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Previous Article | Next Article
Volume 17, Number 1, Issue of January 1, 1997 pp. 241-250
Copyright ©1997 Society for NeuroscienceActin Plays a Role in Both Changes in Cell Shape and Gene- Expression Associated with Schwann Cell Myelination
Cristina Fernandez-Valle1, Douglas Gorman3, Anna M. Gomez1, and Mary Bartlett Bunge1, 2, 3 1 The Chambers Family Electron Microscopy Laboratory,
The Miami Project to Cure Paralysis, and Departments of Neurological Surgery and 2 Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida 33136, and 3 Department of Neurobiology and Anatomy, Washington University School of Medicine, St. Louis, Missouri, 63110
ABSTRACT
Schwann cell (SC) differentiation into a myelinating cell requires concurrent interactions with basal lamina and an axon destined for myelination. As SCs differentiate, they undergo progressive morphological changes and initiate myelin-specific gene expression. We find that disrupting actin polymerization with cytochalasin D (CD) inhibits myelination of SC/neuron co-cultures. Basal lamina is present, neurons are healthy, and the inhibition is reversible. Electron microscopic analysis reveals that actin plays a role at two stages of SC differentiation. At 0.75-1.0 µg/ml CD, SCs do not differentiate and appear as "rounded" cells in contact with axons. This morphology is consistent with disruption of actin filaments and cell shape changes. However, at 0.25 µg/ml CD, SCs partially differentiate; they elongate and segregate axons but generally fail to form one-to-one relationships and spiral around the axon. In situ hybridizations reveal that SCs in CD-treated cultures do not express mRNAs encoding the myelin-specific proteins 2
Key words: Schwann cells; myelination; actin; cytochalasin D; mRNA expression; in situ hybridization,3
INTRODUCTION
In the animal, Schwann cells (SCs) differentiate into a myelinating phenotype on contact with an axon destined for myelination (deWebster, 1971
; Weinberg and Spencer, 1975
Evidence suggests that laminin, present in basal lamina, interacts with SC receptors to initiate signal transduction cascades necessary for differentiation. Addition of soluble laminin, but not type IV collagen or heparan sulfate proteoglycan, to medium lacking ascorbate supports SC myelination (Eldridge et al., 1989
1 integrin antibody suggest that a
1
On binding their native ligands, integrins initiate signal transduction cascades that affect cellular behavior and gene expression in many cell types (Werb et al., 1989
In this study, we demonstrate that actin plays two distinct and necessary roles during SC differentiation; one in mediating cell shape changes and another in influencing myelin gene expression. Abundant expression of mRNAs encoding myelin proteins can be blocked with low cytochalasin D (CD) concentrations that do not inhibit morphological changes occurring early during SC differentiation. This work suggests that F-actin influences myelin-specific gene expression in SCs. Preliminary reports have appeared in abstract form (Gorman and Bunge, 1988a
MATERIALS AND METHODS
Tissue culture Primary SC cultures. SCs were isolated from sciatic nerves removed from E21 or newborn Sprague Dawley rats (Charles River, Raleigh, NC) and expanded in vitro with medium containing DMEM, 10% heat-inactivated fetal bovine serum (FBS, Life Technologies, Grand Island, NY), forskolin, and pituitary extract (BTI, Stoughtan, MA) on poly-L-lysine- (Sigma, St. Louis, MO) coated 100 mm tissue culture dishes (Brockes et al., 1979
Fig. 1. CD inhibits Schwann cell differentiation on collagen. SC-sensory neurons cultures were grown for 1 week in myelination-permissive medium alone (A) or with 1 µg/ml CD (B). Sudan black staining used to visualize myelin revealed that SCs (arrowheads) in control cultures (A) had flattened and elongated along axons and were beginning to form myelin (between arrows). SCs grown in the presence of CD (B) failed to elongate in association with axons, retained a rounded morphology, and did not myelinate axons. Magnification, 800×.
[View Larger Version of this Image (131K GIF file)]
Fig. 2. Schwann cells cultured in myelination-permissive medium rapidly differentiate to myelin-forming cells. This electron micrograph reveals the degree of SC differentiation achieved during 1 week in culture. Four SCs (1-4) elaborated up to 25 myelin lamellae (small asterisks) around axons (large asterisks). Each SC-axon unit is surrounded by a complete basal lamina (arrows). Two SCs (5, 6) at earlier stages of differentiation have begun to spiral around smaller diameter axons. Magnification, 30,000×.
