Gervasi Et Al 2003 Nf in ONR

7/27/2019 Gervasi Et Al 2003 Nf in ONR

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Increased Expression of MultipleNeurofilament mRNAs during

Regeneration of Vertebrate CentralNervous System Axons

CHRISTINE GERVASI, AMAR THYAGARAJAN, AND BEN G. SZARO*

Department of Biological Sciences and the Center for Neuroscience Research, StateUniversity of New YorkUniversity at Albany, Albany, New York 12222

ABSTRACTCharacteristic changes in the expression of neuronal intermediate filaments (nIFs), an

abundant cytoskeletal component of vertebrate axons, accompany successful axon regener-ation. In mammalian regenerating PNS, expression of nIFs that are characteristic of matureneurons becomes suppressed throughout regeneration, whereas that of peripherin, which isabundant in developing axons, increases. Comparable changes are absent from mammalianinjured CNS; but in goldfish and lamprey CNS, expression of several nIFs increases duringaxon regrowth. To obtain a broader view of the nIF response of successfully regeneratingvertebrate CNS, in situ hybridization and video densitometry were used to track multiple nIFmRNAs during optic axon regeneration in Xenopus laevis. As in other successfully regener-ating systems, peripherin expression increased rapidly after injury and expression of thosenIFs characteristic of mature retinal ganglion cells decreased. Unlike the decrease in nIFmRNAs of regenerating PNS, that ofXenopus retinal ganglion cells was transient, with mostnIF 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 ofperipherin and NF-M increased above normal, whereas proportions of xefiltin and NF-Ldecreased and that of XNIF remained the same. The increases in peripherin and NF-MmRNAs were accompanied by increases in protein. These results are consistent with thehypothesis that successful axon regeneration involves changes in nIF subunit compositionconducive to growth and argue that a successful injury response differs between CNS andPNS. J. Comp. Neurol. 461:262275, 2003. 2003 Wiley-Liss, Inc.

Indexing terms: peripherin; intermediate filament; Xenopus laevis; -internexin; optic nerve

Because the optic nerves of fish (Sperry, 1948) and frog(Sperry, 1944; Gaze, 1959) regenerate, they are excellent

systems for studying genes involved in successful recoveryfrom CNS injury. Neuronal intermediate filament (nIF)genes, whose proteins make neurofilaments (NFs), areamong the most abundantly expressed genes that arehighly regulated during regeneration. Vertebrate nIFgenes have been classified according to similarities inamino acid sequence and intron position as either type IIIor type IV intermediate filaments (Steinert and Roop,1988). In Xenopus, as in mammals, all nIFs are classifiedas type IV, with the exception of peripherin, which is typeIII. The stability of NFs and their great tensile strengthhave suggested that the changes in nIF gene expression ateach phase of axon outgrowth may reflect the growing

axons shifting demands for structural stability (Schwartzet al., 1990; Fliegner et al., 1994; Fuchs et al., 1994;

Glasgow et al., 1994). For example, at early stages ofoutgrowth, NFs with immature compositions may facili-tate 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: [email protected]

Received 14 November 2002; Revised 17 February 2003; Accepted 24February 2003

DOI 10.1002/cne.10695

Published online the week of May 5, 2003 in Wiley InterScience (www.interscience.wiley.com).

THE JOURNAL OF COMPARATIVE NEUROLOGY 461:262275 (2003)

2003 WILEY-LISS, INC.


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without inhibiting it (Walker et al., 2001), whereas aftersynaptogenesis NFs of mature composition may furtherenhance axon strength and caliber.

Consistent with this view, two nIF subunits character-istic of immature developing axons [peripherin (Parysekand Goldman, 1988; Troy et al., 1990b) and -internexin(Fliegner et al., 1994)] increase in expression during mam-

malian PNS regeneration (Oblinger et al., 1989; Troy etal., 1990a; McGraw et al., 2002), whereas three subunitsmost abundant in mature axons [low (NF-L), medium(NF-M), and high (NF-H) molecular mass NF triplet pro-teins] decrease (Wong and Oblinger, 1990). Peripherin-and -internexin-like nIFs also increase during goldfishoptic nerve regeneration (Glasgow et al., 1992; Fuchs etal., 1994), but comparable changes in nIF expression failto occur in injured mammalian CNS (Mikucki andOblinger, 1991), arguing that they are linked to successfulrecovery from injury. Moreover, the relative magnitudesof changes in the expression of individual subunits mustbe highly regulated, since substantially altering nIF stoi-chiometries can cause NFs to aggregate and axons todegenerate (Beaulieu et al., 1999, 2000).

The regenerating optic nerve of juvenile Xenopus laevisfrogs is a good system for studying nIF expression duringCNS regeneration, since the time-course of regeneration,as well as changes in the expression of several nIF pro-teins, have already been characterized for it (Szaro et al.,1985; Zhao and Szaro, 1994, 1995, 1997b): Newly regen-erating axons first cross the lesion between 5 and 6 daysafter an orbital nerve crush, and approach the chiasm at 9days. At 15 days the first axons reach the optic tectum andby 18 days they cover it. For several weeks afterward,innervation of the tectum increases, eventually restoringa retinotopic map. For the first week after injury, expres-sion of NF-M and two -internexin-like nIF proteins, xe-filtin and XNIF, is suppressed and then subsequently

increases, but only in those axons that successfully enterthe optic tract.Our current study had two principal objectives. The first

was to determine whether peripherin expression increasesduring Xenopus optic nerve regeneration, as it does afterinjury in the goldfish optic nerve and mammalian PNS. Indeveloping Xenopus retinal ganglion cells (RGCs), periph-erin is the most abundant nIF and it disappears as RGCsmature (Gervasi et al., 2000). Thus, demonstrating thatits expression returns during regeneration would furtherstrengthen the argument that peripherin-like, type IIInIFs are important for axonal growth. The second objec-tive was to estimate the relative magnitudes of changes inmultiple nIFs during regeneration. This would better in-dicate which ones may promote successful regeneration

and would help identify possible differences between re-generating CNS and PNS. Such differences might be ex-pected, since mature, uninjured CNS neurons normallyhave much lower NF contents than do PNS neurons.

We accomplished these objectives using in situ hybrid-ization and video densitometry. In addition, we immuno-stained regenerating axons with antibodies to peripherinand NF-M to determine whether increases in nIF mRNAsproduced increases in axonally transported protein andNF aggregation. As in mammalian regenerating PNS, wefound that during early phases of regeneration, levels ofperipherin mRNA increased and those of other nIFs de-creased. Unlike mammalian PNS, however, levels of sev-eral nIF mRNAs, including NF-M, XNIF, and xefiltin, also

increased above normal well before regeneration was fin-ished. Increased levels of axonally transported peripherinand NF-M did not cause NF aggregates, arguing thatchanges in nIF subunit stoichiometries were balanced toavoid disruption of NFs. At each phase of regeneration,both the level of all nIF mRNAs combined and their rela-tive proportions changed, arguing that successful CNS

axon regeneration involves both a change in the totalamount of nIFs expressed and a unique mix of nIFs con-ducive to growth.

