was seen for EF1- mRNA, a measure of the general increase in RNA levels that occur in eyes following axotomy (Burrell et al., 1978). In addition to the differential increases in peripherin and NF-M mRNAs in the operated eyes in experimental animals, there was an increase of 1.5-fold in both mRNAs (1.49 and 1.51 for peripherin and NF-M, respectively) between the contralateral control eyes in the operated animals and eyes of unoperated, naive animals. This result demonstrated that the different responses to optic nerve crush between peripherin and NF-M were specic to the crush injury, as opposed to general trauma, and also underscored the utility of using the contralateral eye as an internal control to obtain a true picture of this nerve crush response.

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C. GERVASI ET AL. From 9 days on the percentage of RGCs expressing each nIF increased, matching the total percentage of cells in the RGC layer labeling for any type IV nIF. The percentage of cells expressing peripherin (78%), xeltin (93%), XNIF (77%), or NF-M (88%) were indistinguishable from each other (Fig. 3E; single factor ANOVA, df 6, P 0.5), and were comparable to the 89% of RGC layer cells that had labeled for xeltin in unoperated retina. In addition, the overall level of staining also increased in these cells. The remaining RGC layer cells that failed to label for any of these nIFs were likely to be displaced amacrine or glial cells (Dunlop and Beazley, 1984). Thus, during the peak of regeneration, as axons crossed the optic tract and tectum, levels of these nIF mRNAs increased throughout the vast majority, if not all, RGCs. Similarly, although NF-L appeared in fewer RGCs than did the other nIFs, its distribution nevertheless also expanded (Fig. 3E).

Patterns of expression of nIF mRNAs in normal retina

To learn whether changes in nIF expression during regeneration occurred evenly throughout the retina, as opposed to within subpopulations of RGCs, we turned to in situ hybridization. Before we could address this question in regenerating RGCs, it was necessary to characterize in more detail the pattern of expression of each nIF in normal retina. The anatomical distribution of nIF mRNAs in the contralateral, unoperated retinas of regenerating animals were similar to those of normal, unoperated animals and remained constant throughout regeneration (an example of which is shown at 28 days postcrush, Fig. 2AE). Cell counts for unoperated eyes in Figure 3A were pooled from several regenerating animals. RGCs of unoperated eyes contained mostly type IV nIFs and little to no peripherin. Peripherin mRNA was conned primarily to ciliary margin, which contains proliferating neural cells (not shown, but see Gervasi et al., 2000). This staining served as an internal control for hybridization conditions for peripherin. In the RGC layer, only 2% of the cells were labeled for peripherin, and these only weakly (Figs. 2A, 3A). Of the type IV nIF mRNAs, xeltin was the most abundant, labeling 89% of cells, three-fourths of which were moderately to strongly labeled (Figs. 2B, 3A). The remaining type IV nIFs labeled smaller populations of cells. XNIF was found in 42% of cells, of which only onethird were moderately to strongly labeled (Figs. 2C, 3A). The anatomical distributions of NF-M and NF-L were very similar, with 20% and 19% of cells labeling, respectively (Fig. 2D,E). Although their distributions were similar, NF-M was more abundant in labeled cells, labeling more cells moderately to strongly than did NF-L (Fig. 3A). The only subpopulation of RGCs we could unambiguously identify was the largest RGCs, which have cell diameters exceeding twice that of other RGCs. In our study, cells matching this size criterion numbered from 12% of RGCs, which agrees with the published counts of Straznicky and Straznicky (1988). All such RGCs were labeled for xeltin, XNIF, NF-M, and NF-L.

