THE JOURNAL OF COMPARATIVE NEUROLOGY 295:43-51 (1990)
Monoclonal Antibody Rat 401 Recognizes Schwann Cells in Mature and Developing Peripheral Nerve BETH FRIEDMAN, SAM ZAREMBA, AND SUSAN HOCKF'IELD Department of Neurology (B.F.) and Section of Neuroanatomy (S.Z.,S.H.), Yale University School of Medicine, New Haven, Connecticut 06512
ABSTRACT Monoclonal antibody Rat 401 recognizes subsets of cells in the developing central and peripheral nervous systems. Previous studies have shown that in the central nervous system (CNS) Rat 401 immunoreactivity diminishes sharply with cellular differentiation. Here we have examined the time course, cellular localization, and biochemical nature of the Rat 401 antigen in the rat peripheral nerve. In contrast to the CNS, in the periphery Rat 401 immunoreactivity is maintained into adulthood. Rat 401 staining is restricted to Schwann cells in mature peripheral nerve. Myelin-related Schwann cells are intensely immunoreactive, whereas nonmyelin-related Schwann cells are weakly immunoreactive. Unlike many Schwann cell markers, Rat 401 staining is maintained in cultured Schwann cells that lack axon contact. Biochemical analyses show that the antigen recognized by Rat 401 in the peripheral nerve is identical to that in embryonic CNS. The results demonstrate that the capacity for maintained Rat 401 immunoreactivity is restricted to Schwann cells as these cells are stained in adult animals as well as in embryos. In contrast, the same antigens are lost from the CNS a t an early stage of development. Key words: peripheral nervous system, glia, sciatic nerve, differentiation
Nonneuronal cells in the adult mammalian central nervous system (CNS) and the peripheral nervous system (PNS) share a number of molecular characteristics. Astrocytes in the CNS (Eng et al. '71; Bignami et al., '72) and nonmyelin-related Schwann cells in the P N S are immunoreactive to antibodies (reviewed in Lemke, '88). Thus the overlap in the molecular characteristics of CNS and PNS glia may result from shared functions, such as the ensheathment of axons and maintenance of nodal and internodal domains along the mature axolemma (Peters et al., '76; Hirano, '81). During development central and peripheral glia also share several molecular characteristics. CNS astrocytes and P N S Schwann cells are both recognized by antibodies to laminin (Liesi et al., '83; Rogers et al., '86), L1 (Martini and Schachner, '86), and vimentin (Yen and Fields, '81, '85; Bignami et al., '82) at early stages of differentiation. However, one feature that distinguishes CNS from P N S glia is that the temporal regulation of certain molecules is different. For example, laminin and vimentin immunoreactivity are transiently exhibited by immature astrocytes and diminish with astrocyte maturation (Liesi et al., '83, '85; Bovolenta et al., '84). In contrast, Schwann cells shorn7 sustained immunoreactivity for laminin and vimentin even
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in the adult peripheral nerve (Cornbrooks et al., '83; Chiu et al., '88). Previous work in this and in other laboratories has shown that monoclonal antibody Rat 401 recognizes cells in the CNS and the P N S at very early developmental stages (Hockfield and McKay, '85). In the embryonic CNS, Rat 401 positive cells have been demonstrated to be neural precursors (Hockfield and McKay, '85; Frederikson and McKay, '88). However, as these cells begin to express mature neuronal or glial properties, Rat 401 immunoreactivity is sharply reduced (Frederikson and McKay, '88). The aim of the present study was to determine if the Rat 401 antigens display different patterns of' temporal regulation in peripheral glia as compared to CNS glia. Here we show that: (1)the temporal regulation of Rat 401 immunoreactivity in the P N S is distinct from that in the CNS; (2) Rat 401 staining is maintained at high levels into adulthood in the PNS; (3) Rat 401 immunoreactivity in the P N S can be localized to Schwann cells; and (4) Rat 401 recognizes related, if not Accepted December 10,1989. Address reprint requests t o Dr. Susan Hockfield, Section of Neuroanatomy, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.
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identical, molecules in the embryonic CNS and in the adult PNS.
MATERIALS AND METHODS Immunocytochemistry Tissue for immunocytochemistry, cell culture, and biochemistry was obtained from Sprague-Dawley rats at a series of ages from embryonic day 11 (where EO = day of conception) through adulthood. For immunocytochemistry, animals were anesthetized by hypothermia up to postnatal day 5 (P5) or with .042 gm chloral hydrate/100 gm body weight at older ages, and then perfused transcardially with saline (0.9% w/vol) followed by 4% paraformaldehyde (w/vol in 0.1 M phosphate buffer, pH 7.4). Whole embryos and dissected peripheral nerve and spinal cord from postnatal animals were postfixed by overnight immersion in the perfusion fixative. The tissue was then stored in 0.1 M phosphate buffer with 0.2% sodium azide until sectioning. To prepare samples for light microscopic immunocytochemistry, tissue was equilibrated in 30% sucrose in phosphate buffer and frozen on dry ice. Sections of 10-20 Fm were thawed directly onto gelatin-coated slides for immunostaining. Immunocytochemistry of sections on slides or cells cultured on coverslips was carried out by incubation in a humidified chamber with primary antibody with added ' final concentration) to permeabilize the Triton X-100 (0.1% tissue. The primary antibodies utilized were monoclonal antibodies from hybridoma cultures: Rat 401, which visualized embryonic neuroepithelial cells, and Cat 101, which recognizes neurofilaments (Hockfield and McKay, '85). To visualize bound primary antibodies, the tissue was incubated with goat antimouse IgG (Cappel) linked to horseradish peroxidase. The secondary antibody was diluted with Dulbecco's modified Eagle's media (DMEM-Gibco) with 5 % fetal calf serum (Hyclone). The bound secondary antibody was visualized with diaminobenzidine and hydrogen peroxide (Graham and Karnovsky, '66). Immunocytochemistry was also performed on freefloating sections. The main departure from the above protocol was to increase the Triton X-100 to a final concentration of2%. For ultrastructural analysis, tissue was cut on a vibratome a t 50-100 pm and prepared for immunocytochemistry as for light microscopy except Triton X-100 was omitted from antibody incubations to preserve membrane structure. The tissue was osmicated and embedded in Epon resin. Thin sections were examined without poststaining a t 60 kV on a Jeol electron microscope. Nonspecific staining was assessed by incubation of tissue sections with tissue culture media followed by secondary antibody (goat antimouse, Cappel) conjugated to horseradish peroxidase and processed as described above.
Tissue culture Schwann cells for in vitro analysis were prepared from the peripheral nerve, dorsal root ganglia (DRG), and sympathetic ganglia. Schwann cells from postnatal day 5 rat sciatic nerve were prepared with the protocol described by Moretto et al. ('85). These cultures were examined with immunocytochemical staining methods after 4 days in vitro. Schwann cells prepared from postnatal day 1 rat sciatic nerve were maintained for 3-4 weeks in vitro (the generous gift of Dr.
Nurit Kalderon); these cultures were prepared according to the method described by Brockes et al. ('77). Dorsal root ganglia (DRG) cultures (the generous gift of Kathleen Martin) were obtained from rat fetuses a t 15-16 days of gestational age and were maintained in culture for 10 to 14 days. They were prepared according to the method described by Ransom et al. ('77). Cultures of dissociated sympathetic ganglia and sympathetic ganglia explants from E l 5 rats (the generous gift of Dr. Carson Cornbrooks) were maintained for 3 weeks in vitro and prepared as described in Roufa et al. ('86). For immunocytochemical examination of cell cultures, the cultures were rinsed in phosphate-buffered saline (PBS), and then fixed in 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1hour a t room temperature. The cultures were rinsed and stored in 0.1 M phosphate buffer with 0.2% sodium azide. Incubations with primary (Rat 401) and secondary antibodies (goat antimouse IgG-Cappel, at 1:lOO dilution with DMEM) were performed sequentially for 1 hour a t room temperature. Bound HRP-labelled secondary antibody was visualized with diaminobenzidine and the stained cultures were dehydrated and coverslipped with Permount (Fisher) for light microscopic examination.