[View Larger Version of this Image (206K GIF file)]
Fig. 3. CD causes a dose-dependent disruption of morphological differentiation; elongation and spiralization are selectively inhibited by different CD concentrations. SCs were cultured in myelination-permissive medium containing 0.5 (A), 0.75 (B), or 1.0 µg/ml (C) CD for 1 week. At the lowest CD concentration (A), SCs (S) elongated and extended multiple processes into axonal fascicles, thereby segregating axons into smaller groups or isolating them completely. Continuing spiralization around an individual axon was not observed. At the intermediate CD concentration (B), SCs retained the ability to extend processes and segregate axons into smaller groups. However, at the highest CD concentration (C), SCs remained rounded, adhered to axons (asterisks) but did not extend processes into axonal fascicles. Basal lamina (arrows) assembled and attached to the SC surface in all CD doses. Magnification: A, 25,000×, B, 35,000×, C, 15,000×.
[View Larger Version of this Image (194K GIF file)]
Fig. 5. CD's inhibitory effect on myelination is reversible. Cultures were grown in myelination-permissive medium with 0.75 µg/ml CD for 1 week and then without CD for an additional week to determine whether drug toxicity prevented SC function. At removal of CD, SCs spiraled membranes around axons. An SC (S), shown with its nucleus (N), elaborated 20 myelin lamellae (small asterisk) around an axon (large asterisks), and an adjacent SC (S) is beginning to myelinate. Magnification, 25,000×.
[View Larger Version of this Image (116K GIF file)]
SC-neuron co-cultures. Neurons were isolated from cervical dorsal root ganglia of Sprague Dawley rat embryos at 15 d gestation by dissociation with trypsin. Cultures grown on collagen (see Figs. 1, 2, 3, 5) were prepared as described in Eldridge et al. (1987) 
Fig. 4. Lower CD concentrations inhibit myelin formation in cultures grown on laminin instead of collagen. Cultures grown in myelination-permissive medium alone (A, B) or with CD, 0.25 µg/ml (C, D) are illustrated. Sudan black staining (A, C) demonstrates that myelin (arrows) is abundant in control cultures but is generally absent in CD-treated cultures, although SCs elongate along axons. Neuron cell bodies are indicated by arrowheads. EM confirms that in myelination-permissive medium (B), SCs (S) differentiate and form myelin (small asterisks). In CD-treated cultures (D), myelin is absent, but basal lamina is present (arrows). SCs retain the ability to segregate axons (large asterisks) but cannot spiral membrane around the axon to form myelin lamellae in 0.25 µg/ml CD. Magnification: A, C, 166×; B, D, 40,000×.
[View Larger Version of this Image (155K GIF file)]
Fig. 6. Actin organization is progressively disrupted with increasing CD concentrations. Phase (A-C) and fluorescent (D-F) micrographs of cultures grown on laminin for 1 week in myelination-permissive medium alone (A, D) or with 0.25 µg/ml CD (B, E), or 0.5 µg/ml CD (C, F). Cultures were fixed and stained with rhodamine-conjugated phalloidin to visualize the distribution of actin filaments. In the absence of CD, phalloidin staining appears continuous and linear. In the presence of CD, the pattern of staining becomes disrupted, appearing as large fluorescent aggregates. Magnification, 500×.
[View Larger Version of this Image (222K GIF file)]
Fig. 7. CD inhibits SC expression of myelin-specific proteins and mRNAs. Cultures grown in myelination-permissive medium alone (A, C, E, G, I, K) or with 0.25 µg/ml CD (B, D, F, H, J, L) were immunostained with antibodies against CNP (A, B), MAG (E, F), or P0 (I, J) or processed for in situ hybridization using CNP (C, D), MAG (G, H), or P0 RNA probes (K, L) to detect myelin-specific mRNAs. Arrows indicate positively stained Schwann cells. CNP, MAG, and P0 expression was greatly reduced in SCs cultured in the CD. In situ hybridization results indicated that mRNAs encoding the myelin-specific proteins were either not expressed or expressed at very low levels compared with control cultures. Sister cultures were hybridized with sense probes for each mRNA as negative controls. Magnification, 600×.