Quantitative estimates of nIF mRNA expression in regenerating RGCs

To estimate more quantitatively the changes in nIF mRNA levels during regeneration, we used a colorimetric assay based on video densitometric measurements of the intensity of digoxigenin label in the RGC layer under standardized conditions (see Materials and Methods). These data are presented in three different ways, each of which emphasizes a different feature of the changes in nIF expression seen during regeneration: 1) changes in the relative ratio of expression of each nIF between the operated and contralateral control eye (OE/UE); 2) changes in the magnitude of expression of each nIF (OE-UE); and 3) changes in the relative stoichiometries of nIF mRNAs. Changes in the relative ratios of nIF expression between operated and control RGCs. First, we measured the ratio (OE/UE) between nIF mRNA labeling in the RGC layer of operated (OE) and contralateral control (UE) eyes within the same animal (Table 1). This method normalized for any slight variations that occurred in assay conditions and emphasized the relative fold changes in expression during regeneration. Peripherin showed the earliest and greatest fold increase of any of the nIFs, nearly 10-fold during the earliest phase of regeneration (3 6 days), and rising to over 30-fold between 9 and 21 days. In contrast, the levels of the type IV nIF mRNAs dropped during the rst week after nerve crush (3 6 days), but then also rose above normal during the next 2 weeks. Although these increases were signicantly greater than 1-fold (t-test, better than P 0.05), they were relatively smaller than for peripherin, ranging, for example at 9 12 days, from a 1.3-fold increase for xeltin to a 2.6-fold increase for NF-M. In addition, the increase in NF-L expression took longer to occur than that of the other nIFs, appearing at 18 21 days postcrush instead of at 9 12 days. By 5 weeks postcrush, levels of all ve nIFs began to decline toward normal. Changes in the magnitude of expression of each nIF (OE-UE) during regeneration. Although the OE/UE ratio for each nIF mRNA demonstrated that the relative expression changed over time, it obscured the absolute magnitude of these changes, which was better revealed by measuring the differences in expression between operated and contralateral control eyes (OE-UE; Fig. 4). The timecourse and statistical signicance of the changes that occurred during regeneration were comparable with both methods; however, the difference method revealed that

Patterns of nIF expression in regenerating RGCs

Since nIF mRNAs are differentially distributed among normal RGCs, we rst examined whether changes in nIF expression during regeneration were limited to those subpopulations that normally express each nIF. Representative examples of these patterns in sections of regenerating retina are illustrated in Figure 2 for 6 (FJ) and 12 (KO) days. At the earlier stage of regeneration (6 days), which is soon after axons cross the lesion, fewer RGCs than normal expressed xeltin, XNIF, and NF-L, and the level of staining in those cells that were labeled also fell (see cell counts in Fig. 3B,C). In contrast, more RGCs than normal expressed peripherin and NF-M at this time. For peripherin, the level of staining in labeled cells increased, but for NF-M, although the number of labeled cells increased, the overall level of staining was reduced. To explain this result, decreased expression of NF-M in some RGCs must have been accompanied by a reexpression of detectable levels of mRNA in populations of cells that had previously fallen below the threshold of detection in normal retina.

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Fig. 2. Expression of nIF mRNA in the contralateral, unoperated control eye at late stages of regeneration (AE) and in the operated eye 6 (FJ) and 12 (KO) days after optic nerve crush. Neighboring transverse sections were hybridized with digoxigenin-labeled cRNA probes to peripherin (A,F,K), xeltin (B,G,L), XNIF (C,H,M), NF-M (D,I,N), and NF-L (E,J,O). A representative example of a section from

an operated eye hybridized with a sense probe to peripherin showed no label above background (P). i, inner nuclear layer; p, retinal pigmented epithelium (naturally pigmented and not labeled); r, retinal ganglion cell (RGC) layer. Scale bar 100 m in P and applies to all panels.

the formerly very large (OE/UE 30-fold) increase in peripherin expression occurred largely as a consequence of the extremely low levels of peripherin expression in control RGCs. The absolute amount of peripherins increase

(OE-UE) was actually comparable to that seen for NF-M. Similarly, the absolute changes that occurred in NF-L expression, which was the least abundant of the nIF mRNAs in both control and regenerating retina, and which had

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TABLE 1. Relative Increase in nIF mRNA Expression During Optic Nerve Regeneration (OE/UE)1 nIF Probe
Peripherin Xeltin XNIF NF-M NF-L

3 6 Days (n 4)
9.75 4.32 0.65 0.11a 0.37 0.02d 0.71 0.06b 0.61 0.14a

9 12 Days (n 4)
37.34 3.21d 1.31 0.13a 1.58 0.16a 2.56 0.20c 0.84 0.11

18 21 Days (n 5)
31.50 3.92d 1.65 0.16b 2.14 0.30b 2.64 0.22d 1.57 0.28

35 Days (n 2)
9.52 6.27 1.50 0.13 1.45 0.28 1.64 0.40 1.15 0.28

1 For each time period following an orbital nerve crush, the relative increase in nIF mRNA expression was determined by taking the ratio of the intensity of staining in the RGC layer between the operated (OE) and contralateral, unoperated eye (UE) within the same section. Per animal ratios were averaged across all the animals (n) in a given time period to determine the mean ratio SE given above. a,b,c,d Values differed signicantly from 1.0 (two-tailed t-test: P 0.05a; 0.02b; 0.01c; 0.002d).