Biochemistry Samples of E l 9 rat CNS and of spinal cord and sciatic nerve from adult rats were obtained by dissection from anesthetized animals transcardially perfused with saline. Tissue was homogenized (1ml buffer/gm tissue) in 10 mM sodium phosphate pH 6.8, 3%; SDS, 10% glycerol (SDSPAGE buffer) with DNAase (.01 mg/ml; Worthington) and boiled. Boiled samples were vortexed, centrifuged 10 minutes a t 12,000 g, and the supernatants and pellets stored separately at - 7OOC. Extracts were analyzed by immunoadsorbtion and Western blot analysis. Extracts were assayed for protein (BioRad Dye Reagent); amounts indicated in the text were diluted to a volume of 1.4 ml buffer at the following final concentrations: 10 mM TrisCl pH 7.4,0.14 M NaC1,5 mM EDTA, 0.1% SDS, and 0.5% NP-40 (immunoprecipitation buffer). These solutions were immunoadsorbed by mixing overnight in the cold room with goat antimouse IgG Agarose beads (Sigma) that had been preincubated with the appropriate monoclonal antibody. Beads were collected by centrifugation, washed five times with immunoprecipitation buffer, and boiled with SDS-PAGE buffer with 0.01 % Bromophenol Blue and 1% b-mercaptoethanol. The precipitated proteins were separated on 3-8 % acrylamide gradient gels (Laemmli, '70) and transferred to nitrocellulose (Towbin et al., '76) in buffer containing 25 mM Tris, 0.192 M glycine, 0.10: SDS, and 20% methanol. The nitrocellulose was blocked with 1%BSA and then incubated with the appropriate monoclonal antibodies. Antibody binding was detected with alkaline phosphatase conjugated antimouse IgG secondary antibodies (Promega) and visualized with nitroblue tetrazolium and 5-bromo-4-chloro-3indolyl phosphate in 0.1 M Tris C1 pH 8.9,O.l M NaC1, and 5 mM MgC1,.
RESULTS Rat 401 recognizes Schwann cells in adult peripheral nerve Previous work demonstrated that Rat 401 recognizes embryonic CNS and P N S neural tissue (Hockfield and
RAT 401 STAINS SCHWANN CELLS McKay, ’85). The staining in the nerve roots of embryos (E15) localizes to a cell type with patchy basal lamina and an orientation that is parallel to axons (Hockfield and McKay, ’85).These characteristics identify immature Schwann cells that have begun to ensheathe axons in vitro (Bunge et al., ’80) and suggest that the antibody staining of embryonic nerve roots can be largely accounted for by Schwann cell staining. The spinal cord of adult rats, unlike that of embryos, lacks readily detectable Rat 401 immunoreactivity (Hockfield and McKay, ’85). The level of immunoreactivity increases sharply, however, a t the junction between the spinal cord and nerve roots (Fig. 1). The PNS immunoreactivity extends distally into the spinal nerves. In cross sections of sciatic nerve, Rat 401 immunoreactivity produces thin, broken rings of staining that lie exterior to the unstained myelin sheaths (Fig. 2a). The staining appears to be restricted to such “rings” and the adjacent endoneurium is not stained above background levels. The Rat 401 staining pattern in nerve is distinct from that produced by staining with antineurofilament antibodies (Fig. 2b) or by staining with tissue culture media (Fig. 2c). The pattern of immunoreactivity observed at the light microscopic level suggests that Rat 401 recognizes Schwann cells and not axons in peripheral nerve. In order to determine the cellular localization of Rat 401 immunoreactivity at higher resolution, sections from the adult sciatic nerve and the sympathetic trunk were prepared
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for ultrastructural immunocytochemistry (Fig. 3a,b). The sciatic nerve contains populations of both myelinated and unmyelinated axons. Immunoreactivity is most prominent in the cytoplasm of Schwann cells related to myelin sheaths. Myelin and the underlying cytoplasm are also not immunoreactive. The nonmyelin-related Schwann cell in the field shown in Figure 3A is unstained. These results indicate that the widespread Rat 401 staining observed with light microscopic examination of the adult dorsal root (Fig. 1) and adult sciatic nerve (Fig. 2a) results from immunoreactivity of the Schwann cell cytoplasm. The sympathetic trunk is comprised mainly of unmyelinated axons and permits a systematic assessment of Rat 401 immunoreactivity in nonmyelin-related Schwann cells. In order to determine if the adult staining represents immunoreactivity in both myelin and nonmyelin-related Schwann cells, sections from stained sympathetic trunk were examined by electron microscopy. The intensity of staining is generally weaker in the sympathetic trunk than in the sciatic nerve, but is localized to nonmyelin-related Schwann cells (Fig. 3b). The subcellular localization of this Rat 401 staining resembles that seen for myelin-related Schwann cells in the sciatic nerve. Sciatic nerve from 14-day-oldrats contains actively myelinating Schwann cells (Webster and Favilla, ’84). Rat 401 recognizes such Schwann cells that are engaged in axonal myelination (Fig. 312). As a t older ages, the staining is confined to the Schwann cell cytoplasm while the myelin is
Fig. 1. Rat 401 immunoperoxidase stained section of adult rat spinal cord (SC). The adult spinal cord shows faint traces of staining, whereas the attached dorsal root (arrow) shows intense immunoreactivity. Scale bar = 100 urn.
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B. FRIEDMAN ET AL.
Fig. 2. Adult sciatic nerve in coronal section. Sections in A-C were obtained from the same block of tissue. A. Rat 401 staining is restricted to narrow regions (arrows) adjacent to unstained rnyelin. B. Axons stained with Cat 101 exhibit a different staining pattern of filled circular
profiles. C. Negative control section incubated with tissue culture media followed by secondary antibody shows no specific staining pattern. Scale bar = 10pm.
unstained. These results in combination with previous work indicate that Rat 401 recognizes ensheathing, myelinating, and myelin-related Schwann cells in spinal nerves. Nonmyelin-related Schwann cells are stained as well but at lower intensity.
produced extremely intense staining of Schwann cells in these acute cultures. Essentially all small, refractile, process bearing and process free cells were stained (Fig. 4c). By maintaining Schwann cells dissociated from sciatic nerve for much longer times in culture (3-4 weeks), cells devoid of contact with even degenerating fragments of axons can be evaluated. The Schwann cells in these cultures are process bearing and typically bipolar. In these long-term cultures, Rat 401 darkly stained process-bearing Schwann cells. In contrast, the large stress fiber-containing fibroblasts were not stained (Fig. 4D,E). Together, these results indicate that Rat 401 immunoreactivity is selective for Schwann cells. Furthermore, it is not dependent on specific neural architecture or on contact with live axons, axon membranes, or neurons.
Rat 401 recognizes Schwann cells maintained in vitro The in situ studies described above indicate that Rat 401 recognizes adult Schwann cells that ensheathe myelinated and unmyelinated axons. In order to determine if axon contact or some other component of the three-dimensional organization of the nervous system is required for continued Rat 401 expression, Schwann cells were dissociated and maintained in culture prior to antibody staining. Schwann cells were examined under four culture conditions: (1)dorsal root ganglion (DRG) cultures obtained from E15-16 rats and maintained 10 to 14 days in vitro; (2) sympathetic ganglia cultures obtained from E l 5 rats and maintained 21 days in vitro; (3) Schwann cells derived from sciatic nerve of P 5 rats and maintained for 4 days in vitro; and (4) Schwann cells obtained from sciatic nerve of P1 rats and maintained for 21 to 30 days in vitro. The DRG and sympathetic ganglia cultures allow examination of the premyelinating and the prenonmyelinating Schwann cell (Roufa e t al., '86),respectively. In both DRG and sympathetic ganglia cultures, Rat 401 stained cells with Schwann cell morphology (Fig. 4a,b). Other cells such as fibroblasts, neuron clusters, and neurite processes were unstained. The intensity of staining of Schwann cells in the SCG cultures was more variable than in the DRG cultures. Schwann cells that lack contact with live neurons or neuronal processes were obtained by dissociation of sciatic nerves. A t short plating times, many of the Schwann cells are round and do not have extended processes. Rat 401
Rat 401 recognizes antigens with identical molecular weights in the embryonic CNS and adult peripheral nerve The above results indicate that Rat 401 immunoreactivity is maintained in glia of the P N S (Schwann cells) in adulthood, whereas it is known to be sharply reduced in CNS glia a t an early developmental stage (Hockfield and McKay, '85). We next asked whether identical antigens are present in both CNS and PNS but subject to different temporal regulation or if the staining in embryonic CNS and adult PNS represents different sets of molecules that merely share the Rat 401 epitope. Rat 401 positive antigens in adult P N S and embryonic CNS were compared by immunoadsorbtion and Western blot analysis. The immunoadsorbtion step was included in order to isolate Rat 401 antigens from a large amount of tissue extract. This allowed us to analyze antigens that might be in low abundance without grossly overloading the
Fig. 3. Electron micrographs of Rat 401 immunoperoxidase stained nerve. A. Adult rat sciatic nerve. Rat 401 stains a myelin-related Schwann cell (filled arrow). Adjacent nonmyelin-related Schwann cell lacks staining (open arrow). Scale bar = 0.5 pm. B. Adult sympathetic trunk. Rat 401 stains a nonmyelin-related Schwann cell. Staining, as in the myelin-related Schwann cells, is localized to the cytoplasm (arrows).
