Introduction

In the formation of the peripheral nervous system, neuralcrest cells migrate and neurons extend axons through areasrich in extracellular matrix (ECM; for review, Sanes, 1989).Studies in vitro have demonstrated that ECM constituents,in particular fibronectin (FN), support the attachment,spreading and migration of neural crest cells and potentlypromote peripheral neurite outgrowth (Rogers et al., 1983;Tomaselli et al., 1986; Humphries et al., 1988; Dufour etal., 1988). FN has been shown to be localized along thepathways of migrating neural crest cells (Newgreen andThiery, 1980; Krotoski et al., 1986) and reagents, such asRGDS-containing peptides, which disrupt interactions ofcells with fibronectin, inhibit the migration of neural crestcells (Boucaut et al., 1984).

The primary class of cellular FN receptors identified thusfar are members of the integrin family of heterodimers (forreview see Hynes, 1992; Hemler, 1990; Reichardt andTomaselli, 1991). Each integrin heterodimer is composedof an α and β subunit with the ligand specificity determinedby the particular combination of subunits. Four het-erodimers containing the β1 subunit: α5β1, α3β1, α4β1 andαVβ1, have been identified as FN receptors (Pytela et al.,1985; Elices et al., 1990; Takada et al., 1988; Wayner etal., 1988; Vogel et al., 1990)). α5β1, αVβ1 and possibly

α3β1 interact with the RGD-sensitive major cell attachmentsite of FN (Pierschbacher and Ruoslahti, 1984; Elices et al.,1991) while α4β1 appears to interact with several sites inthe C terminus of FN, including two heparin-bindingregions and the alternatively spliced CS1 domain (Wayneret al., 1989; Guan and Hynes, 1990; Mould and Humphries,1991).

Dorsal root ganglion neurons in vitro have been demon-strated to interact with both of these domains, extendingneurites on both the C-terminal heparin-binding fragmentand the RGD-containing 75×103 Mr fragment (Humphrieset al., 1988; Rogers et al., 1985). Similarly neural crest cellsare known to interact with each domain (Dufour et al.,1988). The interactions of both neural crest cells and periph-eral neurons with FN are mediated by integrins containingthe β1 subunit (Bronner-Fraser, 1985; Bozyczko and Hor-witz, 1986; Tomaselli et al., 1986; Duband et al., 1986).These results suggest that the two cell populations utilizeα5β1, αVβ1 or α3β1 to interact with the RGD-sensitive cellbinding domain as well as α4β1 to interact with the C-ter-minal binding sites.

Fibronectin expression is regulated both during embryo-genesis (Roman and McDonald, 1992) and in wound repairin adult mammalian skin (ffrench-Constant and Hynes,1989; ffrench-Constant et al., 1989; Clark, 1990). Duringembryogenesis, the pattern of alternative splicing of FN is

767Development 116, 767-782 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

The extracellular matrix glycoprotein, fibronectin, is apotent promoter of peripheral neurite outgrowth. Inter-actions of peripheral neurons with fibronectin have beenshown to be primarily mediated by the 1 class of inte-grin heterodimers. In the present study, we have exam-ined the expression and regulation of fibronectin and itsintegrin receptor, 5 1, in developing and regeneratingchick peripheral nerve. We show that fibronectin and

5 1 are expressed at comparatively high levels in devel-oping nerve with 5 1 expression on axons and non-neu-ronal cells. With nerve maturation, both proteins areless prominently expressed and the cellular pattern of

5 1 expression becomes more restricted. Following

lesion of mature nerve, both fibronectin and 5 1 arestrongly induced with prominent expression of 5 1 onregenerating neurites and Schwann cells. The elevationin fibronectin levels in the regenerating nerve is highestin the vicinity of the lesion, an area undergoing exten-sive cellular remodeling including Schwann cell migra-tion and growth cone extension. Our results suggest thatfibronectin and its receptor, 5 1, may mediate func-tionally important interactions in the development andregeneration of peripheral nerve.

Key words: fibronectin, integrin, peripheral nerve, chick.

Summary

Regulation of expression of fibronectin and its receptor, 5 1, during

development and regeneration of peripheral nerve

FRANCES LEFCORT2,*, KRISTINE VENSTROM2, JOHN A. McDONALD3 and LOUIS F. REICHARDT1,2

1Neuroscience Program, Department of Physiology and 2Howard Hughes Medical Institute, University of California, San Francisco, 3rd & ParnassusAvenues, U426, San Francisco, CA 94143-0724, USA3Mayo Clinic Scottsdale, 13400 E. Shea Boulevard, Scottsdale, Arizona 85259, USA

*Author for correspondence

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spatially and temporally regulated with inclusion of thealternatively spliced EIIIA and EIIIB regions only duringthe early stages of embryogenesis (ffrench-Constant andHynes, 1989). During cutaneous wound healing in adultskin, these two embryonic splice forms are reexpressed bythe cells at the wound base (ffrench-Constant et al., 1989).The pronounced elevation in FN expression following skininjury is thought to be a critical component of the woundresponse as it provides a provisional matrix that facilitatesthe migration of several cell types into the wound region(for review, see Clark, 1990).

Previous work has shown that the responsiveness of sen-sory neurons to FN, assayed in vitro, is down regulatedduring embryogenesis (Kawasaki et al., 1986; Millaruelo etal., 1988). Similarly, recent work suggests that the tissuedistribution of the α5β1 integrin receptor becomes morerestricted during embryognenesis (Muschler and Horwitz,1991; Roman and McDonald, 1992). However, theexpression and function of FN receptors has been shown toincrease in epidermal cells isolated from healing wounds(Takashima et al., 1986; Grinnell et al., 1987). Several neu-ronal cell surface adhesion molecules whose expressiondecreases during development have been shown to be up-regulated following nerve injury (Daniloff et al., 1986; Mar-tini and Schachter, 1988).