Fig. 3. Frequency of RGC labeling with probes to the ve nIF mRNAs in control and operated eyes. Labeled RGC layer cells were scored according to intensity of labeling as weak, moderate, or strong. Percent labeled cells in each category are represented cumulatively as the mean SE. A: Values pooled from 10 contralateral control eyes at late stages of regeneration represent normal, unoperated eyes. Values in contralateral control (B), and operated eyes (C), 6 days after crush. Values in contralateral control (D) and operated eyes (E), 9 days after crush. The total percentages of labeled RGCs in the operated eye at this time were not signicantly different between peripherin, xeltin, XNIF, and NF-M (single factor ANOVA, df 6, P 0.48). Figure legend in A applies to all panels.

peripherin expression were, on average, balanced by the decreases in expression of the other four nIFs. Once the front of regeneration approached the optic chiasm (9 days and after), the combined levels of all nIF mRNAs approximately doubled and by 35 days these levels declined toward normal. Second, we estimated the relative stoichiometries of the mRNAs. Data from contralateral control eyes were pooled across time points, since these numbers were not statistically different. In these control eyes, the relative proportions of peripherin, xeltin, XNIF, NF-M, and NF-L were approximately 0:5:2:2:1, respectively. During the transitional period (3 6 days), relative proportions of each of the nIFs in the operated eye shifted toward those seen later as axons traversed the optic tract and tectum (9 21 days). During the peak, mid-regeneration phase, the relative proportions of peripherin, xeltin, XNIF, NF-M, and NF-L were approximately 1.5:3:2:3:0.5, respectively. By 35 days, the relative stoichiometries returned to normal. Thus, the relative increases and decreases in nIF expression that occur during regeneration can be seen as a shift in the relative stoichiometries of nIF mRNAs occurring within the context of changes in total nIF mRNA expression. Consequently, during the period when axons grew through the optic tract and tectum, the relative proportions of peripherin and NF-M increased, whereas those of xeltin and NF-L decreased, and that of XNIF remained constant.

shown similar relative fold increases as the other nIFs, were actually smaller than the others. Changes in the relative stoichiometries of each nIF mRNA. NFs are heteropolymers composed of each of the various nIF subunits in varying stoichiometries. We took advantage of the standardized conditions of in situ hybridization to obtain an estimate of the relative proportions of each mRNA present in RGCs during regeneration. This approach revealed that during regeneration there were three distinct proles of nIF expression (Table 2): 1) the normal prole, seen in controls and very late regenerates (35 days); 2) an early transitional regenerating prole, which occurred as axons traversed the lesion and grew down the optic nerve (3 6 days); and 3) a midstage regeneration prole, which happened as axons grew through the optic tract and covered the tectum (9 21 days). We rst estimated the total amount of all nIF mRNAs combined. During the rst 6 days of regeneration, the total amount of nIF mRNAs was about the same in regenerating RGCs as in contralateral controls, as increases in

Increased expression of peripherin and NFM protein without formation of aggregates

To follow changes in peripherin protein expression, we used three separate rabbit antisera: one older antiserum directed against the C-terminus (Gervasi et al., 2000), and two new antisera against the N-terminus. On Western blots of total spinal cord homogenate, the two new antisera recognized a single band, which co-migrated with that stained by the previously characterized antiserum (Fig. 5). Although the three antibodies differed in their tolerance of aldehyde xatives, they produced virtually identical staining patterns. We have illustrated our results for only the C-terminal antibody because it better tolerated aldehyde xation, and thus could be used more reliably on the same animals as those used for in situ hybridization. In addition, because the peripherin peptides used for immunization contained several serine residues that might serve as potential phosphorylation sites, we determined whether treatment of tissues with alkaline phos-

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269

Fig. 4. Differences in nIF mRNA levels between the operated (OE) and unoperated, contralateral control (UE) RGCs, as measured by video densitometry from in situ hybridizations performed under standardized conditions. For each nIF and time period after optic nerve crush, the difference in the mean intensity/pixel was determined between the UE and OE RGCs and then averaged among animals from a given time period (mean SE; n 4 animals for 3 6 days and

9 12 days; 5 for 18 21 days; 2 for 35 days). Levels of nIF mRNA remain relatively stable from 3 6 days postcrush, when regenerating axons cross the lesion and enter the optic nerve, from 9 12 days, when they have reached the optic chiasm, and from 18 21 days, when they reach and cover the tectum. Data within each of these time windows were therefore pooled.

