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The Journal of Neuroscience, October 1, 1998, 18(19):7891-7902
Promyelinating Schwann Cells Express Tst-1/SCIP/Oct-6
Edgardo J.
Arroyo1,
John R.
Bermingham Jr2,
Michael G.
Rosenfeld2, and
Steven S.
Scherer3
Departments of 1 Neuroscience and
3 Neurology, The University of Pennsylvania Medical Center,
Philadelphia, Pennsylvania 19104-6077, and 2 Howard Hughes
Medical Institute, Department of Medicine, University of
California, San Diego, La Jolla, California 92093-0623
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ABSTRACT |
Tst-1/SCIP/Oct-6, a POU domain transcription factor,
is transiently expressed by developing Schwann cells and is required for their normal development into a myelinating phenotype. In tst-1/scip/oct-6-null sciatic nerves, Schwann cells are
transiently arrested at the "promyelinating" stage, when they have
a one-to-one relationship with an axon but before they have elaborated
a myelin sheath. To determine when Schwann cells express
Tst-1/SCIP/Oct-6, we examined  -galactosidase (-gal) expression in
heterozygous tst-1/scip/oct-6 mice, in which one copy of
the tst-1/scip/oct-6 gene has been replaced with the
LacZ gene. -Gal expression from the LacZ gene seems to parallel
Tst-1/SCIP/Oct-6 expression from the endogenous
tst-1/scip/oct-6 gene in developing and regenerating sciatic nerves. Furthermore, electron microscopic examination of
5bromo-4-chloro-3-indolyl--D-galactopyranoside-
(X-gal) and halogenated indolyl--D-galactoside-
(Bluo-gal) stained nerves showed that promyelinating Schwann
cells express the highest levels of -gal, both in developing and in
regenerating nerves. Thus, the expression of -gal, a surrogate
marker of Tst-1/SCIP/Oct-6, peaks at the same stage of Schwann cell
development at which development is arrested in
tst-1/scip/oct-6-null mice, indicating that
Tst-1/SCIP/Oct-6 has a critical role in promyelinating Schwann
cells.
Key words:
myelin; transcription factors; cAMP; POU; axon-Schwann
cell interactions; peripheral nerve; neuropathy
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INTRODUCTION |
Schwann cells are the principal
glial cell type in the PNS, and their development in rodents has been
well described (Webster, 1993 ; Mirsky and Jessen, 1996; Zorick and
Lemke, 1996). They originate from a subpopulation of neural crest cells
that invade peripheral nerves at approximately embryonic day 13 (E13).
These Schwann cell precursors do not have a basal lamina and require
different growth factors than do immature Schwann cells, which first
appear at E15-E16. Immature Schwann cell have a basal lamina, and
their processes separate axons into small bundles. Cells with this
morphological appearance persist until approximately postnatal day 20 (P20), and after this time they segregate small, unmyelinated axons
into separate troughs and are called nonmyelinating Schwann cells. Myelinating Schwann cells are believed to arise from the same population of immature Schwann cells, differentiating in response to as
yet undetermined axonal signal(s). The first morphological manifestation of a myelinating phenotype is a Schwann cell forming a
one-to-one association with an axon, the so-called promyelinating Schwann cell, which then forms a myelin sheath within a few days. The
onset of myelination is temporally dispersed, because the myelinated
axons with the longest internodes and thickest myelin sheaths are
myelinated first and those with the shortest internodes and thinnest
sheaths are myelinated last (Hahn et al., 1987). By P30, the onset of
myelination is complete, and all Schwann cells have one of two
phenotypes: myelinating or nonmyelinating.