The aim of the present study was to characterize theexpression and regulation of FN and one of its putative neu-ronal integrin receptors, α5β1, in the peripheral nerve. Wehave focused on the α5β1 heterodimer because preliminaryexaminations of the other β1-containing FN receptors didnot reveal appropriate cellular expression (αv), or obviousexpression changes (α3) or adequate reagents (α4) toprompt their further study (data not shown). Our resultsshow that FN and α5β1 are more prominently expressed inthe developing chick peripheral nerve than in the maturenerve and, following transection of a mature nerve, thelevels of both fibronectin and the α5 integrin subunit arestrongly increased.

Materials and methods

Surgery Three-week-old White Leghorn chickens were anaesthetized withketamine/xylazine and the medial-ulnar nerve innervating the rightwing was transected. The proximal and distal nerve stumps werejuxtaposed minimizing their separation to facilitate regeneration;the maximal gap distance between the two stumps was no greaterthan 1 mm. At various time points following nerve transection (3days, 1 week, 2 weeks, 4 weeks), the animals were killed by anoverdose of the same anaesthetics followed by CO2 asphyxiation.The regenerated nerve was removed from the animal and dividedinto equal length segments (2-3 mm) proximal and distal to thetransection site (Fig. 1). An equal length segment from the con-tralateral unoperated nerve was removed and served as control.All tissues were frozen immediately and stored at −80°C usuallyovernight before homogenization. For each experiment, nervesfrom 4-7 animals were transected and homogenized together. Thisprocedure was repeated 2-4 times for each experimental timepoint.

Antibodies The anti-fibronectin-CS1 monoclonal antibody (mAb FN-kv1)

was generated using E10 cultured chick brain glial cells as theimmunogen (Harlow and Lane, 1988). Cultured glial cells, scrapedoff in phosphate-buffered saline (PBS: 0.2g/l KCl, 0.2g/l KH2PO4,2.16 g/l Na2HPO4, 8 g/l NaCl; 0.1mM CaCl2, 0.1mM MgCl2) with1 mM PMSF, chymotrypsin, leupeptin, antipain and pepstatinwere injected with RIBI adjuvant (Hamilton, Montana) intoBalb/C mice. Hybridoma supernatants were screened for their abil-ity to block chick E6 retinal cell adhesion to the ‘CS1’ domainof fibronectin (peptide sequence: DELPQLVTLPHPHPNLHG-PEILDVPSTC). The hybridoma secreting FN-kv1 was subclonedby limiting dilution, grown in RPMI with 4% fetal calf serum and1% Nutridoma (Boehringer Manheim Biochemicals, Indianapolis,IN) and injected into Balb-c mice to obtain ascites. The allotypeof the FN-kv1 mAb is mouse IgG1 (Calbiochem Hybridoma Sub-typing kit, San Diego, CA). To purify the ascites Trizma (pH 8-9) was added to 100 mM and NaCl to 2.5 M. The ascites wasthen fractionated on protein-A Sepharose-CL-4B (Pharmacia,Uppsala Sweden) according to Harlow and Lane (1988). The IgGwas dialyzed against PBS (Ca2+- and Mg2+-free) and stored at−20°C. A second monoclonal antibody against fibronectin, VA13,was obtained from the Developmental Studies Hybridoma Bankmaintained by the Department of Pharmacology and MolecularSciences, Johns Hopkins University School of Medicine, Balti-more, MD, and the Department of Biology, University of Iowa,Iowa City, IA (under contract NO1-HD-6-2915 from the NICHD).VA13 recognizes intact FN as well as the 140/160×103 Mr cell-binding fragments generated by elastase digestion. Both mAbsgave identical staining patterns on immunoblots and immunocy-tochemistry and were thus used interchangeably.

Two antibodies were used to recognize the integrin α5 subunit:(1) A2F7, a monoclonal antibody from Drs J. Muschler and A. F.Horwitz (University of Illinois, Urbana; Muschler and Horwitz,1991) and (2) α5-47, a polyclonal antibody (affinity-purified IgG)generated against a 20mer peptide corresponding to the C termi-nus of the human α5 sequence (LPYGTAMEKAQLKPPATSDA;prepared as in Roman et al., 1989). Since the polyclonal antibody,α5-47, provided a much stronger signal for immunocytochemistrythan the A2F7 monoclonal antibody, it alone was used for allimmunocytochemistry.

To identify axons, two neural specific mAbs were used: (1) amAb generated against a β-tubulin isoform (cβ4) specific for neu-rons (Tuji, from Dr A. Frankfurter, University of Virginia; Yag-inuma et al., 1990)) and (2) a cocktail of mAbs against the 68,160, 200×103 Mr neurofilament subunits (Boehringer-Manheim).To label Schwann cells, a polyclonal antibody against the S-100protein (Dakopatts) was used. A polyclonal affinity-purified anti-body raised against mouse laminin (LN) was used to visualize LNisoforms (JW2; Lander et al., 1985).

Immunoblots Nerve segments were homogenized in 10 mM Hepes, pH 7.4 in0.15 M NaCl, 0.32 M sucrose, 2 mM PMSF, 1 mM chymotrypsin,leupeptin, aprotinin and pepstatin, and centrifuged at 10,000

F. Lefcort and others

Fig. 1. At various time points following medial-ulnar nervetransection, nerve segments of equal length (2-3 mm) proximal(P1, P2) and distal (D1, D2) to the transection site were removedand processed as described.