TABLE 2. Relative nIF mRNA Stoichiometries During Optic Nerve Regeneration1 Fraction of the total nIF mRNA content contributed by each nIF (%) Time point
UE3 3 Days 6 Days 9 Days 12 Days 18 Days 21 Days 35 Days

Peripherin
0.0 0.4 4.1 0.3 16.0 0.1 15.5 2.4 19.4 1.5 15.5 1.2 12.4 3.4 5.8 4.5

Xeltin
48.7 4.4 52.5 3.3 39.2 0.9 29.5 5.8 31.0 2.8 32.5 5.6 34.8 2.5 46.0 9.6

XNIF
20.7 2.5 15.3 2.6 11.1 2.7 20.3 6.0 16.0 0.5 19.3 2.5 18.1 1.7 22.1 7.4

NF-M
19.5 2.4 18.9 5.9 28.7 5.2 29.8 5.2 28.9 1.4 25.4 3.2 26.9 3.2 19.0 8.4

NF-L
11.1 1.1 10.2 1.8 5.0 2.4 4.8 1.4 4.7 0.3 7.3 2.2 7.8 1.5 7.3 3.6

Relative2 fold increase in total nIF mRNA (OE/UE)

1.0 0.1 0.9 0.1 2.2 0.5 1.9 0.1 2.1 0.3 2.2 0.3 1.4 0.5

1 For each nIF mRNA at time points after nerve crush, the average staining intensity was rst corrected for slight variations in the length of each probe. The percent contribution to the total nIF mRNA content was then determined by dividing its average staining intensity/pixel by the total average staining intensity/pixel for all nIF mRNAs in that animal. These values were then averaged (mean SE) over all animals at a given time point and tabulated. 2 The relative fold increase in total nIF mRNA (mean SE) was determined by taking the total nIF staining intensity in the operated eye (OE) and dividing it by that of its contralateral, unoperated eye (UE) and then averaging these ratios (OE/UE) among animals. 3 Since values for unoperated, contralateral control eyes (UE) were not signicantly different between 12 and 35 days postcrush (single factor ANOVA, df 8; P 0.05), they were pooled.

phatase altered the patterns of staining of these antisera. Treatment of slides with alkaline phosphatase affected neither the intensity nor the distribution of either peripherin immunostaining or that of a phosphorylationindependent antibody to NF-M (RMO270), but enhanced the staining with the antibody to a dephosphorylated epitope of NF-M (S8), so that it resembled that of RMO270, and obliterated the staining with the S6 anti-

body to phosphorylated NF-M (data not shown). Thus, the patterns of staining with the peripherin antisera reected differences in protein expression rather than differences in levels of protein phosphorylation. In Xenopus, peripherin is expressed in both neurons and radial glia (Gervasi et al., 2000). Peripherin-stained axons could be easily distinguished from glia because axons run longitudinally along the axis of the optic nerve, are thin,

270

C. GERVASI ET AL. perimeter of the optic nerve, close to the glia limitans. Thus, in optic nerve cross-sections at any stage of regeneration, the youngest axons are found along the outside of the nerve, whereas axons that had regenerated earlier are found closer to the degenerating core. In the representative example shown (Fig. 6E, 21 days postcrush), peripherin staining was clearly present along the outer circumference of the nerve, close to the glia limitans, and absent from the older axons close to the core. In contrast, NF-M staining was abundant in the older axons and light to undetectable in younger ones (Fig. 6F). In summary, these results veried that increases in nIF protein expression and transport paralleled those seen at the mRNA level.

DISCUSSION
Fig. 5. Characterization of peripherin antisera on Western blots of total spinal cord homogenates. 1, preimmune serum for lane 2, diluted 1:500. 2, Xenopus peripherin N-terminal head domain peptide, rabbit antiserum #1, diluted 1:500. 3, preimmune serum for lane 4, diluted 1:500. 4, Xenopus peripherin N-terminal head domain peptide rabbit antiserum #2, diluted 1:2,000. 5, Previously characterized Xenopus peripherin C-terminal peptide rabbit antiserum, diluted 1:2,000.