Scale bar = 0.5 pm. C. P14 rat sciatic nerve. Rat 401 stains ruffled cytoplasm of actively myelinating Schwann cells (arrows). Staining at the outer rim of myelin may result from reaction product diffusion. Scale bar = 10 pm. All thin sections were examined without poststaining with lead citrate or uranyl acetate.
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Fig. 4. Rat 401 recognizes Schwann cells in the presence (A and B) and absence (C -E) of neurons. A. R a t 401 stained Schwann cells in DRG cultures have both spindle (arrowhead) and multipolar morphologies. B. Rat 401 stained cells (arrowhead) in cultures from sympathetic ganglia. These cultures show variable intensity in Schwann cell immunoreactivity and weaker staining relative t o the DRG cultures illustrated in A. C. Schwann cells from sciatic nerve maintained in the absence of neurons
for 4 days are strongly immunoreactive with R a t 401 (arrowhead). D, E. Brightfield (D) and phase contrast (E) view of the same culture of Schwann cells (arrows) from sciatic nerve maintained for 3 weeks and passaged in the absence of neurons. Although the Schwann cells are intensely stained, the fibroblasts (arrowheads) are not stained. Bars = 100 pm.
gels. Figure SA shows the Rat 401 positive species obtained from extracts of E l 9 rat CNS (1.2 mg protein) immunoadsorbed with Rat 401. The most prominent species appear as sharp bands at molecular weights of 400 ku and 175 kD. Numerous other bands appear in lower abundance, many at
molecular weights intermediate between the two major bands. Except for the bands of the immunoadsorbed and eluted antibodies themselves (see Fig. 5 legend), none of these antigens appear when identical immunoadsorbed samples were reacted with a control mouse IgG, Cat 301. Nor did
RAT 401 STAINS SCHWA"
A
CELLS
B
Fig. 5. Characterization of Rat 401 reactive antigens. A. 1.2 mg of El9 CNS extract was immunoadsorhed with antimouse IgG agarose heads preincuhated with Rat 401 (lanes 1 and 2) or Cat 301 (lane 3). Immunoadsorhed materials were eluted, electrophoresed, and transferred as described in Methods. Strips were then processed with monoclonal antibodies as follows: Cat 301 (lane l), or Rat 401 (lanes 2 and 3). The lowest molecular weight hand, present in all 3 lanes, also appears when mAB -coated immunoheads (without tissue extract) are analyzed (not shown); consequently it represents the IgG antibody molecule itself. B. Extracts (0.54 mg) from the following tissues were subjected to immunoadsorbtion with Rat 401 followed by Western blot analysis with Rat 401: adult spinal cord (lane 4), E l 9 CNS (lane 5 ) , and adult sciatic nerve (lane 6). All the Rat 401 reactive antigens identified in E l 9 CNS are also present in adult peripheral nerve. This is consistent with the persistent immunoreactivity of Rat 401 in adult peripheral nerve; in contrast adult central nervous tissue contains only trace amounts of the 175 kD Rat 401 antigen, consistent with the marked developmental down-regulation of Rat 401 expression in the CNS.
any of these antigens appear in the inverse control, when extracts were immunoadsorbed with Cat 301, then electrophoresed, transferred and reacted with Rat 401. The 175 kD band presumably matches the major Rat 401 reactive band that has been previously identified as a 180-200 kD species (Hockfield and McKay, '85). The 400 kD band reported here may have emerged as a result of the use of 3-8% gradient gels that would optimize the resolution of heavier molecular weight species. In Figure 5B, extracts (0.54 mg protein) of' embryonic CNS, adult spinal cord, and adult nerve were compared by immunoadsorbtion and Western blot analysis with Rat 401. The major bands a t 400 kD and 175 kD previously identified in embryonic CNS, as well as the minor bands, are also seen in extracts of adult peripherai nerve. No other new Rat 401 immunoreactive bands were evident in extracts of adult peripheral nerve. Adult spinal cord extracts, when subjected to the same analysis, reveal only slight traces of the 175 kD immunoreactive band. These results are consistent with the immunocytochemical findings and provide evidence that the same antigenic species are differentially regulated in the CNS and in the PNS.
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DISCUSSION Here we have studied the time course of Rat 401 immunoreactivity in peripheral glia to determine whether it is similar to that described for central glia. Previous work has shown that Rat 401 immunostaining is sharply reduced in central neuroepithelial cells with maturation (Hockfield and McKay, '85; Frederikson and McKay, '88; Frederikson et al., '88). The results presented here show that Rat 401 strongly stains peripheral glia during active myelination. Furthermore, immunoreactivity persists in both myelin- and nonmyelin-related Schwann cells in the adult peripheral nerve. Rat 401 selectively recognizes Schwann cells maintained in culture in the complete absence of axon contact, as well as in coculture with peripheral neurons. Western blot analysis of immunoprecipitated antigens from the embryonic CNS and the adult sciatic nerve indicates that the apparent molecular weights of immunoreactive antigens in embryonic central and in adult peripheral tissues are identical. Together these results suggest that the temporal regulation of Rat 401 immunoreactivity in Schwann cells is markedly different from that in CNS glia (and neurons). Thus Rat 401 appears to identify antigens that, like vimentin (Chiu et al.,'88) and laminin (Cornbrooks et al., '83) persist in adult Schwann cells but are down-regulated in the CNS at early stages of development (Liesi et al., '83; Bovolenta et al., '84; Liesi, '85). The restriction of intense Rat 401 immunoreactivity to peripheral nerve in the adult rat is demonstrated most dramatically by the staining pattern of the dorsal root entry zone of the spinal cord (Fig. 1).The strong, positive staining of peripheral neural tissue abruptly ends with a border complementary in shape to the glial limitans of the dorsal root entry zone. The paucity of immunocytochemical staining in adult CNS is confirmed by the absence of all but trace levels of biochemically detectable Rat 401 antigens in extracts of adult spinal cord. The normal reduction of Rat 401 immunoreactivity in CNS neural precursors appears to correlate with the expression of cell type specific molecules such as glial fibrillary acidic protein in astrocytes (Frederiksen and McKay, '88). Transformed and immortalized embryonic CNS cells are also capable of sustained Rat 401 immunoreactivity in vitro. However, this immunoreactivity is lost when these cells are forced to differentiate (Frederiksen et al., '88; McKay et al., '88). In contrast, Schwann cells that are fully differentiated are recognized by Rat 401. One intriguing correlate to this persistent immunoreactivity is that adult Schwann cells, unlike adult CNS neurons or glia, are able to dedifferentiate (Abercrombie and ,Johnson, '42; Fekete and Brockes, '86) when they lose contact with axons as a consequence of nerve injury (Holmes and Young, '42; Bradley and Asbury, '70). Furthermore, when axon contact is resumed, during nerve regeneration, Schwann cells can redifferentiate. Thus Rat 401 immunoreactivity may be linked to a still unidentified feature that is expressed by cells that are still capable of differentiation (e.g., CNS neural precursors) and by cells that may readily dedifferentiate and then redifferentiate (e.g., PNS Schwann cells). During early development, Schwann cells are essentially biochemically indistinguishable in premyelinated and prenonmyelinated nerve (Lamperth e t al., '89). Our results provide further evidence for a biochemical homogeneity among these early Schwann cells. Rat 401 produces uniformly intense staining throughout the embryonic PNS, not