769Expression of fibronectin in chick peripheral nerve

revs/minute for 30 minutes. The pellets were resuspended in PBS(pH 7.4) with 1% Triton X-100 and protease inhibitors, vortexedseveral times over 30 minutes, and incubated on ice. After additionof SDS sample buffer (final concentration of 3% SDS; Laemli,1970), the homogenates were incubated on ice for 30 minutes fol-lowed by boiling for 3 minutes. The extracts were then centrifugedfor 10 minutes at 10,000 revs/minute and protein determinationswere made on the supernatants by the Amido Schwartz method.After loading equal protein concentrations, samples were elec-trophoresed on a 6.5% non-reducing polyacrylamide gel(Laemmli, 1970). The gels were then transferred electrophoreti-cally to nitrocellulose for 1 hour at 500 mA. The blots wereblocked in 5% Blotto (5% dry milk powder, 50 mM Tris-HCl, pH7.5, 150 mM NaCl) for 45 minutes and then incubated in primaryantibody for 1 hour (polyclonal antibody) at room temperature orovernight at 4°C (monoclonal antibody). After washing four timesfor 10 minutes each, the blots were incubated for 1 hour in a sec-ondary antibody, goat anti-rabbit or goat anti-mouse (as appro-priate) IgG coupled to alkaline phosphatase (Promega, WI). Afterfurther washing, the alkaline phosphate reaction product was gen-erated by developing at pH 9.5 with the substrates, nitroblue tetra-zolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma). Tomeasure changes in α5 subunit expression, the immunoblots werescanned on a Biorad densitometer (Brugg and Matus, 1988).Immunoprecipitations were performed according to Neugebaueret al., 1991.

Immunocytochemistry Regenerated and control nerves were removed from the animaland either frozen immediately in liquid nitrogen or fixed in 3%paraformaldehyde for 2 hours followed by overnight incubationin 15% sucrose. After embedding in Tissue-Tek OCT (Miles,Inc.), the nerve segments were sectioned at 10-14 µm and theslides stored at −20°C. Both the contralateral control nerve andregenerating nerves were embedded in the same block of OCT;thus each slide contained sections of both nerves. To examinedeveloping nerve, medial nerves were removed from 1-day-oldchicks and treated similarly. The sections taken from nerves thathad not been previously fixed were then fixed in either 3%paraformaldehyde for 20 minutes or −20°C methanol for 10 min-utes. Sections were blocked for 30 minutes in either 5% milkpowder or 5% normal goat serum in Tris-buffered saline plus 0.2%Triton X-100 for 30 minutes followed by overnight incubation at4°C in primary antibody (10 µg/ml). Secondary antibodiesincluded goat anti-rabbit fluorescein (Cappel, 1:100), goat anti-mouse rhodamine (Accurate, 1:100), goat anti-rabbit biotin (Vec-tastain, 1;200), strepavidin fluoroscein (Amersham, 1:100). Sec-tions were then mounted in gelvatol with 2% n-propyl gallate(Sigma) added as an antioxidant and observed on a Zeiss Axio-phot or photomicroscope. All photographs were taken with eitherhypersensitized Kodak Technical pan 2415 (Lumicon; Livermore,CA) or Kodak TMAX 100. To verify the specificity of the anti-body labeling, controls included sections incubated in secondaryantibody alone and for the anti-α5 cytoplasmic IgG, the antibodywas incubated for 1 hour with 10 µg/ml of a 20mer peptide fromthe C terminus of the human α5 sequence before application tosections. Additional experiments were performed to insure thatthere was no cross-reactivity to the cytoplasmic domains of otherintegrin α subunits, focusing on three with highest sequence con-servation in the cytoplasmic domain (α8, αv, α6). Preincubationwith peptides from the C termini of both the chick α8 integrinsubunit (Bossy et al., 1991) and the human α6a subunit, did notinterfere with the α5-47 labeling of nerve. Polyclonal antibodiesagainst the C termini of the α6 and αv integrin subunits generatedvery different staining patterns from that of the anti-α5 IgG inchick peripheral nerve (not shown).

For immunocytochemical comparisons of expression levels ofa particular antigen, sections of the two nerves being compared(e.g. immature vs. mature nerve; control vs. regenerating nerve)were collected on the same slide. After immunolabeling, photo-graphic exposures of equal duration were taken and all negativeswere processed equally.

Results

To identify the chicken α5 subunit, we used two differentantibodies: an affinity-purified polyconal antibody gener-ated against the C terminus of the human α5 sequence (α5-47) and a monoclonal antibody (A2F7) generated againstthe 150×103 Mr band (‘band 1’; Hynes et al., 1989)immunoprecipitated by the CSAT antibody (Bozyczko andHorwitz, 1986). Previous work has confirmed that this150×103 Mr protein from chick fibroblasts binds to a FNcolumn and is a chick homolog of the human α5 subunit(Hynes et al., 1989; Muschler and Horwitz, 1991). Bothantibodies recognized a single band of 150×103 Mr inextracts of chick peripheral nerve (Fig. 2A, lane 1 and 2).Since it was essential to determine that the α5-47 polyclonalantibody was specifically recognizing a chick homolog ofthe human α5 subunit, we tested whether A2F7 would rec-ognize the 150×103 Mr protein immunoprecipitated from achick peripheral nerve extract by the α5-47 affinity-purifiedIgG. A single band was recognized by the mAb A2F7 (Fig.2B, lane 1, which was not recognized in a control immuno-precipation, Fig. 2B, lane 2) indicating that the two anti-bodies interact with the same antigen and that the α5-47IgG recognizes a chick homolog of the human α5 integrinsubunit. Additional evidence that the α5-47 polyclonal anti-body recognizes a chick fibronectin receptor is that α5-47immunohistochemically stains fibrillar apparent ECM con-tact sites and focal contacts (Roman et al., 1989) in chickfibroblasts cultured overnight on fibronectin (data notshown; see Methods for details on additional control exper-iments).