MATERIALS AND METHODS

Optic nerve surgery

Periodic albino Xenopus laevis (Hoperskaya, 1975;Tompkins, 1977) were bred in our laboratory and usedwithin 3 months after metamorphosis. Frogs were anes-thetized by immersion in 0.1% 3-aminobenzoic acid ethyl-ester (Sigma, St. Louis, MO), according to protocols ap-proved by our institutional Animal Care and UseCommittee and conforming to NIH guidelines, and their

left optic nerve was crushed at the orbit as describedpreviously (Zhao and Szaro, 1994). Our present study usedjuvenile frogs from the same stock, reared under similarconditions, and operated upon in the same manner asthose used previously (Zhao and Szaro, 1994). In addition,the reappearance of NF-M immunoreactivity within theregenerating optic nerves of our animals (data not shown)followed the same time-course as in these earlier studies.Consequently, we were confident that the time-course ofregeneration in our study was similar to what had beenpreviously established.

RNase protection assay

Total RNA was isolated from eyes using a Masterpure

RNA Purification kit (Epicenter Technologies, Madison,WI) and pooled as follows: 1) 12 unoperated eyes from sixanimals; 2) 12 eyes whose optic nerve had been crushed 9days before; and 3) 12 unoperated, contralateral controleyes from these same animals. RNase protection assayswere performed using an RPAIII kit (Ambion, Austin, TX)and the following probes, which were synthesized andlabeled by in vitro transcription with 32P: Xenopus periph-erin (spanning nt #14352012; Sharpe et al., 1989); Xeno-

pus 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; Gen-Bank M25697). Aliquots of 10 g of total RNA from eachsample were hybridized simultaneously with 7 104 cpmeach 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. Thesingle-stranded, unprotected RNA was then digested withRNase T1 (100 U/ml) for 1 hour at room temperature. Theprotected RNA fragments were separated on a 5%acrylamide/8 M urea denaturing gel, which was dried andexposed (15 hours) first to X-ray film (XAR-1 film, Kodak,Rochester, NY) with an intensifying screen at 70C, andthen to a phosphoimaging screen (18 and 27 hours;Amersham-Molecular Dynamics, Piscataway, NJ). The ex-posures on the phosphoimaging screen were then read andquantitated using a Storm 860 Image Scanner(Amersham-Molecular Dynamics) running Image Quant(v. 5.0) software. Background was corrected in each lane

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by the histogram peak method. The ratios for each probebetween the operated and contralateral control eye sam-ples were equal for the 18 and 27 hours exposures, whichconfirmed that exposures fell within the linear range ofthe assay.

Preparation of frozen histological sections

At various intervals after crush, the frogs were anesthe-tized as before and fixed by intracardial perfusion with 4%paraformaldehyde/10% sucrose in amphibian Ringers so-lution (113 mM NaCl, 2 mM KCl, 1.1 mM CaCl2, 1.2 mMNaHCO3). Sections were cut transversely at 12 m asdescribed in Gervasi et al. (2000) and thaw-mounted ontocommercially available glass slides treated with aminoal-kylsilane (Polysciences, Warrington, PA). Adjacent sec-tions through the eye were collected successively on sep-arate slides to permit a fair comparison of multiple probeson each animal.

Sample slides containing sections of optic nerve fromeach animal were stained with cresyl violet to confirm thatthe optic nerve had been crushed successfully, i.e., de-

pending on the time of sacrifice, that the optic nerve eithershowed signs of degeneration (3 and 6 days postcrush) orcontained the halo of new axons that is characteristic ofregenerating optic nerves (9 days and later; Zhao andSzaro, 1994). Any animal not meeting these criteria wasexcluded from further analysis.

Preparation of cRNA probes forin situ hybridization

Digoxigenin-labeled cRNA probes were transcribed(Gervasi et al., 2000) using clones generated in our labo-ratory and a Genius 4 Kit (Roche Molecular Biochemicals,Indianapolis, IN). Plasmids containing full-length Xeno-

pus peripherin, NF-L, XNIF, and xefiltin (Zhao and Szaro,

1997a; Gervasi et al., 2000) and a partial length XenopusNF-M (clone 6B-1a of NF-M1, Gervasi and Szaro, 1997)were linearized for in vitro transcription to generatedigoxigenin-labeled antisense cRNA probes of comparablelength and activity (2.1 kb for XNIF, xefiltin and NF-L, 2.0kb for peripherin, 1.7 kb for NF-M). For a negative control,a sense cRNA to peripherin was transcribed. The amountof probe synthesized was determined by measuring fluo-rescence in a TD-700 spectrofluorometer (Turner Designs)using a RiboGreen Quantitation kit (Molecular Probes,Eugene, OR). The quality and size of the probes wereverified on formaldehyde/agarose gels (Davis et al., 1994).To verify that probes were labeled, a known mass wasspotted onto nitrocellulose and then assayed with a horse-radish peroxidase labeled anti-digoxigenin antibody (Ger-

vasi et al., 2000). To improve penetration into the tissue,probes were chemically fragmented into lengths of approx-imately 300 nucleotides (Angerer et al., 1987).

In situ hybridization

In situ hybridization on sectioned tissue was performedessentially as described previously (Zhao and Szaro,1997a). Pilot experiments were used to find conditionsthat reliably produced a signal that minimized saturationof the labeling (intense black staining) and backgroundwhile maximizing the sensitivity of the assay (the numberof RGCs that were labeled at least lightly). In general,increasing developing time and probe concentration above

the conditions described below raised background withoutincreasing the number or the intensity of labeled RGCs.

Slides were hybridized with 0.25 g/ml of cRNA probeat 55C overnight. High stringency washes were per-formed in 0.1 standard saline citrate at 60 64C. Thedigoxigenin signal was visualized by immunostaining slideswith anti-digoxigenin Fab fragments coupled to alkalinephosphatase and then developing them in 4-nitro blue tet-razolium chloride/5-bromo-4-chloro-3-indolyl phosphate(Genius 3 kit; Roche Molecular Biochemicals). For slidesto be compared, the conditions of immunostaining anddevelopment were standardized. Sections were mountedunder glass coverslips in aqueous mounting medium(Shandon Lipshaw, Pittsburgh, PA).