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only in roots, sympathetic trunk, and spinal nerves, but in enteric glia, and the Schwann cells along the olfactory pathway (data not shown). With differentiation, Schwann cell phenotype is strongly regulated by axon contact. Indeed, Schwann cell phenotype can be converted from nonmyelin- to myelin-related simply by forced interaction with axons that are normally myelinated (Aguayo et al., '76). This plasticity is remarkable in the light of the strikingly different biochemical and morphological profiles of mature myelin-related and nonmyelinrelated Schwann cells (Trapp et al., '79; Yen and Fields, '81, '85; Jessen and Mirsky, '84; Jessen et al., '84). Here we show that intense Rat 401 antibody staining is maintained in mature myelin-related Schwann cells. However, whereas mature nonmyelinating Schwann cells are also recognized by Rat 401, the staining ranged from moderate to faint both in situ and in culture. This qualitative difference in staining between mature myelin-related a n d nonmyelinating Schwann cells is a new phenotypic difference between these cell "types." This could provide an unusual example of a limited developmental down regulation of a molecular species in nonmyelin-related Schwann cells. However, biochemical studies are necessary to rigorously quantify differences in antigen concentration that could account for the differences in staining intensity. Another unusual feature of Rat 401 immunoreactivity is that it persists a t a high level when Schwann cells are deprived of axon contact. Another marker for Schwann cells in long-term cultures has been isolated by Dulac and coworkers ('88). Rat 401 immunoreactivity with its cytoplasmic localization differs from their antibody, which recognizes a protein with a cell surface localization (Dulac et al., '88). In contrast to these persistent markers for cultured Schwann cells, laminin (Cornbrooks et al., '83) and Ran-2 (Jessen and Mirsky, '84) are down-regulated in Schwann cell cultures that lack extensive neuronal contact. Reduction of immunoreactivity in dissociated Schwann cells is also typical for myelin-related molecules (Mirsky e t al., '80; LeBlanc et al., '87; Trapp et al., '88). Thus the maintained expression of Rat 401 antigens by dissociated, formerly myelin-related Schwann cells suggests that Rat 401 antigens are probably not closely involved in the production or maintenance of myelin. This characteristic antibody staining of Schwann cells even in the absence of axon contact makes Rat 401 an especially useful marker for Schwann cells in long-term in vitro studies of mammalian tissue. The cross reactivity may, however, be limited as Rat 401 does not appear to stain chick CNS or human peripheral nerve (unpublished observations). The classification of the Rat 401 positive cells in E l l nerve roots remains to be firmly established. However, as early as definitive morphological identification of Schwann cells was possible, at E15, these cells are immunoreactive for Rat 401. A t a minimum, the Rat 401 positive cells in the nerve roots of E l l rats (Hockfield and McKay, '85) are antigenically related to immature and to mature Schwann cells on the basis of shared immunoreactivity to Rat 401 antibodies. Furthermore, as axon contact is not necessary for expression of Rat 401 in cultured Schwann cells, the lack of extensive axon-"Schwann cell" interactions in E l l nerve would not preclude Rat 401 expression in putative Schwann cells. Thus it is tempting to speculate that the early Rat 401 cells in E l l nerve roots are Schwann cells or Schwann cell precursors. If these cells are Schwann cells, then it may become possible to test if de novo outgrowth of mammalian
B. FRIEDMAN ET AL. peripheral axons, like regenerative axonal growth (Aguayo et al., '82), utilizes a substrate formed by Schwann cells.
ACKNOWLEDGMENTS This work was supported by grants from NIH (NS-07877, B.F.) (EY-05855, S.Z.) and KSF (BNS 8644681, S.H.). S.H. is a Klingenstein fellow in the neurosciences. The authors thank Drs. Nurit Kalderon, Carson Cornbrooks, and Kathleen Martin for their generous gifts of cultured Schwann cells.
LITERATURE CITED Abercromhie, M., and J.L. Johnson (1942) The outwandering of cells in tissue cultures of nerves undergoing Wallerian degeneration. J. Exp. Biol. 191266-283. Aguayo, A.J., L.C. Charron, and G.M. Bray (1976) Potential of Schwann cells from unmyelinated nerve to produce myelin: A quantitative ultrastructural and radiographic study. J. Neurocytol. 5:565-573. Aguayo, A.J., P.M. Richardson, S. David, and M. Benfey (1982) Transplantation of neurons and sheath cells- A tool for the study of regeneration. In J.G. Nicholls (ed): Repair and Regeneration of the Nervous System. New York: Springer-Verlag, pp. 91-105. Barber, P.C., and R.M. Lindsay (1982) Schwann cells of the olfactory nerves contain glial fihrillary acidic protein and resemble astrocytes. Neurosci. 7(12) 13077-3090. Bignami, A,, T.R. Raju, and D. Dahl (1982) Localization of vimentin, the nonspecific intermediate filament protein, in embryonal glia and in early differentiating neurons. Dev. Biol. 91:286-295. Bignami, A,, L.F. Eng, D. Uahl, and C.T. Uyeda (1972) Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res. 43r429-435. Bovolenta, P., R.K.H. Liem, C.A. Mason (1984) Development of cerebellar astroglia: Transitions in form and cytoskeletal content. Dev. Biol. 102:24& 259. Bradley, W.G., and A.K. Asbury (1970) Duration of synthesis phase in neurilemma cells in mouse sciatic nerve during degeneration. Exp. Neurol. 26:275-282. Brockes, J.P., K.L. Fields, and M.C. Raff (1977) A surface antigenic marker for rat Schwann cells. Nature 26'61364-366. Bunge, M., A. Williams, P. Wood, J. Uitto, and J. Jeffrey (1980) Comparison of nerve cell and nerve cell plus Schwann cell cultures, with particular emphasis on basal lamina and collagen formation. J. Cell Biol. 84:184202. Chiu, F.X., R.S. Sacchi, L. Claudio, S. Kobayashi, and K. Suzuki (1988) Coexpression of glial fihrillary acidic protein and vimentin in the central and peripheral nervous systems of the twitcher mutant. Glia 1:105-112. Cornbrooks, C.J., D.J. Carey, J.A. McDonald, R. Timpl, and R.P. Bunge (1983) In vivo and in vitro observations on laminin production by Schwann cells. Proc. Nat. Acad. Sci. USA 8Ot3850-3854. Dulac, C., P. Cameron-Curry, C. Ziller, and N.M. Le Douarin (1988) A surface protein expressed by avian myelinating and nonmyelinating Schwann cells but not by satellite or enteric glial cells. Neuron 1:211-220. Eng, L.F., J.J. Vanderhaeghen, A. Bignami, and B. Gerstl (1971) An acidic protein isolated from fibrous astrocytes. Brain Res. 103:613-616. Fekete, D.M., and J.P. Brockes (1987) The aneurogenic limb A puzzle in cell interactions. TINS 10:364-368. Frederiksen, K., and R. McKay (1988) Proliferation and differentiation of neuroepithelial stem cells. J. Neurosci. 8:114&1151. Frederiksen, K., P . J . Jat, N. Valtz, D. Levy, and R McKay (1988) Immortalization of precursor cells from the mammalian CNS. Neuron 1:439-448. Graham, R.C., and M.J. Karnovsky (1966) The early stages of absorption of injected horseradish peroxidase in the proximal tubules of the mouse kidney. Ultrastructural cytochemiatry by a new technique. J. Histochem. Cytochem. 31 112):1394-1398. Hirano, A. (1981) Structure of normal central myelinated fibers. In S.G. Waxman and J.G. Ritchie (eds): Advances in Neurology: Demyelinating Disease, vol. 31. New York: Raven Press, pp. 51-69. Hockfield, S., and R. McKay (1985) Identification of major cell classes in the developing mammalian nervous system. J. Neurosci. 5:3310-3329. Holmes, W., and J.Z. Young (1942) Nerve regeneration after immediate and delayed suture. J. Anat. 77:63-96.