[View Larger Version of this Image (145K GIF file)]
Cytochemistry and EM Immunostaining. Cultures were rinsed with several changes of PBS and fixed in 4% paraformaldehyde for 10 min. For detection of 2
CD treatment. Cultures of SCs and neurons were grown in medium that does not support myelination either alone or together for 2 weeks to expand the SC population on axons. All cultures at this time have equivalent SC densities. Cultures were then fed myelination-permissive medium with or without CD at 0.25, 0.50, 0.75, or 1.0 µg/ml. CD (Sigma) was prepared as a 1 mg/ml stock in DMSO and then further diluted in medium as indicated. In some experiments, matrigel (Collaborative Research, Lexington, MA) was diluted 1:100 in CD-containing myelination-permissive medium. Medium was replenished every other day until myelin became visible in the control cultures (~7-8 d). Cultures were processed for cytochemistry, electron microscopy (EM), or in situ hybridization. Standard cultures typically contained 1000 neurons and ~240,000 SCs. For additional details of the culture procedure, see Kleitman et al. (1991) 20°C. Cultures were rehydrated and incubated for 30 min in 10% goat serum/PBS. The antibodies used were a P0 antiserum (1:100, courtesy of J. Brockes), anti-CNP (1:100, Sigma), anti-MAG (undiluted hybridoma medium, courtesy of M. Schachner). Control staining was carried out by omitting the primary antibody. Cultures were rinsed three times for 5 min each in goat serum/PBS and then incubated in a 1:100 dilution of either rhodamine or fluorescein goat anti-mouse or goat ant-rabbit (Organon Teknika, West Chester, PA) for an additional 30 min. Cultures were rinsed three to five times in PBS, post-fixed in 4% paraformaldehyde/PBS for 5 min, and mounted in Citifluor mounting medium (Citifluor Products, Canterbury, UK) containing Hoechst dye 33342 (Sigma). Cultures were viewed on a Zeiss universal microscope equipped with fluorescence optics.
Phalloidin staining. Cultures were rinsed several times with 0.1 M sodium phosphate buffer (PO4) and fixed in 4% paraformaldehyde for 10 min. To disrupt extracellular matrix to increase phalloidin penetration, cultures were permeabilized in 4% paraformaldehyde/0.1% Triton X-100 for 30 min and then were rinsed with PO4 and incubated for 20 min in PO4 containing 3 µM rhodamine-conjugated phalloidin (Molecular Probes, Eugene, OR). Cultures were rinsed in PO4 and mounted in Citifluor mounting medium (Citifluor Products) containing Hoechst dye 33342 (Sigma).
Sudan black staining and quantitation. Cultures were fixed for 1 hr in 4% paraformaldehyde in PO4, rinsed several times with PO4, and osmicated in 1% osmium tetroxide/PO4 for an additional hour. Co-cultures were dehydrated in 25, 50, and 70% ethanol for 5 min each and stained with 0.5% Sudan black in 70% ethanol for 1 hr. Co-cultures were destained for 10-30 min in 70% ethanol, rehydrated in 50 and 25% ethanol followed by PO4, and then mounted in glycerin jelly. Quantitation was carried out by viewing co-cultures under 400× magnification and counting the number of myelin segments that intersected a reference line per field; 15 fields per co-culture were examined in this manner, and two to five cultures were examined per group. In situ hybridization RNA probe preparation. A rat P0 cDNA was kindly provided by G. Lemke (Lemke and Axel, 1985
EM. Co-cultures were fixed in buffered glutaraldehyde followed by osmium tetroxide, dehydrated in ethanol, and embedded in Embed (EMS, Fort Washington, PA) (Ratner et al., 1986
Hybridization. Cultures were treated as described in Fernandez-Valle et al. (1993)
RESULTS
CD inhibits elongation and myelination in a dose-dependent manner
SC-sensory neuron cultures were maintained in myelination-permissive medium in the absence or presence of various CD concentrations. Cultures were divided in two; one-half was stained with Sudan black, and the other half was processed for EM. In control cultures, myelin was evident after 1 week in myelination-permissive medium (Fig. 1A). Cultures grown for 1 week in the same medium but containing 1 µg/ml CD remained undifferentiated (Fig. 1B). SCs adhered to axon fascicles but were rounded and did not undergo the characteristic elongation into a bipolar cell that heralds initiation of SC differentiation. As the CD concentration was reduced to 0.75 and 0.50 µg/ml, inhibition of cell shape change was reduced, and increasing numbers of SCs displayed morphologies indicative of a differentiating or "promyelinating" phenotype. An occasional myelin segment was observed at these lower CD concentrations. Cultures were systematically scanned, and the number of myelin segments present in each half culture was quantitated (Table 1).