Morphological analysis and intensitymeasurements

Slides processed for in situ hybridization were viewedon a Leitz Laborlux compound microscope with a 25objective (Leitz PL Fluotar, 0.6 NA). Images of retinalsections were captured with a Dage CCD300T-RC videocamera. To set the limits of the range of the intensitymeasurements, the lightest and darkest regions of thesections of the most intensely stained slides were used forsetting the gain and black levels on the camera in order tominimize the number of saturated pixels while maximiz-ing the range over which the intensities could be collected.This was done while also adjusting the level of illumina-tion and the analog contrast on the frame grabber, so thatthe morphological details captured by the computerthrough the camera faithfully reproduced those seen byeye through the microscope. These settings were then keptconstant for all images to be compared. This ensured thatthe light absorbed by the color precipitant present on theslides fell within the linear range of intensities captured

in the computer image and that separate images could bereliably compared.

With the morphometric analysis functions of Meta-morph (v. 4.0, Universal Imaging, West Chester, PA), theaverage labeling intensity per pixel was then measuredwithin the RGC layer and corrected for background (av-erage labeling intensity over the neighboring, unstainedinner nuclear layer). For each retina, 12 such background-corrected measurements were averaged. These measure-ments were then averaged among animals to obtain themean value (SE) for each time point. Measurementswere transferred to an Excel spreadsheet, which was usedfor all further statistical analyses.

To allow for fair comparisons among separate animalsand probes in this analysis, consecutive slides from eachanimal were hybridized with equivalent amounts ofprobes to each of the five nIF mRNAs and processed inparallel. To ensure the consistency of the comparisons,measurements were taken from only the central portion ofthe retina. In all, two animals each at 3, 6, 9, 12, 18, and35 days postcrush and three animals at 21 days postcrushwere used. Several additional animals (one each at 3, 18,21, 28, 35 days postcrush hybridized with probes to all fivenIFs; and one each at 6, 9, 12, and 15 days postcrushhybridized with probes to just peripherin, xefiltin, andXNIF) showed equivalent staining intensities, but werenot included in the quantitative analyses because theywere not processed in parallel.

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Quantitative shifts in the distribution ofnIF mRNAs among RGCs

For determining the percentage of cells within the RGClayer expressing a given nIF mRNA, sections were viewedon a Leitz Laborlux compound microscope with a 25objective. Within a field of view, all cells within the RGC

layer were scored as either unlabeled (i.e., labeled belowbackground), weakly (labeled just above background),moderately (visible distinctly and medium blue in color),or strongly labeled (dark blue and labeled over a signifi-cant area of the cell body; Jacobs et al., 1997). Thesenumbers were normalized against the total number ofcells within the RGC layer for each field of view. Forcomparisons between animals, cells were scored withincomparable regions of the retina and in the same numberof experimental animals as used for the analyses de-scribed above.

Immunocytochemistry

Three separate polyclonal rabbit antisera were used todetect Xenopus peripherin. One of these, made against asynthetic peptide (QVVTESRKEQSSEGE) derived fromthe C-terminus of Xenopus peripherin, had been charac-terized previously in developing Xenopus laevis (Gervasiet al., 2000). The other two were new antisera made inseparate rabbits to a synthetic peptide (RHFGSPSPG-PSSR) derived from the N-terminal head domain of Xeno-

pus peripherin. The peptides were synthesized, conju-gated at their C-terminus to keyhole limpet hemocyanin,and injected into New Zealand rabbits at a commercialfacility (Covance, Richmond, CA). Three additional, pre-viously characterized monoclonal antibodies to XenopusNF-M were also used: RMO270, which recognizes aC-terminal, phosphorylation-independent epitope (Szaroet 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 spinalcord. The secondary antibody was horseradish peroxidaseconjugated, goat antirabbit (Kirkegaard and Perry, Gaith-ersburg, MD) and the chromogen was 4-chloro-1-napthol.

The sections used for immunocytochemistry were fromthe same animals as those used for in situ hybridization,which permitted a better comparison between the distri-butions of protein and mRNA. Immunocytochemistry wasperformed essentially as described in Szaro and Gainer(1988), using a peroxidase-conjugated secondary antibody(2 g/ml, Kirkegaard and Perry), a glucose oxidase reac-tion as a source of peroxide, and diaminobenzidine and

nickel chloride as chromogens.To test whether either epitope masking or phosphory-lation interfered with immunocytochemical staining withthe peripherin antisera, some slides were treated witheither trypsin or phosphatase, respectively. Trypsin orphosphatase was applied after blocking nonspecific bind-ing sites in 10% fetal calf serum. For the trypsin treat-ment, slides were incubated in 1 mg/ml of porcine trypsintype II (Sigma) at 37C for 5 minutes. For the phosphatasetreatment, slides were incubated at 37C for 3 hours in E.coli alkaline phosphatase type III (Sigma) at a concentra-tion of 4 U/ml of buffer (1 mM ZnSO4, 100 mM NaCl, 50mM Tris HCl, pH 8.0). Slides were then incubated in 10%fetal calf serum, followed by a second incubation in 5%

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

RESULTS

RNase protection demonstrated increases inperipherin and NF-M mRNAs during

regeneration

To determine whether peripherin mRNA expression in-creases during regeneration, we performed an RNase pro-

tection assay (Fig. 1) and compared changes in the levelsof peripherin mRNA with those of two other mRNAs:Xenopus NF-M, another nIF protein expressed duringaxon elongation, and Xenopus Elongation Factor 1- (EF1-), a constitutively expressed gene whose expression canbe used to monitor overall levels of transcription in Xeno-

pus. Total RNA was isolated from operated (Fig. 1D) andcontralateral control eyes (Fig. 1C) at 9 days after nervecrush (which coincides with the arrival of axons at thechiasm), as well as from unoperated animals (Fig. 1B).The ratio (OE/UE) of peripherin and NF-M mRNA levelsbetween the operated eyes (OE) and the contralateralcontrol eyes (UE) demonstrated increases of 2.5- and 1.4-fold, respectively. These increases were greater than theslight (1.1-fold) increase that was seen for EF1-mRNA, a

measure of the general increase in RNA levels that occurin eyes following axotomy (Burrell et al., 1978). In addi-tion to the differential increases in peripherin and NF-MmRNAs in the operated eyes in experimental animals,there was an increase of 1.5-fold in both mRNAs (1.49 and1.51 for peripherin and NF-M, respectively) between thecontralateral control eyes in the operated animals andeyes of unoperated, naive animals. This result demon-strated that the different responses to optic nerve crushbetween peripherin and NF-M were specific to the crushinjury, as opposed to general trauma, and also under-scored the utility of using the contralateral eye as aninternal control to obtain a true picture of this nerve crushresponse.