and stain comparatively weakly, whereas glial processes run perpendicularly, are thick, and stain strongly, especially in glial end feet (Fig. 6A,E). In controls, the peripherin antibody only weakly stained an occasional RGC body and some rare axons within the RGC layer, optic nerve, and tract. During regeneration, peripherin-stained axons were readily visible in the retina, optic nerve, and tract on the experimental side of the animal. A representative example of such staining is shown at 28 days postcrush (Fig. 6A,C), which is after axons have covered the tectum, but before a normal retinotopic map is formed (Szaro et al., 1985) and nIF expression has returned to normal. Peripherin-containing bers were abundant in the left, regenerating optic nerve (Fig. 6A, left) and in the right, contralateral optic tract, which is populated by the regenerating axons that cross the chiasm (Fig. 6C). In contrast, the uninjured, contralateral nerve (Fig. 6A, right) and ipsilateral tract exhibited little or no peripherin staining, verifying that the increase in peripherin mRNA expression occurring during regeneration was accompanied by increased peripherin protein within the axon. Moreover, the types of NF aggregates that characterize peripherin staining in injured mammalian CNS (Beaulieu et al., 2002) were not present. NF-M protein expression also increased during regeneration. Staining for NF-M was stronger in the regenerating optic nerve (Fig. 6B, left) and tract (Fig. 6D), than in the contralateral, uninjured control nerve (Fig. 6B, right). This was true for sections stained either with an antibody directed against a phosphorylation-independent epitope of NF-M (RMO270), or with an antibody directed against a dephosphorylated epitope (S8) after sections were treated with alkaline phosphatase, arguing that the intensity difference between injured and control nerves was the result of differences in levels of NF-M protein expression. Immunostaining also demonstrated that during regeneration, increased peripherin expression occurred primarily within relatively younger regenerating axons. In Xenopus, regenerating optic axons grow along the outer

We have demonstrated that during successful optic nerve regeneration, the reemergence of each nIF subunit was marked by increases in mRNA expression that were above normal. Whereas peripherin expression increased soon after nerve crush, marking its return to RGCs, expression of the remaining nIF mRNAs increased only after they initially decreased in response to the injury. By monitoring expression of multiple nIF mRNAs, we demonstrated that both their total levels and relative stoichiometries varied with the phase of regeneration. For example, at the peak of nIF expression, which occurred as axons traversed the optic tract and tectum, RGC nIF mRNAs comprised relatively higher proportions of peripherin and NF-M, lower proportions of NF-L and xeltin, and about the same proportion of XNIF as did mature cells. These results extend prior observations of increased levels of peripherin- and -internexin-like nIF mRNAs during successful CNS regeneration in anamniotes by placing them in context with the other nIF subunits. They also suggest key differences between successfully regenerating CNS and PNS axons, which are discussed below.

Use of in situ hybridization to estimate relative levels of nIF mRNA expression in RGCs
Since nIFs form heteropolymers in vivo, their changes in expression during axon regeneration must undoubtedly alter the structural properties of NFs. Knowing the relative proportions of each nIF subunit is thus the rst step in understanding the function of these changes in NF composition at different stages of axon outgrowth. Since several of the nIF proteins are expressed in multiple cell types within the retina and optic nerve, we turned to in situ hybridization to distinguish the contribution of RGCs from that of other cell types. For example, peripherin is expressed in Mueller glial cells and in replicating stem cells of the ciliary margin (Gervasi et al., 2000), and the other nIFs are found sporadically in other retinal layers (Charnas et al., 1992; Zhao and Szaro, 1997a). Thus, RNase protection of the entire eye would have underrepresented the response within the RGCs themselves, which constitute less than 10% of the cells in the retina. Second, RNase protection would have provided no information on differences among classes of RGCs. We used in situ hybridization rather than immunocytochemistry for comparisons among nIFs, since the physical chemistry of RNA hybridization is more similar among different RNAs than is that of antibodyantigen interac-

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271

Fig. 6. Immunocytochemical localization of peripherin in regenerating axons. Transverse sections through the brains of frogs sacriced 28 days (AD) and 21 days (E,F) after optic nerve crush were immunostained with antibodies to a C-terminal peptide derived from Xenopus peripherin (diluted 1:1000: A,C,E) and to nonphosphorylated NF-M (S8 after phosphatase treatment, 1:1000: B,D; RMO270, 1:100: F). Dorsal side is up and the left side is at the left. A: At the optic chiasm, peripherin-containing bers run longitudinally in the left, crushed optic nerve (arrow). In contrast, in the right, uncrushed optic nerve, peripherin is found only in clusters of glia cells (g). B: On a neighboring section, regenerating bers growing outside the degenerating core (asterisk) stain strongly for NF-M. The degenerating core was not visible in A because of the level of sectioning. C: A more