Table 1. Quantitation of myelin segments in SC/neuron cultures grown on collagen
Conditions No. of fields counted Myelinated segments Per sample Per mm2 MM 53 2050 206 MM + DMSO 63 1934 178 MM + 0.75 µg/ml CD 73 2 0 *MM 0.75 µg/ml CD
70 1305 108 *MM + 0.75 µg/ml 71 164 13 MM, Myelination-permissive medium. * Cultures were grown for 7 d in the first medium and for an additional 7 d in the second medium. Values are derived from single cultures.
Because SCs undergo progressive changes in morphology as they differentiate, the extent to which differentiation was achieved in the presence of CD can be determined using ultrastructural analysis of SC morphology. SCs were studied for indications of axonal adhesion, elongation, one-to-one relations, spiralization, and myelination. In control cultures, the maximum level of differentiation achieved by SCs after 1 week in myelination-permissive medium was the elaboration of 20 to 25 myelin lamellae (Fig. 2). The ability of SCs to differentiate, as evidenced by morphology, was inhibited in a dose-dependent manner by incubation in CD for 1 week. SCs in cultures maintained in 0.5 µg/ml CD had elongated along axons and segregated axonal segments but generally did not form a 1:1 relationship with axons or extend multiple membrane spirals to form myelin (Fig. 3A). SCs in cultures maintained in 0.75 µg/ml also elongated and extended processes into axonal fascicles but again failed to form 1:1 relationships with axons (Fig. 3B). SCs in cultures maintained in the presence of 1 µg/ml CD neither elongated along axons nor extended processes into axonal fascicles (Fig. 3C). Perturbation of actin polymerization dynamics achieved with lower CD concentrations was associated with a failure to form 1:1 relationships with axons but not in elongation, whereas higher CD concentrations inhibited SC elongation and thereby precluded further differentiation.
The ability of SCs to assemble basal lamina when incubated with CD was also studied at the ultrastructural level. We found that basal lamina was present in CD-treated cultures, regardless of the drug concentration used. The amount of basal lamina, however, was less in CD-treated cultures than in controls (compare Figs. 2 and 3 and Fig. 4, B and D). Although extracellular matrix abundance increases as differentiation proceeds and basal lamina is not expected to be continuous until many myelin lamellae have formed, we tested the possibility that inhibition of myelination was attributable to an unrecognized effect of CD on extracellular matrix production. Exogenous basal lamina components in the form of matrigel were added along with CD to cultures grown in myelination-permissive medium. Addition of matrigel did not override CD's inhibitory effect on myelination (data not shown). Therefore, it is unlikely that myelination was inhibited because of nonspecific effects of CD on extracellular matrix secretion or assembly.
To determine whether myelin-specific mRNAs were expressed by SCs in CD-treated cultures using in situ hybridization techniques, the culture substratum was changed from collagen to laminin, which is stable during the procedure. We repeated the experiments presented above to verify that this change in substratum did not alter the results. Three to five cultures per group from at least four separate experiments were treated with various CD concentrations for 7 d and stained with Sudan black, and myelin sheaths were quantitated (Table 2, Fig. 4A,C). Separate cultures were used for EM analysis (Fig. 4B,D). The results demonstrated that the inhibitory effect of CD on myelination was also observed when cultures were grown on laminin and, moreover, that lower CD concentrations led to the results observed when collagen served as the substratum. One-to-one ensheathment of axons was essentially inhibited by 0.25 µg/ml CD. The DMSO-treated controls were myelinated to the same extent as untreated controls. The number of myelin internodes present in control cultures varied between experiments, but the inhibition of myelination by CD was consistent (Table 2). Cultures grown on laminin and treated with either 0.25 or 0.5 µg/ml CD were used for analysis of actin organization and myelin-specific protein and mRNA expression.