Fig. 1. Increases in NF-M and peripherin mRNAs at 9 days post-crush demonstrated by RNase protection. Ten g of total RNA pooledfrom 12 eyes were hybridized simultaneously with 32P-labeled RNAprobes against Xenopus NF-M and peripherin in one tube, and 3 g ofRNA were hybridized with a probe to Elongation Factor 1- (EF1-) ina separate tube (B,C,D). A: Probe hybridized against yeast RNA,demonstrating that the RNase digestion went to completion. B: RNAfrom naive, unoperated eyes. C: RNA from the contralateral controleye, 9 days after optic nerve crush. D: RNA from the operated eye, 9days postcrush. After correcting for a slight increase (10%) in theexpression of EF1- mRNA, the ratios of RNA expression between theoperated and contralateral control eyes (D,C) were 1.3 for NF-M and2.3 for peripherin.



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Patterns of expression of nIF mRNAs innormal retina

To learn whether changes in nIF expression duringregeneration occurred evenly throughout the retina, asopposed to within subpopulations of RGCs, we turned to insitu hybridization. Before we could address this question

in regenerating RGCs, it was necessary to characterize inmore detail the pattern of expression of each nIF in nor-mal retina. The anatomical distribution of nIF mRNAs inthe contralateral, unoperated retinas of regenerating an-imals were similar to those of normal, unoperated animalsand remained constant throughout regeneration (an ex-ample of which is shown at 28 days postcrush, Fig. 2AE).Cell counts for unoperated eyes in Figure 3A were pooledfrom several regenerating animals.

RGCs of unoperated eyes contained mostly type IV nIFsand little to no peripherin. Peripherin mRNA was confinedprimarily to ciliary margin, which contains proliferatingneural cells (not shown, but see Gervasi et al., 2000). Thisstaining served as an internal control for hybridizationconditions for peripherin. In the RGC layer, only 2% of thecells were labeled for peripherin, and these only weakly(Figs. 2A, 3A). Of the type IV nIF mRNAs, xefiltin was themost abundant, labeling 89% of cells, three-fourths ofwhich were moderately to strongly labeled (Figs. 2B, 3A).The remaining type IV nIFs labeled smaller populations ofcells. XNIF was found in 42% of cells, of which only one-third were moderately to strongly labeled (Figs. 2C, 3A).The anatomical distributions of NF-M and NF-L were verysimilar, 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 morecells moderately to strongly than did NF-L (Fig. 3A).

The only subpopulation of RGCs we could unambigu-ously identify was the largest RGCs, which have cell di-

ameters exceeding twice that of other RGCs. In our study,cells matching this size criterion numbered from 12% ofRGCs, which agrees with the published counts ofStraznicky and Straznicky (1988). All such RGCs werelabeled for xefiltin, XNIF, NF-M, and NF-L.

Patterns of nIF expression inregenerating RGCs

Since nIF mRNAs are differentially distributed amongnormal RGCs, we first examined whether changes in nIFexpression during regeneration were limited to those sub-populations that normally express each nIF. Representa-tive examples of these patterns in sections of regeneratingretina are illustrated in Figure 2 for 6 (F J) and 12 (KO)

days.At the earlier stage of regeneration (6 days), which issoon after axons cross the lesion, fewer RGCs than normalexpressed xefiltin, XNIF, and NF-L, and the level of stain-ing in those cells that were labeled also fell (see cell countsin Fig. 3B,C). In contrast, more RGCs than normal ex-pressed peripherin and NF-M at this time. For peripherin,the level of staining in labeled cells increased, but forNF-M, although the number of labeled cells increased, theoverall level of staining was reduced. To explain this re-sult, decreased expression of NF-M in some RGCs musthave been accompanied by a reexpression of detectablelevels of mRNA in populations of cells that had previouslyfallen below the threshold of detection in normal retina.

From 9 days on the percentage of RGCs expressing eachnIF increased, matching the total percentage of cells inthe RGC layer labeling for any type IV nIF. The percent-age of cells expressing peripherin (78%), xefiltin (93%),XNIF (77%), or NF-M (88%) were indistinguishable fromeach 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 xefiltin 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 anyof these nIFs were likely to be displaced amacrine or glialcells (Dunlop and Beazley, 1984). Thus, during the peak ofregeneration, as axons crossed the optic tract and tectum,levels of these nIF mRNAs increased throughout the vastmajority, if not all, RGCs. Similarly, although NF-L ap-peared in fewer RGCs than did the other nIFs, its distri-bution nevertheless also expanded (Fig. 3E).

Quantitative estimates of nIF mRNAexpression in regenerating RGCs

To estimate more quantitatively the changes in nIFmRNA levels during regeneration, we used a colorimetricassay based on video densitometric measurements of theintensity of digoxigenin label in the RGC layer understandardized conditions (see Materials and Methods).These data are presented in three different ways, each ofwhich emphasizes a different feature of the changes in nIFexpression seen during regeneration: 1) changes in therelative ratio of expression of each nIF between the oper-ated and contralateral control eye (OE/UE); 2) changes inthe 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 be-tween operated and control RGCs. First, we measuredthe ratio (OE/UE) between nIF mRNA labeling in theRGC layer of operated (OE) and contralateral control (UE)

eyes within the same animal (Table 1). This method nor-malized for any slight variations that occurred in assayconditions and emphasized the relative fold changes inexpression during regeneration. Peripherin showed theearliest 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 21days. In contrast, the levels of the type IV nIF mRNAsdropped during the first week after nerve crush (3 6days), but then also rose above normal during the next 2weeks. Although these increases were significantlygreater than 1-fold (t-test, better thanP 0.05), they wererelatively smaller than for peripherin, ranging, for exam-ple at 9 12 days, from a 1.3-fold increase for xefiltin to a2.6-fold increase for NF-M. In addition, the increase in

NF-L expression took longer to occur than that of theother nIFs, appearing at 18 21 days postcrush instead ofat 9 12 days. By 5 weeks postcrush, levels of all five nIFsbegan to decline toward normal.