Two different monoclonal antibodies against FN wereused in this study: mAb Va13, which recognizes intact FNand the RGD-cell binding domain, and mAb FN-kv1, whichrecognizes the CS1 domain and intact FN. Both antibodiesrecognized purified bovine FN on a reducing gel (Fig.2C,lanes 1 and 2) and recognized the same bands, around220×103 Mr, on an immunoblot of chick peripheral nerveextract (Fig. 2C, lanes 3 and 4).

Distribution of 5 and FN in normal adult nerveThe α5 subunit associates with the integrin β1 subunit toform the functional FN receptor, α5β1; it has not beenobserved associated with any β integrin subunit other thanβ1. To obtain evidence for the presence of the α5β1 het-erodimer in chick peripheral nerve, we determined byimmunocytochemistry that all cells expressing the α5 sub-unit also expressed the β1 subunit (data not shown); co-pre-cipitation from chick nerve extracts with the α5-47 poly-clonal antibody of an 110×103 Mr protein recognizedspecifically by anti-β1 antibodies provided direct biochem-ical evidence for the association of the two integrin sub-units in peripheral nerve (data not shown). In cross sections

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of mature (4 week) peripheral nerve, both α5 (Fig. 3A) andFN (Fig. 3B) were prominently expressed within the per-ineurium and endoneurium. Overlapping expression of thetwo antigens was evident within both compartments. Exam-ination with higher magnification showed strong expressionof the α5 subunit on myelinated axons and myelinatingSchwann cells (Fig. 3C). Non-myelinated axons and non-myelinating Schwann cells expressed lower levels of the α5subunit. Non-neuronal cells, including capillary endothelialcells and fibroblasts also expressed the α5 subunit.Fibronectin was localized in rings surrounding the Schwanncell endoneurial tubes (Fig. 3B,D) corresponding to the siteof basal lamina deposition (Peters et al., 1976). To confirmthe localization of FN along Schwann cell endoneurialtubes, longitudinal sections of peripheral nerve were doublelabeled with anti-FN and a known Schwann cell marker, S-100 (Fig. 3E,F). The anti-S100 IgG labels Schwann cellcytoplasm (Fig. 3E). FN was distributed just outside andrunning along part of the length of the same endoneurialtube (Fig. 3F). The expression on axons of the integrin α5subunit was also clearly demonstrated in longitudinal sec-tions of peripheral nerve (Fig. 3G,H); being particularly evi-dent under Nomarski optics (Fig. 3H) and revealing a punc-tate distribution at higher magnification withepi-fluorescence optics (Fig. 3G). In developing peripheralnerve, the α5 subunit (Fig. 3I) was prominently expressedon axons, identified with the neuronal-specific anti-neuro-filament antibody (Fig. 3J), and non-neuronal cells.

Changes in expression of FN and the 5 subunit duringperipheral nerve maturationSeveral molecules that promote neurite outgrowth and theirreceptors have been shown to be down regulated as devel-opment proceeds (Martini and Schachner, 1988; Dodd etal., 1988). Reduced expression of these molecules often cor-

relates with the arrival of axons at their targets (Cohen etal., 1986; 1989; Hall et al., 1987; de Curtis et al., 1991).

To determine whether expression of FN and the α5 sub-unit might also be developmentally regulated within theperipheral nerve, we compared their expression levels inhatchlings (day 1) to 4-week-old chicks by immunocyto-chemistry (Fig. 4). We found the distribution of FN to bemore sparse and dispersed in the older nerve (Fig. 4D) com-pared to its more prominent expression in the younger nerve(Fig. 4C) where it was broadly expressed along axon bun-

F. Lefcort and others

Fig. 2. Characterization of antibodies directed against fibronectin and the α5 subunit. (A) Both the mAb A2F7 (lane 1) and the polyclonalantibody α5-47 (lane 2) recognize a 150×103 Mr band in extracts of chick peripheral nerve. (B) The mAb A2F7 recognizes a protein~150×103 Mr immunoprecipitated by the human α5-47 polyclonal antibody (1) but does not recognize any bands immunoprecipated fromthe same chick peripheral nerve extract with a polyclonal antibody against the human β5 subunit (2) (C) Both mAb VA13 and FN-kv1recognize purified bovine FN (lanes 1 and 2 respectively). Similarly, both antibodies recognize chicken FN in an extract from chickmedial nerve (lane 3, Va1-3; lane 4, FN-kv1).

Fig. 3. Distribution of the integrin subunit, α5, and fibronectin inunoperated peripheral nerve. (A-D) Transverse section of a nervefrom a 4-week animal double labeled with anti-α 5-47 (A,C) andanti-fibronectin IgG (B,D). (A,B) Note the overlappinglocalization of the α5 subunit and FN in perineurium (arrowhead).(C,D) Same field as above, but with higher magnification view ofendoneurium. (C) Note that myelinated axons are recognized bythe anti-α5 antibody (C; arrowhead) as are their myelinatingSchwann cells (arrow). (D) Same field as C under optics tovisualize the FN localization surrounding myelinating Schwanncell endoneurial tubes (arrowhead). (E,F) Longitudinal sectionfrom a 4-week adult nerve double labeled with S-100 (E) and FNantibodies (F). Note that FN is localized (F, arrow) adjacent to themyelinating Schwann cell (E, arrow). (G,H) Longitudinal sectionsof normal adult nerve labeled with anti-α5 IgG and (G) with afluorescently linked secondary antibody; note punctate staining ofmyelinated axon (between arrows) and of surrounding myelinatedSchwann cell (arrowhead). (H) Here, the secondary antibody waslinked to horseradish peroxidase and the section visualized withNomarski optics. Note intense labeling of a myelinated axon(arrow) inside its myelin sheath (arrowhead). (I,J) Longitudinalsection of an immature nerve from a 1-day-old chick, doublelabeled with anti-α5 (I) and anti-neurofilament (J). Note that axonsare amongst the cells at this stage that express α5. Bar (A,B) 50µm; (C,D) 10 µm; (E,F) 5 µm; (G)10 µm; (H)5 µm; (I,J) 25 µm.