These morphological changes are accompanied by changes in gene
expression (Mirsky and Jessen, 1996; Scherer and Salzer, 1996). Immature and nonmyelinating Schwann cells have a similar phenotype; both express neural cell adhesion molecule (N-CAM), L1, the
low-affinity neurotrophin receptor/p75 (p75NTR),
growth-associated protein of 43 kDa (GAP-43), and glial fibrillary acidic protein. Myelinating Schwann cells, on the other hand, do
not express these proteins but express a set of proteins that are
components of the myelin sheath, such as protein zero
(P0), peripheral myelin protein of 22 kDa,
myelin basic protein, connexin32, myelin-associated glycoprotein
(MAG), and periaxin. The expression of these myelin-related proteins
and their cognate mRNAs increases substantially as Schwann cells form
myelin sheaths (Stahl et al., 1990; Lee et al., 1997), and this
upregulation requires continuous axon-Schwann cell interactions both
in developing and in regenerating nerves (Gupta et al., 1993; Scherer
et al., 1994). Thus, the maintenance of the myelinating phenotype
depends on axonal interactions, and there is emerging evidence that the
various phenotypes of Schwann cells are related to the expression
of different sets of transcription factors (Blanchard et al., 1996;
Topilko et al., 1996; Zorick and Lemke, 1996; Scherer, 1997b).
Two unrelated transcription factors, Tst-1/SCIP/Oct-6 and
Krox-20, are required for the normal development of myelinating Schwann cells, because their development is either transiently or
permanently arrested at the promyelinating stage in
tst-1/scip/oct-6- and krox-20-null mice,
respectively (Topilko et al., 1994; Bermingham et al., 1996; Jaegle et
al., 1996). The "arrested" promyelinating Schwann cells appear
normal; they have a basal lamina, express MAG and periaxin, but do not
form a myelin sheath. In spite of the similar phenotype of
tst-1/scip/oct-6- and krox-20-null mice, these
two transcription factors have a different temporal profile of
expression; Tst-1/SCIP/Oct-6 mRNA is transiently expressed, peaking at
approximately P1, whereas Krox-20 mRNA is expressed in parallel with
other myelin-related genes, with high levels of expression even in
adults (Monuki et al., 1989; Zorick et al., 1996). The expression of
Tst-1/SCIP/Oct-6 in neonatal nerves and that of Krox-20 in adult nerves
both depend on maintained axon-Schwann cell interactions, because
their mRNA levels and immunoreactivity fall after axotomy (Scherer et
al., 1994; Zorick et al., 1996). In this paper, we used the expression
of the lacZ gene in heterozygous tst-1/scip/oct-6 mice as a
reporter for endogenous Tst-1/SCIP/Oct-6 expression. The product
of the lacZ gene, -galactosidase (-gal), forms an electron-dense
histochemical precipitate, allowing us to determine when Schwann cells
express tst-1/scip/oct-6, both in developing and in lesioned
sciatic nerves.
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MATERIALS AND METHODS |
tst-1/scip/oct-6 heterozygous mice. The creation
and analysis of tst-1/scip/oct-6 mice have been described
(Bermingham et al., 1996). Heterozygous tst-1/scip/oct-6
mice were maintained in a C57Bl/6 background at The University of
Pennsylvania. All mice were genotyped by PCR (Bermingham et al.,
1996).
Sciatic nerve crush and transection. Mice were anesthetized
by intraperitoneal injection of 0.066 M
2,2,2-tribromoethanol diluted in 2-methyl-2-butanol (0.1 ml per 20 gm
of body mass). Using aseptic technique, we exposed the sciatic nerves
of adult anesthetized mice at the sciatic notch. Some nerves were
doubly ligated and transected with fine scissors, and the two nerve
stumps were sutured apart to prevent axonal regeneration. Nerve crush was produced by tightly compressing the sciatic nerve at the sciatic notch with flattened forceps twice, each time for 10 sec; this technique causes axonal degeneration but allows axonal regeneration. At
varying times after nerve injury (8, 12, 24, and 58 d after the
lesion), the animals were deeply anesthetized and processed for -gal
histochemistry or solution assay. Unlesioned nerves were collected from
P1, P10, P30, and young adult (P60-P90) mice.