Table 2. Quantitation of myelin segments in SC-neuron cultures grown on laminin
Condition Experiment 1 Experiment 2 Experiment 3 Experiment 4 MM 1129 ± 56 888 ± 203 2132 ± 620 4247 ± 465 MM + DMSO* 1162, 1260 ND ND ND MM + 0.25 µg/ml CD 7 ± 15 0 5 ± 2 20 ± 2 MM + 0.5 µg/ml CD 3 ± 6 ND ND ND Myelin counts were determined at 250× magnification by sampling the same 15 coordinates in each culture and counting the number of myelin segments that intersected a reference line. Mean and SEM of three to five cultures per group. * Only two cultures were counted. The inhibitory effects of CD are reversible
To determine whether inhibition of myelination observed in cultures incubated in medium containing CD was attributable to cellular toxicity not apparent morphologically, a reversibility experiment was conducted. Cultures were treated with 0.75 µg/ml CD for 7 d and then maintained in myelination-permissive medium without CD for an additional 7 d before Sudan black staining and EM analysis. In these cultures, some SCs had formed myelin and many others had undergone changes in morphology, indicating that differentiation leading to myelination had begun (Fig. 5, Table 1). These changes in cell shape suggest that the SCs were viable and able to respond to myelination-permissive medium by differentiating and that with additional time, the number of myelin segments would have increased. Moreover, adding CD to already myelinated cultures did not lead to adverse changes in cellular morphology or loss of myelin (Table 1). This suggests that CD did not cause a general failure or disruption of cell metabolism, but rather, that when administered at a critical window of time, CD disrupted specific processes necessary for SC differentiation.CD disrupts actin organization
The organization of actin in untreated and CD-treated cultures was ascertained using fluorochrome-conjugated phalloidin staining (Fig. 6). We observed an increasing amount of F-actin disruption with increasing CD concentration. Actin distribution changed from a linear, continuous pattern of staining to an increasingly discontinuous staining pattern that was most noticeable in cultures treated with the higher CD concentration (0.5 µg/ml). In these cultures, phalloidan staining appeared as aggregates of varying size.CD prevents expression of myelin-specific proteins and mRNAs
Expression of myelin-specific proteins was assessed by immunostaining cultures for the presence of three proteins, CNP, MAG, and P0, markers of promyelinating and myelinating SCs. The majority of SCs in cultures incubated with the lowest CD concentration used, 0.25 µg/ml, did not express CNP, MAG, or P0 (Fig. 7). We occasionally observed limited expression and aberrant localization of MAG and P0 in SCs from CD-treated cultures. Expression of mRNAs encoding the myelin-specific proteins in myelinating and CD-treated cultures was determined by in situ hybridization. We were unable to detect myelin mRNAs in CD-treated cultures, whereas all three mRNAs were abundant in control cultures. This indicates that steady-state mRNA levels for CNP, MAG, and P0 in CD-treated cultures were significantly lower than in myelinating cultures, even in the presence of axonal contact and adhesion to basal lamina.
DISCUSSION
Our results suggest that functional actin is needed during SC differentiation not only for changes in cell shape but also for abundant expression of myelin-specific mRNAs. The initial step in morphological differentiation, elongation, was inhibited only when higher CD concentrations (0.75-1.0 µg/ml) known to remove stress fibers in other cell types were used (Yahara et al., 1982
In this study, SCs incubated with the lower CD concentration did not express myelin-specific mRNAs, but elongated and segregated axons, nonetheless. They did not, however, form 1:1 relationships with axons. Establishment of a one-to-one relationship between SC and axon is a necessary prelude to spiralization and compaction of myelin membranes. At this 1:1 stage, an individual SC contacts only one axon and forms an inner and outer mesaxon. This relationship essentially polarizes their membranes; the inner mesaxon contacts the axon and spirals around it, and the outer mesaxon contacts basal lamina (Bunge et al., 1989
Three recent transgenic mice models lacking the transcription factors Krox-20 or SCIP provide indirect evidence for a role for MAG during 1:1 ensheathment (Topilko et al., 1994
It is unclear how disruption of actin polymerization dynamics by CD influences myelin-specific gene expression. Work by others has shown that CD interferes with signal transduction from activated
FOOTNOTES
Received June 7, 1996; revised Sept. 18, 1996; accepted Oct. 18, 1996.
This work was supported by National Institutes of Health Grant NS09923 to R.P.B., the State of Florida, and The Miami Project to Cure Paralysis. We thank A. Feucht, E. Cuervo, and M. Bates for technical assistance; R. Camarena for photographic reproduction; Drs. J. Brockes and M. Schachner for antibodies; Drs. M. Gravel, G. Lemke, and J. Salzer for cDNAs; and Drs. P. Wood and K. Carraway for valuable comments.
Correspondence should be directed to Dr. Cristina Fernandez-Valle at her current address: Department of Molecular Biology, Biol. 330, University of Central Florida, Orlando, Florida 32816-2360.
In memory of Richard Paul Bunge, April 15, 1932-September 10, 1996.
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