771Expression of fibronectin in chick peripheral nerve

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dles. The pattern of the α5 subunit expression changed con-siderably when comparing longitudinal sections from thetwo ages (Fig. 4A,B). In the day 1 nerve (Fig. 4A), all axonswere brightly labeled with the anti-α5 antibody while in the4 week nerve (Fig. 4B) brightly labeled myelinated axonswere separated by bundles of more faintly labeled non-myelinated axons. While at both ages, α5 was expressed onnon-neuronal cells, the proportion of such cells expressingα5 appeared to be greater in the younger nerve (see Fig.3I,J). Between these two timepoints, the nerve undergoes aperiod of extensive cellular remodelling and differentiation,including myelination (Saxod and Bouvet, 1982). The vis-ible decrease in α5 subunit expression within the moremature nerve corresponds primarily to a decreasedexpression on non-myelinated axons and partially to adiminished expression or presence of non-neuronal cells.

Expression of FN and 5 strongly increases during nerveregenerationNerve injury triggers a state of active growth both in termsof neurite extension and Schwann cell differentiation (forreview see Fawcett and Keynes, 1990). To test whetherthere were changes in expression of FN or α5β1 in responseto such an injury, we transected the medial-ulnar nerve in

3 week chickens. To examine regulation of the integrin α5subunit, the relative amounts of α5 expression in both con-trol and experimental nerves were compared onimmunoblots by densitometry. We measured a sixfoldincrease (Fig. 5) in the expression of the α5 subunit inregenerating nerve relative to contralateral control nerve,peaking 1 week following nerve transection. This increaseoccurred both in the segment immediately proximal anddistal to the transection site.

To determine the cell types in which these changesoccurred, longitudinal sections of regenerating and controlnerve at 1 week following nerve transection were labeledwith the anti-α5 and FN antibodies as well as neuronal andSchwann cell markers (Fig. 6). In agreement with our den-sitometry results, we found that nerve injury stronglyinduced expression of the integrin α5 subunit. Compared tothe contralateral control nerve (Fig. 6A), the α5 subunit wasmore highly expressed in the proximal (P2 and P1) anddistal (D2 and D1) regions of the regenerating nerve as wellas in the site of injury (Fig. 6H). In this nerve, the distalfront of the regenerating axons had not yet reached the D1region (see Fig. 8). Thus the expression of the α5 subunitobserved distal to the transection site (D1 and D2 regions)must reflect expression on non-neuronal cells. In the more

F. Lefcort and others

Fig. 4. Immunohistochemical comparison of expression levels of α5 and FN in nerves from a 1-day-old (A,C) versus 4-week-old (B,D)chick. (A,B) Anti-α5 labeling; (C,D) anti-FN labeling. All exposure times and development times (for the same given antigen) were ofequal duration in order to compare fairly the levels of fluorescence intensity between the two ages. Note that for both antigens, their levelsdecrease with nerve maturation. Bar, 35 µm.

773Expression of fibronectin in chick peripheral nerve

distal D2 region, the distribution and morphology of the α5labeled cells closely resembled proliferating Schwann cellsthat align to form the Bands of Bungner during Walleriandegeneration (Allt, 1976; for review see Fawcett andKeynes, 1990). These cells could not be positively identi-fied with anti-S100 antibodies since immature proliferatingSchwann cells express little of this antigen (Neuberger andCornbrooks, 1989; Jessen et al., 1989). Proliferating fibrob-lasts and endothelial cells may also contribute to the stain-ing pattern. At the site of injury, the integrin α5 subunit co-localized with axons and sprouting growth cones asindicated by the overlap of neurofilament (Fig. 6I) and α5expression (Fig. 6H). Since there were also cells in thisregion that expressed α5 but not the neuronal markers (neu-rofilaments or β-tubulin), the non-neuronal cells with whichthe axonal sprouts and growth cones were associated (mostlikely Schwann cells, see Hall, 1986; Fawcett and Keynes,1990) must also express the α5 subunit. Thus, it appearsthat the number of cells expressing α5 increased in theregenerating nerve, both proximal and distal to the tran-section site.

The distribution of fibronectin was also examined in theregenerating nerve (Fig. 7). Compared to the contralateralcontrol nerve (Fig. 7A), one week after nerve transection adramatic increase in the level of fibronectin expression wasobserved in the regenerating nerve (Fig. 7B-F). Interest-ingly, while the P2 and D2 regions showed a slight increasecompared to the control section, the area surrounding and

including the transection site (P1, P1-D1, D1; Fig. 7C-D)showed a very strong increase compared to control nerve.In fact, the region with the highest expression was very dis-crete: it began in the area surrounding the nerve transec-tion site in the vicinity of the distal front of the regenerat-ing growth cones (distal edge of P1) and increaseddramatically in the region connecting P1 to D1 (P-D), ter-minating in the proximal portion of the D1 region. Thisvery circumscribed pattern of FN expression with respectto the disposition of the regenerating growth cones is moreclearly illustrated in Fig. 8, where several mm of the regen-erating nerve are reproduced. The region of highest FNintensity (Fig. 8B) coincided with the ‘bridge’ region con-necting the proximal and distal stumps (Longo et al., 1984;Martini et al., 1990). The growth cones of the regeneratingneurites (labelled in Fig. 8A with anti-β-tubulin antibody)had penetrated into the region of elevated FN expression,which extended distally in advance of the growth cones.