-Gal solution assay. Nerves were frozen in liquid
nitrogen, stored at  80°C, and then crushed with a steel mortar and
pestle into a fine powder, mixed with lysis buffer in Eppendorf tubes (0.1 M potassium phosphate, pH 7.8, 0.2% Triton X-100, 0.5 mM dithiothreitol, and 0.2 mM PMSF), and
sonicated on ice five times for 10 sec each. After a 30 min
centrifugation, the supernatant was incubated for 60 min at 48°C to
reduce the background level of -gal activity. The amount of protein
present in each sample was measured using the Bio-Rad (Hercules,
CA) DC protein assay, and equal amounts of protein were assayed
in triplicate for -gal activity with the Galacton Light-Plus kit
(Tropix) according to the manufacturer's instructions. For each
sample, the protein concentration was diluted with lysis buffer to
obtain a similar final concentration of protein (0.61 µg/µl for
development and 0.49 µg/µl for nerve injury). Ten microliters of
each sample were placed in a luminometer cuvette, mixed with an
additional 10 µl of lysis buffer, and then mixed with 200 µl of
reaction buffer [0.1 M phosphate buffer (PB), pH
8.0, and 1 mM MgCl]. After 15-30 min, 300 µl of Light
Emission Accelerator reagent was added, and the cuvettes were placed in
a luminometer. After allowing 10 sec for equilibration, the relative
light units (RLU) were recorded in triplicate samples. For each
experiment, a standard curve of RLU was constructed using purified
-gal (Sigma, St. Louis, MO).
5-Bromo-4-chloro-3-indolyl--D-galactopyranoside
and halogenated indolyl--D-galactoside
histochemistry. Anesthetized mice were perfused with 0.9% NaCl
and then with 0.5% glutaraldehyde in 0.1 M PB, pH 7.4. In
unlesioned animals, a piece of sciatic nerve, centered on the sciatic
notch, was removed. In lesioned animals, the distal nerve stump was
removed from the site of transection or crush to the popliteal fossa.
To avoid the site of the lesion, we removed ~5 mm of the proximal end
(nearest the site of the lesion) from crushed nerves; we removed 1-2
mm from transected nerves. The nerves were fixed for 1 hr in 0.5%
glutaraldehyde in 0.1 M PB, rinsed in 0.1 M PB
plus 2 mM MgCl2, and then incubated overnight at 37°C in either
5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-gal)
staining solution [35 mM
K3Fe(CN)6, 35 mM
K3Fe(CN)6·3H2O,
0.02% NP-40, 0.01% sodium deoxycholate, 2 mM
MgCl2, 0.1 M[R PB, AND 1 MG/ML X-GAL]
OR HALOGENATED INDOLYL--[SCAP]D-galactoside (Bluo-gal)
staining solution [3.1 mM
K3Fe(CN)6, 3.1 mM
K4Fe(CN)6·3H2O, 1 mM MgCl2, 0.1 M PB, and 0.4 mg/ml Bluo-gal]. The nerves were rinsed three times with 0.1 M PB, fixed in 3.6% glutaraldehyde (in 0.1 M
PB) for 24 hr, rinsed twice in 0.1 M PB, osmicated, dehydrated in graded ethanols, and rapidly infiltrated in propylene oxide and epoxy to minimize the solubilization of X-gal and Bluo-gal. Semithin (500 nm) and thin (75 nm) transverse sections were cut from
the sciatic notch region of unlesioned nerves and from the proximal end
of the distal nerve stump from lesioned nerves. Sections were mounted
on 200 mesh grids, stained with uranyl acetate and lead citrate, and
examined with a Zeiss EM10 electron microscope. For every sample of
nerve, a single section was selected for analysis. All cells with a
complete nucleus (not obscured by grid bars) in the plane of the
section were photographed at 8000-25,000× and were analyzed on the
resulting prints according to their relationships with axons and the
presence or absence of X-gal or Bluo-gal crystals. To demonstrate the
crystals more clearly, we took the photographs (see Figs. 3, 4, 6) from
sections that were not counterstained with lead citrate and uranyl
acetate.