Changes in the magnitude of expression of each nIF(OE-UE) during regeneration. Although the OE/UEratio for each nIF mRNA demonstrated that the relativeexpression changed over time, it obscured the absolutemagnitude of these changes, which was better revealed bymeasuring the differences in expression between operatedand contralateral control eyes (OE-UE; Fig. 4). The time-course and statistical significance of the changes that oc-curred during regeneration were comparable with bothmethods; however, the difference method revealed that



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the formerly very large (OE/UE 30-fold) increase inperipherin expression occurred largely as a consequence ofthe extremely low levels of peripherin expression in con-trol 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-Lexpression, which was the least abundant of the nIF mRNAsin both control and regenerating retina, and which had

Fig. 2. Expression of nIF mRNA in the contralateral, unoperatedcontrol eye at late stages of regeneration (AE) and in the operatedeye 6 (FJ) and 12 (KO) days after optic nerve crush. Neighboringtransverse sections were hybridized with digoxigenin-labeled cRNAprobes to peripherin (A,F,K), xefiltin (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 showedno label above background (P). i, inner nuclear layer; p, retinal pig-mented epithelium (naturally pigmented and not labeled); r, retinalganglion cell (RGC) layer. Scale bar 100 m in P and applies to allpanels.



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shown similar relative fold increases as the other nIFs,were actually smaller than the others.

Changes in the relative stoichiometries of each nIFmRNA. NFs are heteropolymers composed of each of thevarious nIF subunits in varying stoichiometries. We tookadvantage of the standardized conditions of in situ hybrid-

ization to obtain an estimate of the relative proportions ofeach mRNA present in RGCs during regeneration. Thisapproach revealed that during regeneration there werethree distinct profiles of nIF expression (Table 2): 1) thenormal profile, seen in controls and very late regenerates(35 days); 2) an early transitional regenerating profile,which occurred as axons traversed the lesion and grewdown the optic nerve (3 6 days); and 3) a midstage regen-eration profile, which happened as axons grew throughthe optic tract and covered the tectum (9 21 days).

We first estimated the total amount of all nIF mRNAscombined. During the first 6 days of regeneration, thetotal amount of nIF mRNAs was about the same in regen-erating RGCs as in contralateral controls, as increases in

peripherin expression were, on average, balanced by thedecreases in expression of the other four nIFs. Once thefront of regeneration approached the optic chiasm (9 daysand after), the combined levels of all nIF mRNAs approx-imately doubled and by 35 days these levels declined to-ward normal.

Second, we estimated the relative stoichiometries of themRNAs. Data from contralateral control eyes were pooledacross time points, since these numbers were not statisti-cally different. In these control eyes, the relative propor-tions of peripherin, xefiltin, XNIF, NF-M, and NF-L wereapproximately 0:5:2:2:1, respectively. During the transi-tional period (3 6 days), relative proportions of each of thenIFs in the operated eye shifted toward those seen later asaxons traversed the optic tract and tectum (9 21 days).During the peak, mid-regeneration phase, the relativeproportions of peripherin, xefiltin, XNIF, NF-M, and NF-Lwere approximately 1.5:3:2:3:0.5, respectively. By 35 days,the relative stoichiometries returned to normal. Thus, therelative increases and decreases in nIF expression thatoccur during regeneration can be seen as a shift in therelative stoichiometries of nIF mRNAs occurring withinthe context of changes in total nIF mRNA expression.Consequently, during the period when axons grewthrough the optic tract and tectum, the relative propor-tions of peripherin and NF-M increased, whereas those ofxefiltin and NF-L decreased, and that of XNIF remainedconstant.

Increased expression of peripherin and NF-M protein without formation of aggregates

To follow changes in peripherin protein expression, weused three separate rabbit antisera: one older antiserumdirected against the C-terminus (Gervasi et al., 2000), andtwo new antisera against the N-terminus. On Western

blots of total spinal cord homogenate, the two new anti-sera recognized a single band, which co-migrated withthat stained by the previously characterized antiserum(Fig. 5). Although the three antibodies differed in theirtolerance of aldehyde fixatives, they produced virtuallyidentical staining patterns. We have illustrated our re-sults for only the C-terminal antibody because it bettertolerated aldehyde fixation, and thus could be used morereliably on the same animals as those used for in situhybridization.

In addition, because the peripherin peptides used forimmunization contained several serine residues thatmight serve as potential phosphorylation sites, we deter-mined whether treatment of tissues with alkaline phos-

TABLE 1. Relative Increase in nIF mRNA Expression DuringOptic Nerve Regeneration (OE/UE)1

nIFProbe

3 6 Days(n 4)

9 12 Days(n 4)

18 21 Days(n 5)

35 Days(n 2)

Per ipheri n 9.75 4.32 37.34 3.21d 31.50 3.92d 9.52 6.27Xefiltin 0.65 0.11a 1.31 0.13a 1.65 0.16b 1.50 0.13XNIF 0.37 0.02d 1.58 0.16a 2.14 0.30b 1.45 0.28NF-M 0.71 0.06b 2.56 0.20c 2.64 0.22d 1.64 0.40NF-L 0.61 0.14a 0.84 0.11 1.57 0.28 1.15 0.28

1For each time period following an orbital nerve crush, the relative increase in nIFmRNA expression was determined by taking the ratio of the intensity of staining in theRGC layer between the operated (OE) and contralateral, unoperated eye (UE) withinthe same section. Per animal ratios were averaged across all the animals (n) in a giventime period to determine the mean ratio SE given above.a,b,c,dValues differed significantly 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 five nIFmRNAs in control and operated eyes. Labeled RGC layer cells werescored according to intensity of labeling as weak, moderate, or strong.Percent labeled cells in each category are represented cumulatively asthe mean SE. A: Values pooled from 10 contralateral control eyes at

late stages of regeneration represent normal, unoperated eyes. Valuesin contralateral control (B), and operated eyes (C), 6 days after crush.

Values in contralateral control (D) and operated eyes (E), 9 days aftercrush. The total percentages of labeled RGCs in the operated eye atthis time were not significantly different between peripherin, xefiltin,

XNIF, and NF-M (single factor ANOVA, df 6, P 0.48). Figurelegend in A applies to all panels.



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phatase altered the patterns of staining of these antisera.Treatment of slides with alkaline phosphatase affectedneither the intensity nor the distribution of either periph-erin immunostaining or that of a phosphorylation-independent antibody to NF-M (RMO270), but enhancedthe staining with the antibody to a dephosphorylatedepitope of NF-M (S8), so that it resembled that ofRMO270, and obliterated the staining with the S6 anti-

body to phosphorylated NF-M (data not shown). Thus, thepatterns of staining with the peripherin antisera reflecteddifferences in protein expression rather than differencesin levels of protein phosphorylation.