posterior section of the same animal as in A and B shows peripherincontaining bers in the contralateral optic tract of the diencephalon (arrow). D: A neighboring section to that in C shows the distribution of NF-M for comparison. E: In the operated optic nerve close to the chiasm, peripherin is present in the youngest regenerating axons (arrows), which are located close to the glia limitans, but is absent in the oldest regenerating axons, which are found close to the degenerating core (asterisk). F: On a section adjacent to that shown in E, NF-M is present in older regenerating axons, but is absent from young regenerating axons close to the glial limitans (dots). g, glia; oc, optic chiasm; ot, optic tract; p, pial surface; v, ventricle. Scale bars 300 m in D (applies also to AC) and 100 m in F (applies also to E).

tions. To achieve greater uniformity when comparing levels of expression among nIFs, we used probes of comparable length, G/C content, and specic activity, and processed the slides under the same conditions. Levels of mRNA expression were estimated by video densitometry, a method that has previously been validated for digoxigenin-labeled cRNA probes (Hill et al., 1993; Matus-Leibovitch et al., 1995; Zhang et al., 1995). With this method, the main source of determinative error arises from levels of expression falling outside the linear range of the assay. We reduced this error by adjusting the conditions for hybridization and development to minimize the number of cells with saturated levels of staining and to maximize the number of cells staining above background, while maintaining conditions among the various probes. Since increasing development times increased background staining but had little effect on the number of labeled cells, we believe that our conditions were optimal. Nevertheless, because of these limitations, our measurements are best regarded as good semiquantitative estimates rather than

rigorous quantitative measures of levels of nIF mRNA expression.

Expression of nIF mRNAs in the unoperated eye

In situ hybridization enabled us to compare expression levels of the various nIFs among different RGCs. By far the most abundant nIF mRNA was that of xeltin. Even so, about 6 10% of cells remained consistently unlabeled, even during regeneration when xeltin expression rises. Between metamorphosis and adulthood, the number of nonganglionic cells (glia and amacrine cells) in the Xenopus RGC layer rises from 3% (Dunlop and Beazley, 1984) to 15-25% (Graydon and Giorgi, 1984; Dunlop and Beazley, 1984). The 6 10% of cells in the RGC layer that failed to label for xeltin in our juvenile frogs therefore most likely represent nonganglionic cells. Conversely, since in Xenopus, 1% of RGCs are displaced into the inner nuclear layer (Toth and Straznicky, 1989), some of the labeled cells in that layer may represent displaced ganglion cells.

272 Compared to the number of RGCs that expressed xeltin in normal retina, the numbers expressing the other type IV nIFs were much smaller. These cells may represent distinct populations of RGCs, which in Xenopus are identied by their size: Large, with diameters of 16 24 m (1% of adult RGCs); medium, with diameters of 1117 m (8-9% of adult RGCs); and small, with diameters of 4 10 m (90% of RGCs; Straznicky and Straznicky, 1988). Our count of 1.1% large RGCs, which expressed all four type IV nIFs, is consistent with these data. Since in Xenopus retina RGC soma size correlates well with axonal caliber (Dunlop and Beazley, 1984; Straznicky and Straznicky, 1988), and in all vertebrates the largest caliber axons have the greatest nIF contents, the expression of all four type IV nIFs in these cells may reect their need for large amounts of these cytoskeletal proteins to maintain axon caliber.