We performed a statistical analysis on the number of X-gal or Bluo-gal
crystals in different kinds of Schwann cells in developing and
regenerating nerves. Because the X-gal and Bluo-gal histochemistry cannot be assumed to be uniform between different nerves, all comparisons were made within the same sample of nerve. Crystals appeared to be rectangular in shape, but not preferentially oriented with respect to the nerve fibers, and were typically found at least in
part within membranes. To count crystals, we examined the electron
micrographs of individual Schwann cells in which the nucleus was in the
plane of sections, and we considered an uninterrupted electron dense
structure to be a single crystal. In developing nerves, we compared the
number of crystals in bundling, in promyelinating, and in myelinating
Schwann cells by the Wilcoxon rank-sum test (JMP IN) (Hollander and
Wolfe, 1973); in regenerating nerves, we compared the number of
crystals in promyelinating; in myelinating; in bundling,
promyelinating > 1, and nonmyelinating; and in perineurial-like
cells.
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RESULTS |
The developmental expression of -gal parallels that of
endogenous Tst-1/SCIP/Oct-6 protein
Because the lacZ gene essentially replaced the
tst-1/scip/oct-6 gene without disrupting its promoter
(Bermingham et al., 1996), the level of -gal protein in heterozygous
tst-1/scip/oct-6 (tst-1/scip/oct-6 +/) nerves
should parallel the expression of the Tst-1/SCIP/Oct-6 protein itself.
To determine this directly, we analyzed tst-1/scip/oct-6 +/ sciatic nerves from mice of different ages with a sensitive, luminometric assay of -gal. Figure 1
shows the result of one set of assays. The RLU was high at P1, peaked
at P5, and rapidly diminished after P10, so that by P20 the levels were
comparable with that in adults. We repeated this assay using a
different set of tst-1/scip/oct-6 +/ sciatic nerves and
found similar results (data not shown). The RLUs of sciatic nerves from
age-matched wild-type littermates, in contrast, were low at every age
tested, ranging from ~1500 to ~4000, always less than the lowest
values found in tst-1/scip/oct-6 +/ mice at any age. Thus,
the overall pattern of -gal expression in developing
tst-1/scip/oct-6 +/ nerves matches that of
Tst-1/SCIP/Oct-6 mRNA in developing rat sciatic nerves (Monuki et al.,
1989; Scherer et al., 1994; Zorick et al., 1996).
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Figure 1.
Solution assay of -gal in developing
tst-1/scip/oct-6 +/ sciatic nerves. The
heights of the bars represent the mean
RLUs per milligram of protein for P1, P5, P10, P15,
P20, P37, and adult mouse sciatic nerves. The RLUs were measured in
triplicate for each sample; error bars represent the SEM.
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tst-1/scip/oct-6 +/ promyelinating Schwann cells
express -gal
The above data justify using -gal expression as a surrogate
marker of the endogenous tst-1/scip/oct-6 gene expression.
Because myelination appears normal in tst-1/scip/oct-6 +/
mice (Bermingham et al., 1996; Jaegle et al., 1996), we examined
tst-1/scip/oct-6 +/ nerves after staining them with X-gal
or Bluo-gal. In the presence of -gal, these sugars produce a blue
precipitate that becomes electron dense after osmication. We examined
sciatic nerves from P1, P10, and P30 tst-1/scip/oct-6 +/
mice, a range of ages that encompasses the onset of myelination and the
peak of Tst-1/SCIP/Oct-6 expression. P1 and P10 nerves were grossly
blue, but P30 nerves were only lightly stained, in agreement with the
luminometric -gal assays, demonstrating again that -gal
expression falls during postnatal development.
To determine precisely which Schwann cells express -gal, we
performed electron microscopy on these sciatic nerves, because this
allowed axon-Schwann cell relationships to be more accurately determined than can be done by light microscopy. Every nucleated Schwann cell that was visible in a single transverse section was photographed and classified by its morphological relationship to axons
(Webster, 1993), and the presence or absence of X-gal or Bluo-gal
crystals was noted. As shown schematically in Figure 2, we classified Schwann cells as
follows: denervated (not associated with any axon), bundling
(associated with more than one axon, typically many axons, but not
individually ensheathing any of them), promyelinating (ensheathing one
axon), promyelinating more than one axon (promyelinating > 1;
individually ensheathing more than one axon), myelinating
(wrapping an individual axon more than once or forming compact
myelin), or nonmyelinating (ensheathing more than one axon). At P1 and
P10, we classified Schwann cells that were associated with
nonmyelinated axons as either bundling or promyelinating, whereas at
P30, all of these were considered to be nonmyelinating Schwann cells
according to morphological criteria (Peters and Muir, 1959; Kobayashi
and Suzuki, 1990; Webster, 1993).