In Xenopus, peripherin is expressed in both neurons andradial glia (Gervasi et al., 2000). Peripherin-stained axonscould be easily distinguished from glia because axons runlongitudinally along the axis of the optic nerve, are thin,

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 stan-dardized conditions. For each nIF and time period after optic nervecrush, the difference in the mean intensity/pixel was determinedbetween the UE and OE RGCs and then averaged among animalsfrom 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 mRNAremain relatively stable from 3 6 days postcrush, when regeneratingaxons cross the lesion and enter the optic nerve, from 9 12 days,when they have reached the optic chiasm, and from 18 21 days, whenthey reach and cover the tectum. Data within each of these timewindows were therefore pooled.

TABLE 2. Relative nIF mRNA Stoichiometries During Optic Nerve Regeneration1

Fraction of the total nIF mRNA content contributed by each nIF (%)

Timepoint Peripherin Xefiltin XNIF NF-M NF-L

Relative2 fold increase intotal nIF mRNA (OE/UE)

UE3 0.0 0.4 48.7 4.4 20.7 2.5 19.5 2.4 11.1 1.13 Days 4.1 0.3 52.5 3.3 15.3 2.6 18.9 5.9 10.2 1.8 1.0 0.16 Days 16.0 0.1 39.2 0.9 11.1 2.7 28.7 5.2 5.0 2.4 0.9 0.19 Days 15.5 2.4 29.5 5.8 20.3 6.0 29.8 5.2 4.8 1.4 2.2 0.512 Days 19.4 1.5 31.0 2.8 16.0 0.5 28.9 1.4 4.7 0.3 1.9 0.118 Days 15.5 1.2 32.5 5.6 19.3 2.5 25.4 3.2 7.3 2.2 2.1 0.321 Days 12.4 3.4 34.8 2.5 18.1 1.7 26.9 3.2 7.8 1.5 2.2 0.335 Days 5.8 4.5 46.0 9.6 22.1 7.4 19.0 8.4 7.3 3.6 1.4 0.5

1For each nIF mRNA at time points after nerve crush, the average staining intensity was first corrected for slight variations in the length of each probe. The percent contributionto 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 itscontralateral, unoperated eye (UE) and then averaging these ratios (OE/UE) among animals.3Since values for unoperated, contralateral control eyes (UE) were not significantly different between 12 and 35 days postcrush (single factor ANOVA, df 8; P 0.05), they werepooled.



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and stain comparatively weakly, whereas glial processesrun perpendicularly, are thick, and stain strongly, espe-cially in glial end feet (Fig. 6A,E). In controls, the periph-erin antibody only weakly stained an occasional RGC bodyand some rare axons within the RGC layer, optic nerve,and tract. During regeneration, peripherin-stained axonswere readily visible in the retina, optic nerve, and tract onthe experimental side of the animal. A representativeexample 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 etal., 1985) and nIF expression has returned to normal.Peripherin-containing fibers were abundant in the left,regenerating optic nerve (Fig. 6A, left) and in the right,contralateral optic tract, which is populated by the regen-erating axons that cross the chiasm (Fig. 6C). In contrast,the uninjured, contralateral nerve (Fig. 6A, right) andipsilateral tract exhibited little or no peripherin staining,verifying that the increase in peripherin mRNA expres-sion occurring during regeneration was accompanied byincreased peripherin protein within the axon. Moreover,the types of NF aggregates that characterize peripherinstaining in injured mammalian CNS (Beaulieu et al.,2002) were not present.

NF-M protein expression also increased during regen-

eration. Staining for NF-M was stronger in the regener-ating optic nerve (Fig. 6B, left) and tract (Fig. 6D), than inthe contralateral, uninjured control nerve (Fig. 6B, right).This was true for sections stained either with an antibodydirected against a phosphorylation-independent epitope ofNF-M (RMO270), or with an antibody directed against adephosphorylated epitope (S8) after sections were treatedwith alkaline phosphatase, arguing that the intensity dif-ference between injured and control nerves was the resultof differences in levels of NF-M protein expression.

Immunostaining also demonstrated that during regen-eration, increased peripherin expression occurred primar-ily within relatively younger regenerating axons. In Xeno-

pus, regenerating optic axons grow along the outer

perimeter of the optic nerve, close to the glia limitans.Thus, in optic nerve cross-sections at any stage of regen-eration, the youngest axons are found along the outside ofthe nerve, whereas axons that had regenerated earlier arefound closer to the degenerating core. In the representa-tive example shown (Fig. 6E, 21 days postcrush), periph-erin staining was clearly present along the outer circum-

ference of the nerve, close to the glia limitans, and absentfrom the older axons close to the core. In contrast, NF-Mstaining was abundant in the older axons and light toundetectable in younger ones (Fig. 6F). In summary, theseresults verified that increases in nIF protein expressionand transport paralleled those seen at the mRNA level.

DISCUSSION

We have demonstrated that during successful opticnerve regeneration, the reemergence of each nIF subunitwas marked by increases in mRNA expression that wereabove normal. Whereas peripherin expression increasedsoon after nerve crush, marking its return to RGCs, ex-

pression of the remaining nIF mRNAs increased only afterthey initially decreased in response to the injury. By mon-itoring expression of multiple nIF mRNAs, we demon-strated that both their total levels and relative stoichiom-etries varied with the phase of regeneration. For example,at the peak of nIF expression, which occurred as axonstraversed the optic tract and tectum, RGC nIF mRNAscomprised relatively higher proportions of peripherin andNF-M, lower proportions of NF-L and xefiltin, and aboutthe same proportion of XNIF as did mature cells. Theseresults extend prior observations of increased levels ofperipherin- and -internexin-like nIF mRNAs during suc-cessful CNS regeneration in anamniotes by placing themin context with the other nIF subunits. They also suggestkey differences between successfully regenerating CNS

and PNS axons, which are discussed below.

Use of in situ hybridization toestimate relative levels of

nIF mRNA expression in RGCs

Since nIFs form heteropolymers in vivo, their changesin expression during axon regeneration must undoubtedlyalter the structural properties of NFs. Knowing the rela-tive proportions of each nIF subunit is thus the first stepin understanding the function of these changes in NFcomposition at different stages of axon outgrowth. Sinceseveral of the nIF proteins are expressed in multiple celltypes within the retina and optic nerve, we turned to insitu hybridization to distinguish the contribution of RGCs

from that of other cell types. For example, peripherin isexpressed in Mueller glial cells and in replicating stemcells of the ciliary margin (Gervasi et al., 2000), and theother 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 underrep-resented the response within the RGCs themselves, whichconstitute less than 10% of the cells in the retina. Second,RNase protection would have provided no information ondifferences among classes of RGCs.