To determine whether there was a general increase inexpression of ECM molecules in the bridge region, weexamined the distribution of laminin, a representative con-stituent of basal lamina, in regenerating nerve (Fig. 9).Based on antibody perturbation studies, laminin isoformshave been implicated functionally in peripheral nerveregeneration (Sandrock and Mathews, 1987a,b). Interest-ingly, while laminin immunoreactivity was very promi-nently distributed proximally and distal to the cut site,mostly on the surface of Schwann cells (Fig. 9B,D) or inthe endoneurial basal lamina (Fig. 9A; see also Sanes et al.,1990), there was little detectable laminin expression in thebridge region (Fig. 9C).

Two weeks following nerve transection, results illus-trated in Figs 10 and 11 show that the expression of FNwas still elevated in the regenerating nerve (compare Fig.10A to panels B,C,E and G). The region of most intenseexpression still corresponded to the original transection site.Compared to one week after transection, the elevated FNexpression extended further distally down the nerve intosegment D2 (Fig. 10G). In addition, the regenerating axonshad now traversed the bridge region and extended distallyfor several mm into the D2 region (Figs 10, 11). Both inthe vicinity of the transection (Figs 10D,F, 11A,C,E) andfurther distally (D2 region, Figs 10H, 11B,D,F), α5 labeledregenerating axons were observed in regions with elevatedFN expression. In the D2 segment, axons grew with straighttrajectories, indicating that they were growing throughendoneurial tubes (Ide et al., 1983; Longo et al., 1984; Faw-cett and Keynes, 1990). However, in the bridge region, neu-rites were observed in several orientations (Figs 10F,11C,E), an indication that they had not yet reached the par-allel array of endoneurial tubes. Since regenerating neuritesexpressed α5 (Figs 6, 11), growth cones appear able tointeract with FN in both the bridge and more distal regions.

Discussion

We report here on the spatiotemporal expression pattern offibronectin and one of its receptors, the α5β1 integrin het-erodimer, during development and regeneration of periph-eral nerve. We and others have not found any instances

Fig. 5. Quantification of changes in α5 expression in theregenerating nerve compared to the contralateral control nerve(=100% on y-axis). For each experiment, 4-7 nerves werehomogenized together. Values expressed for the first twotimepoints are the mean (± s.e.m.) of 2-4 experiments.

774 F. Lefcort and others

775Expression of fibronectin in chick peripheral nerve

where the α5 subunit is expressed in the absence of β1; inimmunoprecipitations α5 is invariably associated with β1and has not been detected in association with any other βsubunit. Thus we believe in peripheral nerve the distribu-tion of the α5 subunit reflects that of the α5β1 receptor. Wehave found that (1) both FN and α5β1 are prominentlyexpressed in developing nerve with strong α5 subunitexpression on neurons and non-neuronal cells, (2) theprevalence of both α5 and fibronectin decreases with mat-uration of the peripheral nerve, (3) in response to injury tothe mature nerve, both FN and α5 are strongly induced, (4)the elevated FN expression at the site of injury suggeststhat it is functionally important for reestablishing connec-tions between the severed nerve segments and (5) the dis-

tribution of the fibronectin receptor, α5β1, suggests that itis utilized by both axons and Schwann cells during nerveregeneration.

As others have noted (Palm and Furcht, 1983; Longo etal., 1984; Sanes, 1989), in mature nerve, we observedfibronectin distributed around the endoneurial tubes, theregion known to correspond to the endoneurial basal lamina(Peters et al., 1976; Bunge et al., 1989a) and in the per-ineurium. Thus α5β1 receptors on the outer Schwann cellmembrane seem likely to interact with fibronectin in thebasal lamina. Since the α5β1 receptor has been demon-strated to be required for optimal FN matrix assembly(McDonald et al., 1987; Roman et al., 1989), its expressionon Schwann cells is consistent with such a role in the nerve.

Fig. 7. Fibronectin expression in regenerating nerve one week post-transection. All photographic exposure times were equivalent and allnegatives were processed equally. (A) Contralateral control nerve stained with anti-fibronectin antibody. (B-F) Regenerating nervestained with anti-fibronectin antibody; (B) P2 region; (C) P1 region; (D) the ‘bridge’ region connecting P1 and D1 regions of nerve; (E)D1 region; (F) D2 region. Bar, 45 µm.

776 F. Lefcort and others

777Expression of fibronectin in chick peripheral nerve

Non-myelinated axons and non-myelinating Schwann cells,which also expressed the α5 subunit but at lower levels,have been observed in direct contact with the endoneurialbasal lamina (Kuecherer-Ehret et al., 1990) and thus arealso likely to interact with FN. In the mature nerve, FNwould not be accessible to the α5β1 receptors on myeli-nated axons. It is thus possible that, in addition to FN, α5β1may also interact with an unidentified ligand on the inte-rior surface of myelinating Schwann cells. Many other inte-grins have been shown to have more than one ligand andthese include integral membrane proteins (for example, seeElices et al., 1990).