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Figure 2.
Graphical summary of -gal expression in Schwann
cells in tst-1/scip/oct-6 +/ sciatic nerves. At each
developmental age (P1, P10, and P30), the heights of the
bars show the percentage of Schwann cells of the
following morphologies: denervated, bundling, promyelinating > 1, promyelinating, myelinating, and nonmyelinating. The lightly
shaded portions of the bars indicate the
percentage of -gal-negative cells, whereas the darkly shaded
portions represent the percentage of -gal-positive Schwann
cells (visualized with X-gal and Bluo-gal at P1 and with X-gal at P10
and P30). The fraction over each bar
shows the number of positive Schwann cells relative to the total number
of Schwann cells of that morphology. n, Number of nerves
analyzed at each age.
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In P1 tst-1/scip/oct-6 +/ or tst-1/scip/oct-6
+/+ nerves, most Schwann cells were bundling and promyelinating, as
reported by Bermingham et al. (1996). Light microscopic examination of X-gal-treated nerves, however, revealed that blue crystals were only
seen in tst-1/scip/oct-6 +/ nerves, mainly in what
appeared to be promyelinating Schwann cells. By the use of electron
microscopy, the X-gal crystals were electron dense and concentrated in
the nuclear membrane (Feltri et al., 1992). Most X-gal-positive cells were promyelinating Schwann cells, although some bundling and myelinating Schwann cells were also positive (Fig. 2). In addition to
Schwann cells, there was a small population of fibroblasts and
macrophages; these cells were X-gal-negative (data not shown). In
addition to X-gal, we also used Bluo-gal to produce bigger and more
conspicuous crystals, which were also found in other membranous
components of the cell, especially the plasma membrane (Fig.
3). Although Bluo-gal appeared to be more
sensitive than X-gal, staining a higher proportion of promyelinating
and myelinating Schwann cells, the results were essentially the same
(Fig. 2).
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Figure 3.
Electron microscopy of P1
tst-1/scip/oct-6 +/ sciatic nerve stained with
Bluo-gal. A, The nuclei of three bundling
(b) Schwann cells and one promyelinating
(p) Schwann cell. B,
C, Higher magnifications of the promyelinating Schwann
cell seen in A. Some of the Bluo-gal crystals are
indicated (arrowheads); the symbols plus
and minus mark the presence or absence, respectively, of
crystals in classified cells. To demonstrate the crystals more clearly,
we took these photographs from a section that was not counterstained
with lead citrate and uranyl acetate. Scale bars: A, 1.0 µm; B, C, 0.5 µm.
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In P10 tst-1/scip/oct-6 +/ and tst-1/scip/oct-6
+/+ nerves, the number of myelinating Schwann cells increased
substantially compared with that in P1 nerves (Fig. 2). In
tst-1/scip/oct-6 +/ nerves, a high proportion of
myelinating and promyelinating Schwann cells had X-gal crystals,
especially the latter. As in P1 nerves, fibroblasts and macrophages did
not have X-gal crystals, and Schwann cells in
tst-1/scip/oct-6 +/+ nerves did not have X-gal crystals.
P30 tst-1/scip/oct-6 +/ and tst-1/scip/oct-6
+/+ nerves contained only myelinating and nonmyelinating Schwann cells
(Figs. 2, 4). In addition to the
occasional X-gal- or Bluo-gal-positive Schwann cell (Figs. 2, 4), we
did not note any differences between tst-1/scip/oct-6 +/
and tst-1/scip/oct-6 +/+ nerves (Bermingham et al., 1996;
Jaegle et al., 1996).
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Figure 4.