We used in situ hybridization rather than immunocyto-chemistry for comparisons among nIFs, since the physicalchemistry of RNA hybridization is more similar amongdifferent RNAs than is that of antibodyantigen interac-

Fig. 5. Characterization of peripherin antisera on Western blots oftotal spinal cord homogenates. 1, preimmune serum for lane 2, diluted1:500. 2, Xenopus peripherin N-terminal head domain peptide, rabbitantiserum #1, diluted 1:500. 3, preimmune serum for lane 4, diluted1:500. 4, Xenopus peripherin N-terminal head domain peptide rabbit

antiserum #2, diluted 1:2,000. 5, Previously characterized Xenopusperipherin C-terminal peptide rabbit antiserum, diluted 1:2,000.



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tions. To achieve greater uniformity when comparing lev-els of expression among nIFs, we used probes of comparablelength, G/C content, and specific activity, and processedthe slides under the same conditions. Levels of mRNAexpression were estimated by video densitometry, a methodthat 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 mainsource of determinative error arises from levels of expres-sion falling outside the linear range of the assay. Wereduced this error by adjusting the conditions for hybrid-ization and development to minimize the number of cellswith saturated levels of staining and to maximize thenumber of cells staining above background, while main-taining conditions among the various probes. Since in-creasing development times increased background stain-ing but had little effect on the number of labeled cells, webelieve that our conditions were optimal. Nevertheless,because of these limitations, our measurements are bestregarded as good semiquantitative estimates rather than

rigorous quantitative measures of levels of nIF mRNAexpression.

Expression of nIF mRNAs in theunoperated eye

In situ hybridization enabled us to compare expression

levels of the various nIFs among different RGCs. By farthe most abundant nIF mRNA was that of xefiltin. Evenso, about 6 10% of cells remained consistently unlabeled,even during regeneration when xefiltin expression rises.Between metamorphosis and adulthood, the number ofnonganglionic cells (glia and amacrine cells) in the Xeno-

pus RGC layer rises from 3% (Dunlop and Beazley, 1984)to 15-25% (Graydon and Giorgi, 1984; Dunlop and Beaz-ley, 1984). The 6 10% of cells in the RGC layer that failedto label for xefiltin in our juvenile frogs therefore mostlikely represent nonganglionic cells. Conversely, since in

Xenopus, 1% of RGCs are displaced into the inner nuclearlayer (Toth and Straznicky, 1989), some of the labeledcells in that layer may represent displaced ganglion cells.

Fig. 6. Immunocytochemical localization of peripherin in regener-ating axons. Transverse sections through the brains of frogs sacrificed28 days (AD) and 21 days (E,F) after optic nerve crush were immu-nostained with antibodies to a C-terminal peptide derived from Xeno-

pus peripherin (diluted 1:1000: A,C,E) and to nonphosphorylatedNF-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 fibers run longitudinally in the left,crushed optic nerve (arrow). In contrast, in the right, uncrushed opticnerve, peripherin is found only in clusters of glia cells (g). B: On aneighboring section, regeneratingfibers growing outside the degener-ating core (asterisk) stain strongly for NF-M. The degenerating corewas 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 peripherin-containing fibers in the contralateral optic tract of the diencephalon(arrow). D: A neighboring section to that in C shows the distributionof NF-M for comparison. E: In the operated optic nerve close to thechiasm, peripherin is present in the youngest regenerating axons(arrows), which are located close to the glia limitans, but is absent inthe oldest regenerating axons, which are found close to the degener-

ating core (asterisk). F: On a section adjacent to that shown in E,NF-M is present in older regenerating axons, but is absent from youngregenerating axons close to the glial limitans (dots). g, glia; oc, opticchiasm; ot, optic tract; p, pial surface; v, ventricle. Scale bars 300m in D (applies also to AC) and 100 m in F (applies also to E).



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Compared to the number of RGCs that expressed xefil-tin in normal retina, the numbers expressing the othertype IV nIFs were much smaller. These cells may repre-sent distinct populations of RGCs, which in Xenopus areidentified by their size: Large, with diameters of 16 24m (1% of adult RGCs); medium, with diameters of 1117m (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 fourtype IV nIFs, is consistent with these data. Since in Xe-nopus retina RGC soma size correlates well with axonalcaliber (Dunlop and Beazley, 1984; Straznicky andStraznicky, 1988), and in all vertebrates the largest cali-ber axons have the greatest nIF contents, the expressionof all four type IV nIFs in these cells may reflect their needfor large amounts of these cytoskeletal proteins to main-tain axon caliber.

Increased expression of peripherin mRNAduring regeneration

Our finding that Xenopus peripherin expression in-creased dramatically soon after nerve injury extends sim-ilar findings on the expression of peripherin and its or-thologs during axon development and regeneration. Forexample, in both fish and Xenopus embryos, peripherinorthologs are present at the onset of axonogenesis andtheir expression precedes that of all type IV nIF proteins(Canger et al., 1998; Goldstone and Sharpe, 1998; Leake etal., 1999; Gervasi et al., 2000). Moreover, in mature fishand amphibian retina, where new neurons are continuallyadded throughout life (Johns and Easter, 1977), periph-erin orthologs are found only in the youngest RGCs, whichcan be identified by their proximity to the ciliary margin(Fuchs et al., 1994; Gervasi et al., 2000). In mammalianperipheral axon development, peripherin is also expressedat 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 proteinexpressed throughout the entire nervous system, and isalso abundant in growth cones. It later disappears frommost of the CNS as axons mature (Gervasi et al., 2000;Undamatla and Szaro, 2001). The correlation betweenperipherin expression and axon outgrowth is especiallystrong during regeneration, when its expression increasesdramatically in regenerating fish 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 XenopusRGCs during axonal regeneration further underscores theimportance of peripherin to growing axons.

Regulation of type IV nIF mRNAs

Our study adds to the growing body of evidence thattype IV nIFs may also play a substantial role in support-ing the normal growth of developing and regeneratingaxons. The behavior of the type IV nIFs in Xenopus regen-erating optic nerve resembles that reported in successfullyregenerating CNS axons in other systems. For example, intransected spinal cord axons of lamprey, NF-M expressionis initially suppressed and then rises in only those axonsthat successfully regenerate, while remaining suppressedin those that do not (Jacobs et al., 1997). Similarly, in fishoptic nerve expression of the xefiltin ortholog, gefiltin, isalso initially suppressed, then rises above normal during

the peak period of regeneration, and then falls to normallevels after regeneration is complete (Glasgow et al., 1994;Asch et al., 1998). This return to normal depends on sig-nals from the optic tectum (Niloff et al., 1998), and mayinvolve the successful formation of a retinotopic map,since in the lizard Ctenophorus ornatus, where vision failsto return after optic nerve crush despite the regrowth of

axons and the reformation of synaptic connections in vi-sual targets (Beazley et al., 1997; Stirling et al., 1999),levels of gefiltin expression remain permanently elevated(Rodger et al., 2001). Thus, an initial suppression of typeIV nIF expression, followed by an increase above normalduring the peak period of regrowth may be general fea-tures of successful CNS regeneration.