In contrast to their more restricted expression in the oldernerve, fibronectin and α5β1 were much more prominentlyexpressed in a relatively immature nerve. In the hatchlingnerve, FN was rather continuously expressed along axonbundles whereas in the older nerve it was sparse and moredispersed within the endoneurium. In the younger nerve,the α5 subunit seemed ubiquitously expressed throughoutthe nerve with strong expression on all axons. In the oldernerve, strong axonal α5 expression was restricted to myeli-nated axons in contrast to the considerably weakerexpression on non-myelinated axons. Between these twotime points (day 1 versus 4 weeks), the nerve undergoes aperiod of extensive myelination; at hatching, fewer than 4%of axons are myelinated, while by 6 weeks, 40% of theaxons are myelinated. (In the adult, the maximum numberof myelinated fibers reaches 60%; Saxod and Verna, 1979.)In the nerves of hatchlings (day 1), none of the broader(myelinated) axons observed in the older nerves were evi-dent; instead most of the axons were thin and appeared inbroad bundles, characteristic of an immature nerve whereseveral (non-myelinated) axons are often associated with anindividual Schwann cell (cf. Webster and Favilla, 1984).Thus our data suggest that the diminished expression of α5observed on the neuronal cells with nerve maturation is pri-marily due to a decreased expression on axons that remainnon-myelinated. The prominent expression of α5β1 at thisearly time point also coincides with a period of pronouncedSchwann cell proliferation which later ceases (Webster andFavilla, 1984; Asbury, 1967). As Schwann cells mature theyalter their expression of several proteins including cell sur-face adhesion molecules (Daston and Ratner, 1991; Neu-berger and Cornbrooks, 1989; Jessen et al., 1989).

Fibronectin and the fibronectin receptor 5 1 are inducedin response to nerve injuryOur results show that FN is strongly induced in peripheralnerve in response to injury. Both one and two weeks fol-lowing transection, fibronectin expression was moststrongly increased in the vicinity of the site of injury.Laminin, another representative ECM constituent, is virtu-ally absent from this region, although the expression oftenascin increases distally following nerve injury (Martini

Fig. 9. Anti-laminin labelling of section adjacent to one depictedin Fig. 8. Note how while laminin is expressed both proximally(A,B) and distally (D) to the transection site, it is virtually absentfrom the ‘bridge’ region (C:PD). In the P2 region (A), laminin islocated surrounding the endoneurial tubes. Bar, 25 µm.

778

et al., 1990). Thus fibronectin, but not laminin, is distrib-uted appropriately to play a significant role as a promotorof both Schwann cell migration and axonal regenerationthrough this bridge region.

The elevated FN in this area may be deposited fromplasma or be synthesized locally by fibroblasts and endo-thelial cells in the bridge region (Longo et al., 1984; Wool-ley et al., 1990; Cornbrooks et al., 1983). This bridge regionis not simply an acellular matrix characteristic of a fibrinclot; labelling with nuclear markers (e.g., DAPI, data notshown) demonstrates a rather uniform cellular arraythroughout this region connecting the two severed stumpsand most likely corresponds to the fibroblasts reforming theouter nerve sheath (peri or epineurium; Bunge et al.,1989b). Since, in adult nerve, FN is localized along theexternal surface of Schwann cell endoneurial tubes, it isconceivable that FN might be synthesized by Schwann cellsin vivo or alternatively synthesized by other neighboringnon-neuronal cells and incorporated by Schwann cells intotheir overlying matrix (McDonald et al., 1987; Roman etal., 1989). In vitro, Schwann cell synthesis of FN has beenobserved in the presence of cAMP analogs and ascorbate(Baron Van Evercooren et al., 1986).

The fibronectin receptor α5β1 was also strongly inducedin regenerating peripheral nerve on both axons andSchwann cells. Notable increases in α5β1 expression wereobserved both proximal and distal to the site of injury. Prox-imal to the transection, a major proportion of the increasein α5β1 appeared to be neuronal. This reflected both ele-vated α5β1 expression on individual axons (compare theintensity of Fig. 6H to 6A), and an increased density ofneurites due to axonal sprouting, which is triggered bynerve injury (Cajal, 1928; Diamond et al., 1987). The grow-ing tips of regenerating axons strongly expressed α5β1 (Fig.6H,I). Further, α5 subunit was also induced in non-neuronalcells both proximal and distal to the transection site (Fig.6F,G). This was particularly striking in the D2 region (about

3-6 mm distal to the transection site) where at one weekfollowing transection, Schwann cells were proliferating andaligning to form the bands of Bungner (Fawcett andKeynes, 1990; Allt, 1976). Thus the induction of α5 sub-unit in this segment must be on Schwann cells since theyconstitute the vast majority of cells at this time. α5β1, then,appears to be reexpressed on dedifferentiating Schwanncells as well as on regenerating axons. Overall, theexpression patterns of the α5 subunit in the developing andregenerating nerve were similar; in both, expression wasstrong on several cell types.

Previous studies have demonstrated that fibronectinlevels are greatly elevated during wound healing in adultrat skin (ffrench-Constant et al., 1989; Grinnell et al., 1981),resembling its embryonic expression pattern. The authorssuggest that the reexpression of FN, whose localizationduring development coincides with a period of active cellmigration, would facilitate the enhanced migration thatoccurs as part of the wound response. For successful nerveregeneration, migration of Schwann cells into the woundregion is critical; in their absence axons fail to regenerate(Hall, 1986; Fawcett and Keynes, 1990). Since FN has beenshown to act not only as a substratum for Schwann cellmigration, but also as a chemoattractant and mitogen forSchwann cells (Baron Van-Evercoorren et al., 1982), itseems possible that the elevated FN expression in the tran-section region may be an integral component of successfulnerve regeneration by inducing the proliferation and migra-tion of Schwann cells to the site of injury.