RAT 401 STAINS SCHWANN CELLS Jessen, K.R., and R. Mirsky (1984) Nonmyelin forming Schwann cells coexpress surface proteins and intermediate filaments not found in myelin forming cells: A study of Ran-2, A5E3 antigen and glial fibrillary acidic protein. J . Neurocytol. 13:923-934. Jessen, K.R., R. Thorpe, and R. Mirsky (1984) Molecular identity, distrihution and heterogeneity of glial fibrillary acidic protein: An immunoblotting and immunohistochemical study of Schwann cells, satellite cells, enteric glia and astrocytes. J. Neurocytol. 13:187-200. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227t680-682. Lamperth, L., L. Manuelidis, and H. deF. Webster (1989) Nonmyelinforming perineuronal Schwann cells in rat trigeminal ganglia express PO myelin glycoprotein during postnatal development, Molec. Brain Res. 5:177-181. LeBlanc, A.C., J.F. Poduslo, and C. Mezei (1987) Gene expression in the presence or absence of myelin assembly. Mol. Brain Res. 257-67. Lemke, G. (1988) Unwrapping the genes of myelin. Neuron 1:535-543. Liesi, P. (1985) Laminin-immunoreactive glia distinguish regenerative adult CNS systems from non-regenerative ones. EMBO J. 4:2505-2511. Liesi, P., D. Dahl, and A. Vaheri (1983) Laminin is produced by early rat astrocytes in primary culture. J. Cell Biol. 96:920-924. McKay, R., K. Frederiksen, P.-J. Jat, and D. Levy (1988) Reconstructing the brain from immortal cell lines. Prog. Brain Res. 781647449. Martini, R., and M. Schachner (1986) Inimunoelectronmicroscopic localization of neural cell adhesion molecules (Ll, N-CAM, and MAG) and their shared carbohydrate epitope and myelin basic protein in developing sciatic nerve. J. Cell Biol. 1032439-2448. Moretto, G., S.U. Kim, D.H. Shin, D.E. Pleasure, and N. Rizzuro (19&1) Long-term cultures of human adult Schwann cells isolated from autopsy materials. Acta Neuropathol. 64:15-21. Mirsky, R., J. Winter, E.R. Ahney, R.M. Pruss, J. Gavrilovic, and M.C. Raff (1980) Myelin specific proteins and glycolipids in rat Schwann cells and oligodendrocytes in culture. J. Cell Biol. 84t483-494.
51 Peters, A., S.Palay, and H.DeF. Wehster (1976) The Fine Structure of the Nervous System: The Neurons and the Supporting Cells. Philadelphia: W.B. Saunders. Ransom. B.R.. E. Neale, M. Henkart. P.N. Bullock, and P.G. Nelson (1977) Mouse spinal cord in culture. 1: Morphology and intrinsic neuronal electrophysiologic properties. J. Neurophysiol. 40:1132-1150. Rogers, S.L., K.J. Edson, P.C. Letourneau, and S.C. McLoon (1986) Distribution of laminin in the developing peripheral nervous system of the chick. Dev. Biol. 113:429-435. Roufa, D., M.B. Bunge, M.1. Johnson, and C.J. Cornhrooks (19%) Variation in content and function of nonneuronal cells in the outgrowth of sympathetic ganglia from embryos of differing age. J. Neurosci. 6790802. Trapp, B.D., P. Hauer, and G. Lemke (1988) Axonal regulation of myelin protein mRNA levels in actively myelinating Schwann cells. J. Neurosci. 8(91:3515-3521. Trapp, B.D., L.J. hlclntyre, R.H. Quarles, N.H. Sternherger, and H deF. Webster (1979) Immunocytochemical localization of rat peripheral nerVOUY system proteins. P 2 is not a component of all peripheral nervous system myelin sheaths. Proc. Nat. Acad. Sci. USA 76:3552-3556. Towbin, H., T. Staehlin, and J. Gordon (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Nat. Acad. Soc. USA 76:43504354. Webster, H.deF., and J.T. Favilla (1984) Development of peripheral nerve fibers. In P.J. Dyck, P.K. Thomas, E.H. Lamhert and R. Bunge (eds): Peripheral Neuropathy. Philadelphia: Saunders, pp. 329-359. Yen, S.-H., and K.L. Fields (1981) Antibodies to neurofilament, glial filament and fibroblast intermediate filament proteins bind to different cell types of the nervous system. J. Cell Biol. 88:115-126. Yen, S.-H., and K.L. Fields (1985) A protein related to glial filaments in Schwann cells. Ann. NY Acad. Sci. 455538-551.
Monoclonal Antibody Rat 401 Recognizes Schwann Cells in Mature and Developing Peripheral Nerve BETH FRIEDMAN, SAM ZAREMBA, AND SUSAN HOCKF'IELD Department of Neurology (B.F.) and Section of Neuroanatomy (S.Z.,S.H.), Yale University School of Medicine, New Haven, Connecticut 06512
ABSTRACT Monoclonal antibody Rat 401 recognizes subsets of cells in the developing central and peripheral nervous systems. Previous studies have shown that in the central nervous system (CNS) Rat 401 immunoreactivity diminishes sharply with cellular differentiation. Here we have examined the time course, cellular localization, and biochemical nature of the Rat 401 antigen in the rat peripheral nerve. In contrast to the CNS, in the periphery Rat 401 immunoreactivity is maintained into adulthood. Rat 401 staining is restricted to Schwann cells in mature peripheral nerve. Myelin-related Schwann cells are intensely immunoreactive, whereas nonmyelin-related Schwann cells are weakly immunoreactive. Unlike many Schwann cell markers, Rat 401 staining is maintained in cultured Schwann cells that lack axon contact. Biochemical analyses show that the antigen recognized by Rat 401 in the peripheral nerve is identical to that in embryonic CNS. The results demonstrate that the capacity for maintained Rat 401 immunoreactivity is restricted to Schwann cells as these cells are stained in adult animals as well as in embryos. In contrast, the same antigens are lost from the CNS a t an early stage of development. Key words: peripheral nervous system, glia, sciatic nerve, differentiation
Nonneuronal cells in the adult mammalian central nervous system (CNS) and the peripheral nervous system (PNS) share a number of molecular characteristics. Astrocytes in the CNS (Eng et al. '71; Bignami et al., '72) and nonmyelin-related Schwann cells in the P N S are immunoreactive to antibodies (reviewed in Lemke, '88). Thus the overlap in the molecular characteristics of CNS and PNS glia may result from shared functions, such as the ensheathment of axons and maintenance of nodal and internodal domains along the mature axolemma (Peters et al., '76; Hirano, '81). During development central and peripheral glia also share several molecular characteristics. CNS astrocytes and P N S Schwann cells are both recognized by antibodies to laminin (Liesi et al., '83; Rogers et al., '86), L1 (Martini and Schachner, '86), and vimentin (Yen and Fields, '81, '85; Bignami et al., '82) at early stages of differentiation. However, one feature that distinguishes CNS from P N S glia is that the temporal regulation of certain molecules is different. For example, laminin and vimentin immunoreactivity are transiently exhibited by immature astrocytes and diminish with astrocyte maturation (Liesi et al., '83, '85; Bovolenta et al., '84). In contrast, Schwann cells shorn7 sustained immunoreactivity for laminin and vimentin even
o 1990 WTLEY-LISS, INC.
in the adult peripheral nerve (Cornbrooks et al., '83; Chiu et al., '88). Previous work in this and in other laboratories has shown that monoclonal antibody Rat 401 recognizes cells in the CNS and the P N S at very early developmental stages (Hockfield and McKay, '85). In the embryonic CNS, Rat 401 positive cells have been demonstrated to be neural precursors (Hockfield and McKay, '85; Frederikson and McKay, '88). However, as these cells begin to express mature neuronal or glial properties, Rat 401 immunoreactivity is sharply reduced (Frederikson and McKay, '88). The aim of the present study was to determine if the Rat 401 antigens display different patterns of' temporal regulation in peripheral glia as compared to CNS glia. Here we show that: (1)the temporal regulation of Rat 401 immunoreactivity in the P N S is distinct from that in the CNS; (2) Rat 401 staining is maintained at high levels into adulthood in the PNS; (3) Rat 401 immunoreactivity in the P N S can be localized to Schwann cells; and (4) Rat 401 recognizes related, if not Accepted December 10,1989. Address reprint requests t o Dr. Susan Hockfield, Section of Neuroanatomy, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510.