Electron microscopy of P30
tst-1/scip/oct-6 +/ sciatic nerve stained with
Bluo-gal. Two Schwann cells, one myelinating (m)
and one nonmyelinating (n), are shown. The
arrowhead indicates a Bluo-crystal. The symbols
plus and minus mark the presence or absence,
respectively, of crystals in classified cells. To demonstrate the
crystals more clearly, we took these photographs from a section that
was not counterstained with lead citrate and uranyl acetate. Scale bar,
1.0 µm.
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The above results, taken together, demonstrate a dynamic,
maturation-dependent expression of -gal in
tst-1/scip/oct-6 +/ Schwann cells. The proportion of cells
expressing -gal indicates that some bundling Schwann cells express
low levels of -gal but that -gal expression increases
dramatically in promyelinating Schwann cells and then decreases after
the onset of myelination. To analyze further the stage-specific
expression of -gal, we counted the number of X-gal or Bluo-gal
crystals in Schwann cells of P1 and P10 tst-1/scip/oct-6
+/ nerves. Because the degree of staining of individual nerves with
X-gal or Bluo-gal histochemistry cannot be assumed to be the same,
comparisons were made within individual nerves. These results are
summarized in Table 1. In P1 nerves,
there were more crystals in promyelinating Schwann cells than in
bundling Schwann cells; the number of myelinating Schwann cells was too
low to analyze. In P10 nerves, there were more crystals in
promyelinating Schwann cells than in either bundling or myelinating
Schwann cells. Thus, promyelinating Schwann cells had both the highest
proportion and the highest number of X-gal or Bluo-gal crystals,
indicating that these cells express the highest levels of -gal and,
by inference, Tst1/SCIP/Oct-6.
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Table 1.
Quantitative analysis of X-gal or Bluo-gal expression in
individual Schwann cells of P1 and P10
tst-1/scip/oct-6 ± sciatic nerves
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-gal expression in lesioned adult nerves
Lesioning adult nerves disrupts axon-Schwann cell interactions
and leads to the dedifferentiation of Schwann cells, most pronounced in
myelinating Schwann cells, which cease expressing myelin-related genes
and begin expressing markers typical of immature Schwann cells, such as
N-CAM, L1, p75NTR, and GAP-43 (Mirsky and Jessen,
1996; Scherer, 1997b). In permanently transected nerves, in which axons
are deliberately prevented from regenerating, Schwann cells remain
dedifferentiated indefinitely. In crushed nerves, axons readily
regenerate, and some are remyelinated by Schwann cells, whose
differentiation appears to recapitulate that seen in development,
including the re-expression of Tst-1/SCIP/Oct-6 mRNA and protein
(Scherer et al., 1994; Zorick et al., 1996).
To confirm and extend these findings, we performed -gal assays on
crushed and permanently transected sciatic nerves from adult
tst-1/scip/oct-6 +/ and tst-1/scip/oct-6 +/+
mice. We analyzed the amount of -gal activity in nerve segments
distal to the site of injury at 1, 4, 8, 12, 24, and 58 d after
crushing and after transection (Fig. 5).
In tst-1/scip/oct-6 +/ mice, the amount of -gal
activity in the distal nerve stumps of crushed nerves increased
substantially between 1 and 12 d, followed by a decline. The
amount of -gal activity in the distal nerve stumps of transected nerves, in contrast, increased only slightly over this same time frame.
The higher levels of -gal activity in crushed nerves coincide with
the re-ensheathment and remyelination of regenerating axons and,
overall, agree with the previous studies of Tst-1/SCIP/Oct-6 expression
in lesioned adult rat sciatic nerves (Monuki et al., 1990; Scherer et
al., 1994; Zorick et al., 1996). The -gal activity in both
transected and crushed adult tst-1/scip/oct-6 +/+ nerves was
low, comparable with that in unlesioned adult
tst-1/scip/oct-6 +/ nerves (data not shown), further
demonstrating that the increase in transected and crushed
tst-1/scip/oct-6 +/ nerves is not the result of
nerve-injury per se but rather is the result of increased Tst-1/SCIP/Oct-6 expression.
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Figure 5.