Thus fibronectin might facilitate nerve regeneration viatwo mechanisms. (1) One mechanism could be indirect, byinducing an influx of Schwann cells into the wound region.Since Schwann cell surfaces contain several adhesion mol-ecules that potently promote neurite outgrowth (Bixby etal., 1988; Tomaselli et al., 1986), these cells would providean attractive substratum for axonal outgrowth. (2) A secondmechanism could be direct by virtue of itself being a

F. Lefcort and others

Fig. 10. Double labeling of nerve 2 weeks following transection with anti-α5 and anti-FN antibodies. All negatives were exposed andprocessed equally. FN labeling depicted in (A,B,C,E,G) and α5 labeling depicted in (D,F,H). (A) Control nerve. (B) P2 region ofregenerating nerve. (C,D) P1 region, note elevated expression of FN in area containing (D) α5 labeled cells. (E) D1 region, high FNexpression has extended further distally by 2 weeks. In same field numerous α5 labeled cells are observed (F). Elevated FN expression isnow seen in D2 region by this time (G; compare to Fig. 7F), a region where regenerating axons are growing straight through theendoneurial tubes; note numerous α5 labeled cells, most of them being axons (H). Bar, 25 µm.

779Expression of fibronectin in chick peripheral nerve

demonstrated attractive glycoprotein for neuronal adhesionand neurite extension (for review, see Reichardt andTomaselli, 1991). Its elevated expression in the vicinity of

the regenerating growth cones and axons strongly implicateFN as a substratum for regenerating axons. At one weekfollowing transection, FN levels were highest in the vicin-

Fig. 11. 2 weeks post-transection, regenerating axons express α5 and grow in regions of elevated FN expression. Double-labeled sectionsof regenerating nerve 2 weeks post-transection with anti-α5 (A,B) and anti-neurofilament abs (C,D). In a proximal portion of the D1region (A,C) just distal to the site of transection, regenerating neurites labeled with neurofilament abs (C) all express the α5 subunit (A).The curvilinear growth is typical of the morphology of regenerating neurites in this region near the site of transection. Further distally(B,D) in the D2 region, axons have aligned themselves along the endoneurial tubes and hence grow relatively straight . Note how heretoo, all axons (D) express α5 (B). (E,F) Double labeling of an adjacent section with anti-neurofilament and anti-FN antibodies reactedwith the same secondary antibody so that both antigens could be photographed on the same negative and yet distinguished by therelatively cylindrical neurofilament staining versus the punctate FN distribution. These photographs illustrate that FN is expressed in thesame area as regenerating neurites both in the D1 region (E) and further distally in the D2 region (F), although FN expression is higher inthe D1 region than in the D2 region. Bar, 20 µm.

780

ity of the regenerating growth cones and decreased furtherdistally where the axons had not yet extended. By twoweeks following nerve transection, the regenerating axonshad grown into the distal stump and were now regrowingthrough the Schwann cell endoneurial tubes. In this distalregion, axons are known to grow between the Schwann cellsurface and its basal lamima in contact with both (Ide etal., 1983; Fawcett and Keynes, 1990). By this time, FNexpression was elevated surrounding the reformedendoneurial tubes perhaps in response to axonal contact(Bunge et al., 1989a). Significantly, at both time points, wealways observed prominent expression of α5β1 on regener-ating neurites. The elevated expression of α5β1 on bothSchwann cells and regenerating growth cones could facili-tate their motility through a FN-rich region since previouswork has positively correlated enhanced motility on FNwith elevated expression of the α5β1 receptor (Schreiner etal., 1989).

Peripheral nerve transection induces an inflammatoryresponse (Brown et al., 1991). For sensory axons, thisresponse is thought to be an essential element for their suc-cessful regeneration. Macrophages recruited into the woundarea secrete interleukin I (Il-1) which causes an elevationin NGF secretion by the non-neuronal cells in the nervethereby trophically supporting the sensory neurons(Heumann et al., 1987; Lindholm et al., 1987; Brown et al.,1991). The increase in α5β1 in the nerve may similarly beregulated by cytokines such as Il-1. Another cytokine cen-trally involved in wound healing, TGF-β1 (for review seeClark, 1990), has been found to induce Schwann cell pro-liferation (Ridley et al., 1989). Further, levels of TGFβ1mRNA significantly increase during nerve regeneration(Scherer and Jakolew, 1991). Interestingly, TGF−β1 hasbeen found to upregulate expression of both FN and α5β1on fibroblasts in vitro (Ignotz and Massague, 1987). Thus,there are several growth and differentiation factors that maydirectly upregulate the α5β1 and/or FN expression in regen-erating peripheral nerve. In addition to regulating integrinexpression levels, cytokines may also directly activate theα5β1 receptor (for review, see Hynes, 1992).

Our results suggest that one feature of the woundresponse induced by nerve transection, elevated FNexpression, may be an integral component in the promotionof Schwann cell migration and axonal extension through acomplicated terrain undergoing extensive cellular remodel-ing. The prominent expression of its receptor, α5β1, onSchwann cells and on regenerating and developing neuritessuggests that this receptor-ligand interaction is importantfor successful peripheral nerve development and regenera-tion.

We thank T. Broekelmann for assistance in production of theα5-47 antibody, J. Muschler and A. F. Horwitz for generouslyproviding the A2F7 monoclonal antibody, A. Frankfurter for thegift of the TuJi monoclonal antibody, M. Kirschner and T. Mitchi-son for the use of their microscopes, and M. Meyerson for experttyping assistance. We gratefully acknowledge D. Clary, I. deCurtis and K. Jones for critically reading the manuscript and infor-mative discussions. Grant support included NIH#1F32NSO8451-01A1 to F. L., NS19090 to L. F. R., 2PO1HL29594 and1RO1HL43418 to J. A. M.; L. F. R. is an investigator of theHoward Hughes Medical Institute.

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(Accepted 20 August 1992)

F. Lefcort and others