B. FRIEDMAN ET AL.
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identical, molecules in the embryonic CNS and in the adult PNS.
MATERIALS AND METHODS Immunocytochemistry Tissue for immunocytochemistry, cell culture, and biochemistry was obtained from Sprague-Dawley rats at a series of ages from embryonic day 11 (where EO = day of conception) through adulthood. For immunocytochemistry, animals were anesthetized by hypothermia up to postnatal day 5 (P5) or with .042 gm chloral hydrate/100 gm body weight at older ages, and then perfused transcardially with saline (0.9% w/vol) followed by 4% paraformaldehyde (w/vol in 0.1 M phosphate buffer, pH 7.4). Whole embryos and dissected peripheral nerve and spinal cord from postnatal animals were postfixed by overnight immersion in the perfusion fixative. The tissue was then stored in 0.1 M phosphate buffer with 0.2% sodium azide until sectioning. To prepare samples for light microscopic immunocytochemistry, tissue was equilibrated in 30% sucrose in phosphate buffer and frozen on dry ice. Sections of 10-20 Fm were thawed directly onto gelatin-coated slides for immunostaining. Immunocytochemistry of sections on slides or cells cultured on coverslips was carried out by incubation in a humidified chamber with primary antibody with added ' final concentration) to permeabilize the Triton X-100 (0.1% tissue. The primary antibodies utilized were monoclonal antibodies from hybridoma cultures: Rat 401, which visualized embryonic neuroepithelial cells, and Cat 101, which recognizes neurofilaments (Hockfield and McKay, '85). To visualize bound primary antibodies, the tissue was incubated with goat antimouse IgG (Cappel) linked to horseradish peroxidase. The secondary antibody was diluted with Dulbecco's modified Eagle's media (DMEM-Gibco) with 5 % fetal calf serum (Hyclone). The bound secondary antibody was visualized with diaminobenzidine and hydrogen peroxide (Graham and Karnovsky, '66). Immunocytochemistry was also performed on freefloating sections. The main departure from the above protocol was to increase the Triton X-100 to a final concentration of2%. For ultrastructural analysis, tissue was cut on a vibratome a t 50-100 pm and prepared for immunocytochemistry as for light microscopy except Triton X-100 was omitted from antibody incubations to preserve membrane structure. The tissue was osmicated and embedded in Epon resin. Thin sections were examined without poststaining a t 60 kV on a Jeol electron microscope. Nonspecific staining was assessed by incubation of tissue sections with tissue culture media followed by secondary antibody (goat antimouse, Cappel) conjugated to horseradish peroxidase and processed as described above.
Tissue culture Schwann cells for in vitro analysis were prepared from the peripheral nerve, dorsal root ganglia (DRG), and sympathetic ganglia. Schwann cells from postnatal day 5 rat sciatic nerve were prepared with the protocol described by Moretto et al. ('85). These cultures were examined with immunocytochemical staining methods after 4 days in vitro. Schwann cells prepared from postnatal day 1 rat sciatic nerve were maintained for 3-4 weeks in vitro (the generous gift of Dr.
Nurit Kalderon); these cultures were prepared according to the method described by Brockes et al. ('77). Dorsal root ganglia (DRG) cultures (the generous gift of Kathleen Martin) were obtained from rat fetuses a t 15-16 days of gestational age and were maintained in culture for 10 to 14 days. They were prepared according to the method described by Ransom et al. ('77). Cultures of dissociated sympathetic ganglia and sympathetic ganglia explants from E l 5 rats (the generous gift of Dr. Carson Cornbrooks) were maintained for 3 weeks in vitro and prepared as described in Roufa et al. ('86). For immunocytochemical examination of cell cultures, the cultures were rinsed in phosphate-buffered saline (PBS), and then fixed in 4 % paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 1hour a t room temperature. The cultures were rinsed and stored in 0.1 M phosphate buffer with 0.2% sodium azide. Incubations with primary (Rat 401) and secondary antibodies (goat antimouse IgG-Cappel, at 1:lOO dilution with DMEM) were performed sequentially for 1 hour a t room temperature. Bound HRP-labelled secondary antibody was visualized with diaminobenzidine and the stained cultures were dehydrated and coverslipped with Permount (Fisher) for light microscopic examination.
Biochemistry Samples of E l 9 rat CNS and of spinal cord and sciatic nerve from adult rats were obtained by dissection from anesthetized animals transcardially perfused with saline. Tissue was homogenized (1ml buffer/gm tissue) in 10 mM sodium phosphate pH 6.8, 3%; SDS, 10% glycerol (SDSPAGE buffer) with DNAase (.01 mg/ml; Worthington) and boiled. Boiled samples were vortexed, centrifuged 10 minutes a t 12,000 g, and the supernatants and pellets stored separately at - 7OOC. Extracts were analyzed by immunoadsorbtion and Western blot analysis. Extracts were assayed for protein (BioRad Dye Reagent); amounts indicated in the text were diluted to a volume of 1.4 ml buffer at the following final concentrations: 10 mM TrisCl pH 7.4,0.14 M NaC1,5 mM EDTA, 0.1% SDS, and 0.5% NP-40 (immunoprecipitation buffer). These solutions were immunoadsorbed by mixing overnight in the cold room with goat antimouse IgG Agarose beads (Sigma) that had been preincubated with the appropriate monoclonal antibody. Beads were collected by centrifugation, washed five times with immunoprecipitation buffer, and boiled with SDS-PAGE buffer with 0.01 % Bromophenol Blue and 1% b-mercaptoethanol. The precipitated proteins were separated on 3-8 % acrylamide gradient gels (Laemmli, '70) and transferred to nitrocellulose (Towbin et al., '76) in buffer containing 25 mM Tris, 0.192 M glycine, 0.10: SDS, and 20% methanol. The nitrocellulose was blocked with 1%BSA and then incubated with the appropriate monoclonal antibodies. Antibody binding was detected with alkaline phosphatase conjugated antimouse IgG secondary antibodies (Promega) and visualized with nitroblue tetrazolium and 5-bromo-4-chloro-3indolyl phosphate in 0.1 M Tris C1 pH 8.9,O.l M NaC1, and 5 mM MgC1,.
RESULTS Rat 401 recognizes Schwann cells in adult peripheral nerve Previous work demonstrated that Rat 401 recognizes embryonic CNS and P N S neural tissue (Hockfield and
RAT 401 STAINS SCHWANN CELLS McKay, ’85). The staining in the nerve roots of embryos (E15) localizes to a cell type with patchy basal lamina and an orientation that is parallel to axons (Hockfield and McKay, ’85).These characteristics identify immature Schwann cells that have begun to ensheathe axons in vitro (Bunge et al., ’80) and suggest that the antibody staining of embryonic nerve roots can be largely accounted for by Schwann cell staining. The spinal cord of adult rats, unlike that of embryos, lacks readily detectable Rat 401 immunoreactivity (Hockfield and McKay, ’85). The level of immunoreactivity increases sharply, however, a t the junction between the spinal cord and nerve roots (Fig. 1). The PNS immunoreactivity extends distally into the spinal nerves. In cross sections of sciatic nerve, Rat 401 immunoreactivity produces thin, broken rings of staining that lie exterior to the unstained myelin sheaths (Fig. 2a). The staining appears to be restricted to such “rings” and the adjacent endoneurium is not stained above background levels. The Rat 401 staining pattern in nerve is distinct from that produced by staining with antineurofilament antibodies (Fig. 2b) or by staining with tissue culture media (Fig. 2c). The pattern of immunoreactivity observed at the light microscopic level suggests that Rat 401 recognizes Schwann cells and not axons in peripheral nerve. In order to determine the cellular localization of Rat 401 immunoreactivity at higher resolution, sections from the adult sciatic nerve and the sympathetic trunk were prepared
45
for ultrastructural immunocytochemistry (Fig. 3a,b). The sciatic nerve contains populations of both myelinated and unmyelinated axons. Immunoreactivity is most prominent in the cytoplasm of Schwann cells related to myelin sheaths. Myelin and the underlying cytoplasm are also not immunoreactive. The nonmyelin-related Schwann cell in the field shown in Figure 3A is unstained. These results indicate that the widespread Rat 401 staining observed with light microscopic examination of the adult dorsal root (Fig. 1) and adult sciatic nerve (Fig. 2a) results from immunoreactivity of the Schwann cell cytoplasm. The sympathetic trunk is comprised mainly of unmyelinated axons and permits a systematic assessment of Rat 401 immunoreactivity in nonmyelin-related Schwann cells. In order to determine if the adult staining represents immunoreactivity in both myelin and nonmyelin-related Schwann cells, sections from stained sympathetic trunk were examined by electron microscopy. The intensity of staining is generally weaker in the sympathetic trunk than in the sciatic nerve, but is localized to nonmyelin-related Schwann cells (Fig. 3b). The subcellular localization of this Rat 401 staining resembles that seen for myelin-related Schwann cells in the sciatic nerve. Sciatic nerve from 14-day-oldrats contains actively myelinating Schwann cells (Webster and Favilla, ’84). Rat 401 recognizes such Schwann cells that are engaged in axonal myelination (Fig. 312). As a t older ages, the staining is confined to the Schwann cell cytoplasm while the myelin is
Fig. 1. Rat 401 immunoperoxidase stained section of adult rat spinal cord (SC). The adult spinal cord shows faint traces of staining, whereas the attached dorsal root (arrow) shows intense immunoreactivity. Scale bar = 100 urn.