In further support of the idea that type IV nIFs areimportant for successful regeneration, the NF compositionof CNS axons that fail to regenerate is often markedlydifferent from that of axons that do, although the precisepattern 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). Al-though these studies implicate appropriate expression oftype IV nIFs in the successful regenerative response toaxonal injury, their expression is apparently not essentialfor axonal regrowth per se. For example, injured mamma-lian optic axons induced to grow through a peripheralnerve graft (McKerracher et al., 1993a), as well as Xeno-

pus optic axons that fail to enter the optic tract but growinstead to the contralateral eye (Zhao and Szaro, 1995),fail to increase their expression of NF-M despite theirability to traverse relatively long distances. Also, trans-genic mice lacking NFs nonetheless develop a nervoussystem and can regenerate their peripheral axons afteraxotomy (Zhu et al., 1997; Williamson et al., 1998; Elder etal., 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 notessential for life itself, may nonetheless be needed for thedevelopment of a fully viable and competitive organism inthe wild. This idea is supported both by the observationthat transgenic mice lacking NF-L regenerate peripheralaxons more slowly than normal (Zhu et al., 1997) and byexperiments in Xenopus embryos that demonstrate thatloss of type IV nIFs inhibits axons from entering the phaseof rapid axon elongation, which follows early neurite out-growth (Walker et al., 2001).

Although other studies have demonstrated that expres-sion of type IV mRNAs increases during successful regen-eration of CNS axons, ours is the first to compare therelative expression levels of multiple nIFs simultaneously.Our results demonstrated that increased expression of

type IV nIFs in successfully regenerating vertebrateRGCs is not limited to gefiltin and its orthologs, but ex-tends to other type IV nIFs as well. In addition, the tem-poral progression of increases in nIF expression duringregeneration clearly resembles that seen during develop-ment, arguing further that this progression is stronglylinked to axonal outgrowth. That changes in mRNA ex-pression accompany those seen in protein expression alsoargues that mRNA levels are likely to be a major deter-minant of nIF protein levels during successful RGC regen-eration.

The increased expression of NF-M during axonal elon-gation differs between regenerating anamniotic RGCs andmammalian PNS neurons. Whereas levels of NF-M ex-



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pression initially fall in both systems, they rise sooner,while axons are still elongating, in Xenopus RGCs than inmammalian PNS, where they remain suppressed untilafter 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 myelinatedPNS axons after synaptogenesis, an expansion that isdriven by increased nIF expression. Since in frog the vastmajority of optic axons are unmyelinated and RGC axoncaliber is only weakly correlated with NF content, theremay be no need for mature RGCs to maintain high levelsof NF-M mRNA expression. Thus, in both cases levels ofNF-M after injury may change to promote axonal re-growth. In frog RGCs these levels may be higher thannormal, whereas in regenerating PNS neurons they maybe lower.

Implications for the function of nIFproteins in axon development

Given the role of NFs as prominent structural compo-nents of the cytoskeleton, it is tempting to conclude thatthe increased expression of their subunit mRNAs duringregeneration simply reflects an increased need to supplythese structural proteins to build a new axon. Since nIFproteins are relatively long lived and stable, normal axonsmay simply not need to synthesize as much mRNA tomaintain high levels of protein in the axon. However, thishypothesis cannot explain the higher than normal levelsof NF-M protein that were observed in regenerating ax-ons. Instead, our estimates of the relative stoichiometriesof the various nIF mRNAs during regeneration suggestthat growing axons contain a different composition of nIFsto support various phases of axonal regrowth.

Although the composition of mRNAs in the RGCs may

not precisely reflect the composition of individual NFs inthe optic nerve, the tight regulation of mRNA compositionduring Xenopus optic nerve regeneration suggests thatcertain combinations of nIFs are characteristic of eachphase of axon regeneration. On this basis, optic nerveregeneration in Xenopus can be divided into three distinctphases: 1) an initial period of axonal growth through theoptic nerve, which is characterized by increased expres-sion of peripherin and decreased expression of type IVnIFs, so that the total nIF mRNA content of RGCs is closeto normal; 2) a second period occurring as axons cross theoptic tract and tectum, which is characterized by a dou-bling of the total level of nIF mRNA and a shift in nIFcomposition to become relatively enriched for peripherinand NF-M; and 3) a return to normal levels of expression

and relative mixtures of the various nIF mRNAs. Thesephases fit comfortably within the general scheme ofchanges in nIF expression reported for other developingand regenerating systems in which newly extended andgrowing axons are enriched for type III nIFs, elongatingaxons add type IV nIFs, such as NF-M and -internexin,and maturing axons express additional type IV nIFs andextensively phosphorylated NF-M and -H (Dahl et al.,1986; Bennett, 1987; Carden et al., 1987; Szaro et al.,1989).

Although this is the first report of stoichiometricchanges in nIF mRNAs during successful CNS regenera-tion, other studies have indicated that the relative mix ofnIFs is important for proper neuronal development. In

transgenic mice, for example, overexpression of NF-Mand/or NF-H inhibits dendritic arborization of motor neu-rons, an effect that is alleviated by a concomitant increasein NF-L (Kong et al., 1998), suggesting that dendriticarborization is influenced by the ratio of NF-M and NF-Hto NF-L. Similarly, reducing levels of NF-H in mice leadsto 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 anincrease in -internexin and a loss of sensory axons(Lariviere et al., 2002). In addition, changing nIF stoichi-ometry affects axon diameter (Wong et al., 1995; Marsza-lek et al., 1996; Xu et al., 1996; Elder et al., 1998) andoverexpression of NF-M results in faster rates of slowaxonal transport, which are also generally faster in grow-ing than in mature axons (Xu and Tung, 2000). The ab-sence from Xenopus regenerating optic axons of NF aggre-gates similar to those seen in mice that express abnormallevels of individual nIF subunits argues that the stoichi-ometric changes in Xenopus are part of a coordinatedresponse to injury.

Our data also support the idea that axonal growth andmaturation are supported by NFs with subunit composi-tions especially suited for each stage of development.Since each NF subunit functions only in context with itsco-assembled partners, experiments aimed at revealingNF function during axon development should test thecontribution made by NFs of biologically relevant compo-sitions rather than that made by each subunit separately.

ACKNOWLEDGMENTS

We thank Drs. Suzannah and David Tieman for the useof photographic equipment. We also thank Drs. SuzannahTieman and John Schmidt for critical comments on thearticle.

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