Solution assay of -gal in adult
tst-1/scip/oct-6 +/ sciatic nerves. The
heights of the bars represent the mean
RLUs per milligram of protein from the distal stumps of nerves 1, 4, 8, 12, 24, and 58 d after transection or after
crushing. Note that the RLUs of the crushed nerves is higher than that
in the transected nerves during the period of ensheathment and
myelination (8-24 d after crushing).
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Promyelinating and denervated Schwann cells express -gal in
lesioned nerves
To determine which Schwann cells express Tst-1/SCIP/Oct-6 in
lesioned adult tst-1/scip/oct-6 +/ nerves, we examined the
distal nerve stumps of lesioned nerves (8, 12, 24, and 58 d after
the lesion) by electron microscopy after Bluo-gal histochemistry. At
8 d after transection, there were a few axonal remnants, and all
of the myelin sheaths were degenerating. Between 8 and 58 d after
transection, the amount of myelin debris progressively diminished, and
it was increasing found in macrophages rather than in Schwann cells.
Thus, by 58 d after transection, denervated Schwann cells and
macrophages were the predominant cell types in lesioned nerves. At 24 and 58 d, lesioned nerves also contained perineurial-like cells,
which typically had a discontinuous basal lamina, caveoli, and
elongated processes that often partially surrounded Schwann cells (Fig.
6); these looked different than denervated Schwann cells, which were primarily oval in shape and surrounded by a complete basal lamina (Fig.
7A). These perineurial-like cells have been noted previously in lesioned nerves, but their cellular
origin (Schwann cell vs endoneurial fibroblast) has not been resolved
(Morris et al., 1972). Even though nerves had been deliberately
transected to prevent axonal regeneration, at 58 d there were a
few axons that had regenerated (Fig. 8);
these were associated with Schwann cells.
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Figure 6.
Electron micrograph of a perineurial-like cell
from the distal stump 24 d after crushing. This cell shows the
typical features, including a patchy basal lamina (fat
arrows), caveoli (arrowheads), and two long
processes that ensheathe Schwann cells (s) and
axons (a). Scale bar, 1.0 µm.
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Figure 7.
Electron microscopy of lesioned adult
tst-1/scip/oct-6 +/ sciatic nerves stained with
Bluo-gal. A, A denervated (d)
Schwann cell at 12 d after transection.
B-D, Promyelinating
(p); bundling, promyelinating > 1, and
nonmyelinating (b,p>1,n);
and myelinating (m) Schwann cells at 12 d
after crushing, respectively. Some Bluo-gal crystals are indicated
(arrowheads). The symbols plus and
minus indicate the presence or absence, respectively, of
crystals in classified cells. To demonstrate the crystals more clearly,
we took these photographs from sections that were not counterstained
with lead citrate and uranyl acetate. Scale bars, 1.0 µm.
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Figure 8.
Graphical summary of -gal expression by Schwann
cells in lesioned adult tst-1/scip/oct-6 +/ sciatic
nerves. The bars show the percentage of Schwann cells of
each morphology at 8, 12, 24, and 58 d after transection
(upper) and after crushing (lower); the
lightly and darkly shaded bars indicate
the percentage of Bluo-gal-negative and -positive Schwann cells,
respectively. The fraction over each bar
shows the number of positive Schwann cells relative to the total number
of Schwann cells of that morphology. n, Number of
animals analyzed. The Schwann cell morphologies are as follows:
d, denervated; bp>1n,
bundling, and promyelinating > 1, and nonmyelinating;
p, promyelinating; and m,
myelinating.
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|
In crushed adult tst-1/scip/oct-6 +/ nerves, axons and
myelin sheaths degenerated in a similar manner to that in transected nerves, but axonal regeneration occurred. At 8 d, most Schwann cells were denervated, but some were associated with bundles of regenerating axons or with single regenerating axons in a one-to-one promyelinating configuration. At 12 and 24 d, a higher proportion of Schwann cells was associated with regenerating axons, which were
more numerous than at 8 d, and some Schwann cells had myelinated regenerating axons (Fig. 7B-D). By 58 d, nerves had
assumed a more mature appearance, with more myelinating and
nonmyelinating Schwann cells and fewer denervated and bundling Schwann
cells than at 24 d. Thus, the morphological aspects of
axon-Schwann cell interactions that culminate in myelination appear to
be similar in developing and regenerating nerves.