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Fig. 2. Adult sciatic nerve in coronal section. Sections in A-C were obtained from the same block of tissue. A. Rat 401 staining is restricted to narrow regions (arrows) adjacent to unstained rnyelin. B. Axons stained with Cat 101 exhibit a different staining pattern of filled circular
profiles. C. Negative control section incubated with tissue culture media followed by secondary antibody shows no specific staining pattern. Scale bar = 10pm.
unstained. These results in combination with previous work indicate that Rat 401 recognizes ensheathing, myelinating, and myelin-related Schwann cells in spinal nerves. Nonmyelin-related Schwann cells are stained as well but at lower intensity.
produced extremely intense staining of Schwann cells in these acute cultures. Essentially all small, refractile, process bearing and process free cells were stained (Fig. 4c). By maintaining Schwann cells dissociated from sciatic nerve for much longer times in culture (3-4 weeks), cells devoid of contact with even degenerating fragments of axons can be evaluated. The Schwann cells in these cultures are process bearing and typically bipolar. In these long-term cultures, Rat 401 darkly stained process-bearing Schwann cells. In contrast, the large stress fiber-containing fibroblasts were not stained (Fig. 4D,E). Together, these results indicate that Rat 401 immunoreactivity is selective for Schwann cells. Furthermore, it is not dependent on specific neural architecture or on contact with live axons, axon membranes, or neurons.
Rat 401 recognizes Schwann cells maintained in vitro The in situ studies described above indicate that Rat 401 recognizes adult Schwann cells that ensheathe myelinated and unmyelinated axons. In order to determine if axon contact or some other component of the three-dimensional organization of the nervous system is required for continued Rat 401 expression, Schwann cells were dissociated and maintained in culture prior to antibody staining. Schwann cells were examined under four culture conditions: (1)dorsal root ganglion (DRG) cultures obtained from E15-16 rats and maintained 10 to 14 days in vitro; (2) sympathetic ganglia cultures obtained from E l 5 rats and maintained 21 days in vitro; (3) Schwann cells derived from sciatic nerve of P 5 rats and maintained for 4 days in vitro; and (4) Schwann cells obtained from sciatic nerve of P1 rats and maintained for 21 to 30 days in vitro. The DRG and sympathetic ganglia cultures allow examination of the premyelinating and the prenonmyelinating Schwann cell (Roufa e t al., '86),respectively. In both DRG and sympathetic ganglia cultures, Rat 401 stained cells with Schwann cell morphology (Fig. 4a,b). Other cells such as fibroblasts, neuron clusters, and neurite processes were unstained. The intensity of staining of Schwann cells in the SCG cultures was more variable than in the DRG cultures. Schwann cells that lack contact with live neurons or neuronal processes were obtained by dissociation of sciatic nerves. A t short plating times, many of the Schwann cells are round and do not have extended processes. Rat 401
Rat 401 recognizes antigens with identical molecular weights in the embryonic CNS and adult peripheral nerve The above results indicate that Rat 401 immunoreactivity is maintained in glia of the P N S (Schwann cells) in adulthood, whereas it is known to be sharply reduced in CNS glia a t an early developmental stage (Hockfield and McKay, '85). We next asked whether identical antigens are present in both CNS and PNS but subject to different temporal regulation or if the staining in embryonic CNS and adult PNS represents different sets of molecules that merely share the Rat 401 epitope. Rat 401 positive antigens in adult P N S and embryonic CNS were compared by immunoadsorbtion and Western blot analysis. The immunoadsorbtion step was included in order to isolate Rat 401 antigens from a large amount of tissue extract. This allowed us to analyze antigens that might be in low abundance without grossly overloading the
Fig. 3. Electron micrographs of Rat 401 immunoperoxidase stained nerve. A. Adult rat sciatic nerve. Rat 401 stains a myelin-related Schwann cell (filled arrow). Adjacent nonmyelin-related Schwann cell lacks staining (open arrow). Scale bar = 0.5 pm. B. Adult sympathetic trunk. Rat 401 stains a nonmyelin-related Schwann cell. Staining, as in the myelin-related Schwann cells, is localized to the cytoplasm (arrows).
Scale bar = 0.5 pm. C. P14 rat sciatic nerve. Rat 401 stains ruffled cytoplasm of actively myelinating Schwann cells (arrows). Staining at the outer rim of myelin may result from reaction product diffusion. Scale bar = 10 pm. All thin sections were examined without poststaining with lead citrate or uranyl acetate.
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Fig. 4. Rat 401 recognizes Schwann cells in the presence (A and B) and absence (C -E) of neurons. A. R a t 401 stained Schwann cells in DRG cultures have both spindle (arrowhead) and multipolar morphologies. B. Rat 401 stained cells (arrowhead) in cultures from sympathetic ganglia. These cultures show variable intensity in Schwann cell immunoreactivity and weaker staining relative t o the DRG cultures illustrated in A. C. Schwann cells from sciatic nerve maintained in the absence of neurons
for 4 days are strongly immunoreactive with R a t 401 (arrowhead). D, E. Brightfield (D) and phase contrast (E) view of the same culture of Schwann cells (arrows) from sciatic nerve maintained for 3 weeks and passaged in the absence of neurons. Although the Schwann cells are intensely stained, the fibroblasts (arrowheads) are not stained. Bars = 100 pm.
gels. Figure SA shows the Rat 401 positive species obtained from extracts of E l 9 rat CNS (1.2 mg protein) immunoadsorbed with Rat 401. The most prominent species appear as sharp bands at molecular weights of 400 ku and 175 kD. Numerous other bands appear in lower abundance, many at
molecular weights intermediate between the two major bands. Except for the bands of the immunoadsorbed and eluted antibodies themselves (see Fig. 5 legend), none of these antigens appear when identical immunoadsorbed samples were reacted with a control mouse IgG, Cat 301. Nor did
RAT 401 STAINS SCHWA"
A
CELLS
B
Fig. 5. Characterization of Rat 401 reactive antigens. A. 1.2 mg of El9 CNS extract was immunoadsorhed with antimouse IgG agarose heads preincuhated with Rat 401 (lanes 1 and 2) or Cat 301 (lane 3). Immunoadsorhed materials were eluted, electrophoresed, and transferred as described in Methods. Strips were then processed with monoclonal antibodies as follows: Cat 301 (lane l), or Rat 401 (lanes 2 and 3). The lowest molecular weight hand, present in all 3 lanes, also appears when mAB -coated immunoheads (without tissue extract) are analyzed (not shown); consequently it represents the IgG antibody molecule itself. B. Extracts (0.54 mg) from the following tissues were subjected to immunoadsorbtion with Rat 401 followed by Western blot analysis with Rat 401: adult spinal cord (lane 4), E l 9 CNS (lane 5 ) , and adult sciatic nerve (lane 6). All the Rat 401 reactive antigens identified in E l 9 CNS are also present in adult peripheral nerve. This is consistent with the persistent immunoreactivity of Rat 401 in adult peripheral nerve; in contrast adult central nervous tissue contains only trace amounts of the 175 kD Rat 401 antigen, consistent with the marked developmental down-regulation of Rat 401 expression in the CNS.
any of these antigens appear in the inverse control, when extracts were immunoadsorbed with Cat 301, then electrophoresed, transferred and reacted with Rat 401. The 175 kD band presumably matches the major Rat 401 reactive band that has been previously identified as a 180-200 kD species (Hockfield and McKay, '85). The 400 kD band reported here may have emerged as a result of the use of 3-8% gradient gels that would optimize the resolution of heavier molecular weight species. In Figure 5B, extracts (0.54 mg protein) of' embryonic CNS, adult spinal cord, and adult nerve were compared by immunoadsorbtion and Western blot analysis with Rat 401. The major bands a t 400 kD and 175 kD previously identified in embryonic CNS, as well as the minor bands, are also seen in extracts of adult peripherai nerve. No other new Rat 401 immunoreactive bands were evident in extracts of adult peripheral nerve. Adult spinal cord extracts, when subjected to the same analysis, reveal only slight traces of the 175 kD immunoreactive band. These results are consistent with the immunocytochemical findings and provide evidence that the same antigenic species are differentially regulated in the CNS and in the PNS.