C. GERVASI ET AL. the peak period of regeneration, and then falls to normal levels after regeneration is complete (Glasgow et al., 1994; Asch et al., 1998). This return to normal depends on signals from the optic tectum (Niloff et al., 1998), and may involve the successful formation of a retinotopic map, since in the lizard Ctenophorus ornatus, where vision fails to return after optic nerve crush despite the regrowth of axons and the reformation of synaptic connections in visual targets (Beazley et al., 1997; Stirling et al., 1999), levels of geltin expression remain permanently elevated (Rodger et al., 2001). Thus, an initial suppression of type IV nIF expression, followed by an increase above normal during the peak period of regrowth may be general features of successful CNS regeneration. In further support of the idea that type IV nIFs are important for successful regeneration, the NF composition of CNS axons that fail to regenerate is often markedly different from that of axons that do, although the precise pattern of expression of nIFs varies among these systems (Oblinger and Lasek, 1988; Mikucki and Oblinger, 1991; Hoffman et al., 1993; McKerracher et al., 1993a,b). Although these studies implicate appropriate expression of type IV nIFs in the successful regenerative response to axonal injury, their expression is apparently not essential for axonal regrowth per se. For example, injured mammalian optic axons induced to grow through a peripheral nerve graft (McKerracher et al., 1993a), as well as Xenopus optic axons that fail to enter the optic tract but grow instead to the contralateral eye (Zhao and Szaro, 1995), fail to increase their expression of NF-M despite their ability to traverse relatively long distances. Also, transgenic mice lacking NFs nonetheless develop a nervous system and can regenerate their peripheral axons after axotomy (Zhu et al., 1997; Williamson et al., 1998; Elder et al., 1999; Jacomy et al., 1999; Levavasseur et al., 1999). Instead, type IV nIFs may be important for maintaining certain aspects of normal axonal growth, which, while not essential for life itself, may nonetheless be needed for the development of a fully viable and competitive organism in the wild. This idea is supported both by the observation that transgenic mice lacking NF-L regenerate peripheral axons more slowly than normal (Zhu et al., 1997) and by experiments in Xenopus embryos that demonstrate that loss of type IV nIFs inhibits axons from entering the phase of rapid axon elongation, which follows early neurite outgrowth (Walker et al., 2001). Although other studies have demonstrated that expression of type IV mRNAs increases during successful regeneration of CNS axons, ours is the rst to compare the relative expression levels of multiple nIFs simultaneously. Our results demonstrated that increased expression of type IV nIFs in successfully regenerating vertebrate RGCs is not limited to geltin and its orthologs, but extends to other type IV nIFs as well. In addition, the temporal progression of increases in nIF expression during regeneration clearly resembles that seen during development, arguing further that this progression is strongly linked to axonal outgrowth. That changes in mRNA expression accompany those seen in protein expression also argues that mRNA levels are likely to be a major determinant of nIF protein levels during successful RGC regeneration. The increased expression of NF-M during axonal elongation differs between regenerating anamniotic RGCs and mammalian PNS neurons. Whereas levels of NF-M ex-

Increased expression of peripherin mRNA during regeneration

Our nding that Xenopus peripherin expression increased dramatically soon after nerve injury extends similar ndings on the expression of peripherin and its orthologs during axon development and regeneration. For example, in both sh and Xenopus embryos, peripherin orthologs are present at the onset of axonogenesis and their expression precedes that of all type IV nIF proteins (Canger et al., 1998; Goldstone and Sharpe, 1998; Leake et al., 1999; Gervasi et al., 2000). Moreover, in mature sh and amphibian retina, where new neurons are continually added throughout life (Johns and Easter, 1977), peripherin orthologs are found only in the youngest RGCs, which can be identied by their proximity to the ciliary margin (Fuchs et al., 1994; Gervasi et al., 2000). In mammalian peripheral axon development, peripherin is also expressed at early stages of neurite outgrowth (Escurat et al., 1990; Troy et al., 1990b). During Xenopus axon development, peripherin is the earliest and most abundant nIF protein expressed throughout the entire nervous system, and is also abundant in growth cones. It later disappears from most of the CNS as axons mature (Gervasi et al., 2000; Undamatla and Szaro, 2001). The correlation between peripherin expression and axon outgrowth is especially strong during regeneration, when its expression increases dramatically in regenerating sh optic (Glasgow et al., 1992, 1994; Fuchs et al., 1994; Hall, 1994; Asch et al., 1998) and mammalian peripheral axons (Oblinger et al., 1989; Wong and Oblinger, 1990; Troy et al., 1990a). Thus, the dramatic return of peripherin expression to Xenopus RGCs during axonal regeneration further underscores the importance of peripherin to growing axons.

Regulation of type IV nIF mRNAs

Our study adds to the growing body of evidence that type IV nIFs may also play a substantial role in supporting the normal growth of developing and regenerating axons. The behavior of the type IV nIFs in Xenopus regenerating optic nerve resembles that reported in successfully regenerating CNS axons in other systems. For example, in transected spinal cord axons of lamprey, NF-M expression is initially suppressed and then rises in only those axons that successfully regenerate, while remaining suppressed in those that do not (Jacobs et al., 1997). Similarly, in sh optic nerve expression of the xeltin ortholog, geltin, is also initially suppressed, then rises above normal during