49
DISCUSSION Here we have studied the time course of Rat 401 immunoreactivity in peripheral glia to determine whether it is similar to that described for central glia. Previous work has shown that Rat 401 immunostaining is sharply reduced in central neuroepithelial cells with maturation (Hockfield and McKay, '85; Frederikson and McKay, '88; Frederikson et al., '88). The results presented here show that Rat 401 strongly stains peripheral glia during active myelination. Furthermore, immunoreactivity persists in both myelin- and nonmyelin-related Schwann cells in the adult peripheral nerve. Rat 401 selectively recognizes Schwann cells maintained in culture in the complete absence of axon contact, as well as in coculture with peripheral neurons. Western blot analysis of immunoprecipitated antigens from the embryonic CNS and the adult sciatic nerve indicates that the apparent molecular weights of immunoreactive antigens in embryonic central and in adult peripheral tissues are identical. Together these results suggest that the temporal regulation of Rat 401 immunoreactivity in Schwann cells is markedly different from that in CNS glia (and neurons). Thus Rat 401 appears to identify antigens that, like vimentin (Chiu et al.,'88) and laminin (Cornbrooks et al., '83) persist in adult Schwann cells but are down-regulated in the CNS at early stages of development (Liesi et al., '83; Bovolenta et al., '84; Liesi, '85). The restriction of intense Rat 401 immunoreactivity to peripheral nerve in the adult rat is demonstrated most dramatically by the staining pattern of the dorsal root entry zone of the spinal cord (Fig. 1).The strong, positive staining of peripheral neural tissue abruptly ends with a border complementary in shape to the glial limitans of the dorsal root entry zone. The paucity of immunocytochemical staining in adult CNS is confirmed by the absence of all but trace levels of biochemically detectable Rat 401 antigens in extracts of adult spinal cord. The normal reduction of Rat 401 immunoreactivity in CNS neural precursors appears to correlate with the expression of cell type specific molecules such as glial fibrillary acidic protein in astrocytes (Frederiksen and McKay, '88). Transformed and immortalized embryonic CNS cells are also capable of sustained Rat 401 immunoreactivity in vitro. However, this immunoreactivity is lost when these cells are forced to differentiate (Frederiksen et al., '88; McKay et al., '88). In contrast, Schwann cells that are fully differentiated are recognized by Rat 401. One intriguing correlate to this persistent immunoreactivity is that adult Schwann cells, unlike adult CNS neurons or glia, are able to dedifferentiate (Abercrombie and ,Johnson, '42; Fekete and Brockes, '86) when they lose contact with axons as a consequence of nerve injury (Holmes and Young, '42; Bradley and Asbury, '70). Furthermore, when axon contact is resumed, during nerve regeneration, Schwann cells can redifferentiate. Thus Rat 401 immunoreactivity may be linked to a still unidentified feature that is expressed by cells that are still capable of differentiation (e.g., CNS neural precursors) and by cells that may readily dedifferentiate and then redifferentiate (e.g., PNS Schwann cells). During early development, Schwann cells are essentially biochemically indistinguishable in premyelinated and prenonmyelinated nerve (Lamperth e t al., '89). Our results provide further evidence for a biochemical homogeneity among these early Schwann cells. Rat 401 produces uniformly intense staining throughout the embryonic PNS, not
50
only in roots, sympathetic trunk, and spinal nerves, but in enteric glia, and the Schwann cells along the olfactory pathway (data not shown). With differentiation, Schwann cell phenotype is strongly regulated by axon contact. Indeed, Schwann cell phenotype can be converted from nonmyelin- to myelin-related simply by forced interaction with axons that are normally myelinated (Aguayo et al., '76). This plasticity is remarkable in the light of the strikingly different biochemical and morphological profiles of mature myelin-related and nonmyelinrelated Schwann cells (Trapp et al., '79; Yen and Fields, '81, '85; Jessen and Mirsky, '84; Jessen et al., '84). Here we show that intense Rat 401 antibody staining is maintained in mature myelin-related Schwann cells. However, whereas mature nonmyelinating Schwann cells are also recognized by Rat 401, the staining ranged from moderate to faint both in situ and in culture. This qualitative difference in staining between mature myelin-related a n d nonmyelinating Schwann cells is a new phenotypic difference between these cell "types." This could provide an unusual example of a limited developmental down regulation of a molecular species in nonmyelin-related Schwann cells. However, biochemical studies are necessary to rigorously quantify differences in antigen concentration that could account for the differences in staining intensity. Another unusual feature of Rat 401 immunoreactivity is that it persists a t a high level when Schwann cells are deprived of axon contact. Another marker for Schwann cells in long-term cultures has been isolated by Dulac and coworkers ('88). Rat 401 immunoreactivity with its cytoplasmic localization differs from their antibody, which recognizes a protein with a cell surface localization (Dulac et al., '88). In contrast to these persistent markers for cultured Schwann cells, laminin (Cornbrooks et al., '83) and Ran-2 (Jessen and Mirsky, '84) are down-regulated in Schwann cell cultures that lack extensive neuronal contact. Reduction of immunoreactivity in dissociated Schwann cells is also typical for myelin-related molecules (Mirsky e t al., '80; LeBlanc et al., '87; Trapp et al., '88). Thus the maintained expression of Rat 401 antigens by dissociated, formerly myelin-related Schwann cells suggests that Rat 401 antigens are probably not closely involved in the production or maintenance of myelin. This characteristic antibody staining of Schwann cells even in the absence of axon contact makes Rat 401 an especially useful marker for Schwann cells in long-term in vitro studies of mammalian tissue. The cross reactivity may, however, be limited as Rat 401 does not appear to stain chick CNS or human peripheral nerve (unpublished observations). The classification of the Rat 401 positive cells in E l l nerve roots remains to be firmly established. However, as early as definitive morphological identification of Schwann cells was possible, at E15, these cells are immunoreactive for Rat 401. A t a minimum, the Rat 401 positive cells in the nerve roots of E l l rats (Hockfield and McKay, '85) are antigenically related to immature and to mature Schwann cells on the basis of shared immunoreactivity to Rat 401 antibodies. Furthermore, as axon contact is not necessary for expression of Rat 401 in cultured Schwann cells, the lack of extensive axon-"Schwann cell" interactions in E l l nerve would not preclude Rat 401 expression in putative Schwann cells. Thus it is tempting to speculate that the early Rat 401 cells in E l l nerve roots are Schwann cells or Schwann cell precursors. If these cells are Schwann cells, then it may become possible to test if de novo outgrowth of mammalian
B. FRIEDMAN ET AL. peripheral axons, like regenerative axonal growth (Aguayo et al., '82), utilizes a substrate formed by Schwann cells.
ACKNOWLEDGMENTS This work was supported by grants from NIH (NS-07877, B.F.) (EY-05855, S.Z.) and KSF (BNS 8644681, S.H.). S.H. is a Klingenstein fellow in the neurosciences. The authors thank Drs. Nurit Kalderon, Carson Cornbrooks, and Kathleen Martin for their generous gifts of cultured Schwann cells.
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