PERIPHERIN IN REGENERATING FROG OPTIC NERVE pression initially fall in both systems, they rise sooner, while axons are still elongating, in Xenopus RGCs than in mammalian PNS, where they remain suppressed until after regeneration is completed (Goldstein et al., 1988; Oblinger and Lasek, 1988; Hoffman and Cleveland, 1988; Muma et al., 1990; Wong and Oblinger, 1990; Troy et al., 1990a). One possibility is that this difference is tied to the expansion of axon caliber that occurs in large myelinated PNS axons after synaptogenesis, an expansion that is driven by increased nIF expression. Since in frog the vast majority of optic axons are unmyelinated and RGC axon caliber is only weakly correlated with NF content, there may be no need for mature RGCs to maintain high levels of NF-M mRNA expression. Thus, in both cases levels of NF-M after injury may change to promote axonal regrowth. In frog RGCs these levels may be higher than normal, whereas in regenerating PNS neurons they may be lower.

273 transgenic mice, for example, overexpression of NF-M and/or NF-H inhibits dendritic arborization of motor neurons, an effect that is alleviated by a concomitant increase in NF-L (Kong et al., 1998), suggesting that dendritic arborization is inuenced by the ratio of NF-M and NF-H to NF-L. Similarly, reducing levels of NF-H in mice leads to an increase in NF-M expression and a reduction by 1319% in the numbers of motor and sensory neurons (Rao et al., 1998), and knocking out peripherin leads to an increase in -internexin and a loss of sensory axons (Lariviere et al., 2002). In addition, changing nIF stoichiometry affects axon diameter (Wong et al., 1995; Marszalek et al., 1996; Xu et al., 1996; Elder et al., 1998) and overexpression of NF-M results in faster rates of slow axonal transport, which are also generally faster in growing than in mature axons (Xu and Tung, 2000). The absence from Xenopus regenerating optic axons of NF aggregates similar to those seen in mice that express abnormal levels of individual nIF subunits argues that the stoichiometric changes in Xenopus are part of a coordinated response to injury. Our data also support the idea that axonal growth and maturation are supported by NFs with subunit compositions especially suited for each stage of development. Since each NF subunit functions only in context with its co-assembled partners, experiments aimed at revealing NF function during axon development should test the contribution made by NFs of biologically relevant compositions rather than that made by each subunit separately.

Implications for the function of nIF proteins in axon development

Given the role of NFs as prominent structural components of the cytoskeleton, it is tempting to conclude that the increased expression of their subunit mRNAs during regeneration simply reects an increased need to supply these structural proteins to build a new axon. Since nIF proteins are relatively long lived and stable, normal axons may simply not need to synthesize as much mRNA to maintain high levels of protein in the axon. However, this hypothesis cannot explain the higher than normal levels of NF-M protein that were observed in regenerating axons. Instead, our estimates of the relative stoichiometries of the various nIF mRNAs during regeneration suggest that growing axons contain a different composition of nIFs to support various phases of axonal regrowth. Although the composition of mRNAs in the RGCs may not precisely reect the composition of individual NFs in the optic nerve, the tight regulation of mRNA composition during Xenopus optic nerve regeneration suggests that certain combinations of nIFs are characteristic of each phase of axon regeneration. On this basis, optic nerve regeneration in Xenopus can be divided into three distinct phases: 1) an initial period of axonal growth through the optic nerve, which is characterized by increased expression of peripherin and decreased expression of type IV nIFs, so that the total nIF mRNA content of RGCs is close to normal; 2) a second period occurring as axons cross the optic tract and tectum, which is characterized by a doubling of the total level of nIF mRNA and a shift in nIF composition to become relatively enriched for peripherin and NF-M; and 3) a return to normal levels of expression and relative mixtures of the various nIF mRNAs. These phases t comfortably within the general scheme of changes in nIF expression reported for other developing and regenerating systems in which newly extended and growing axons are enriched for type III nIFs, elongating axons add type IV nIFs, such as NF-M and -internexin, and maturing axons express additional type IV nIFs and extensively phosphorylated NF-M and -H (Dahl et al., 1986; Bennett, 1987; Carden et al., 1987; Szaro et al., 1989). Although this is the rst report of stoichiometric changes in nIF mRNAs during successful CNS regeneration, other studies have indicated that the relative mix of nIFs is important for proper neuronal development. In

ACKNOWLEDGMENTS
We thank Drs. Suzannah and David Tieman for the use of photographic equipment. We also thank Drs. Suzannah Tieman and John Schmidt for critical comments on the article.

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