To determine which cells expressed -gal, we determined the
proportion of cells with Bluo-gal crystals in a single, transverse section of lesioned adult tst-1/scip/oct-6 +/ nerves.
After transection, the proportion of denervated Schwann cells with
Bluo-gal crystals varied between 3 and 31% (Fig. 8). Whether these
different proportions at various times after transection represent
significant differences, however, is moot, because the overall level of
-gal activity in the distal stumps of transected nerves increased
only slightly after transection (Fig. 5). In addition to being found in
Schwann cells, Bluo-gal crystals were also found in some macrophages
and rarely in perineurial-like cells.
Because the axon-Schwann cell interactions that lead to myelination in
regenerating nerves appear to recapitulate those in developing nerves,
it seemed likely that promyelinating Schwann cells in crushed nerves
would also have the highest levels of -gal. To determine whether
this was case, we examined the Schwann cells in the distal stumps of
crushed adult tst-1/scip/oct-6 +/ nerves for the presence
of X-gal or Bluo-gal crystals. For this analysis, we lumped
together bundling, promyelinating > 1, and nonmyelinating Schwann
cells together into a single category (b,p>1,n) (Fig. 7C), because it is difficult to distinguish these
potentially different kinds of Schwann cells in lesioned nerves and,
particularly, because single Schwann cells may ensheathe misdirected
axons from sensory, motor, and autonomic neurons (King and
Thomas, 1971; Scherer and Easter, 1984). As summarized in Figure 8 and
Table 2, a high proportion of
promyelinating Schwann cells contained Bluo-gal crystals in crushed
nerves, and promyelinating Schwann cells had significantly more
Bluo-gal crystals than did denervated or
b,p>1,n Schwann cells at all time
points. In some nerves, promyelinating Schwann cells had more crystals
than did myelinating Schwann cells. Although the number of
perineurial-like cells was variable, a low proportion of these cells
also had Bluo-gal crystals. These data, taken together, show that the
timing of higher -gal activity in crushed nerves is related to the
ensheathment and myelination of regenerating axons and not to Wallerian
degeneration per se. During the peak of -gal expression, at
approximately 12 d after crushing, promyelinating and myelinating
Schwann cells account for the bulk of -gal expression and, hence,
are the cells that express the highest levels of Tst-1/SCIP/Oct-6. In
further support of these conclusions, we did not find crystals in the
lesioned adult tst-1/scip/oct-6 +/+ nerves at 12 d
after transection and after crushing (data not shown).
View this table:
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Table 2.
Quantitative analysis of X-gal and Bluo-gal expression in
individual Schwann cells from tst-1/scip/oct-6 sciatic
nerves 8, 12, and 24 days after crushing
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DISCUSSION |
In this paper, we demonstrate for the first time that
Tst-1/SCIP/Oct-6 is principally expressed by promyelinating Schwann cells. Whereas the timing of Tst-1/SCIP/Oct-6 expression in developing and lesioned peripheral nerves was consistent with the idea that it
functions in proliferating Schwann cells (Monuki et al., 1990), subsequent anatomical studies suggested that Tst-1/SCIP/Oct-6 expression may be independent of proliferation and that it functions in
promyelinating Schwann cells (Scherer et al., 1994). The observation that Schwann cells are temporarily arrested at the promyelinating stage
in tst-1/scip/oct-6-null mice is further evidence that it plays an essential role at these cells (Bermingham et al., 1996; Jaegle
et al., 1996).
Does -gal expression reflect Tst-1/SCIP/Oct-6 expression?
We used the expression of -gal in tst-1/scip/oct-6
+/ mice as a means of identifying when Schwann cells express
Tst-1/SCIP/Oct-6. This analysis assumes that the expression of one copy
of tst-1/scip/oct-6 is sufficient for the normal development
of myelinating Schwann cells. This assumption seems to be justified as
tst-1/scip/oct-6 +/ mice are long-lived and without a
discernable phenotype (Bermingham et al., 1996; Jaegle et al., 1996),
and we have not seen any difference in the degree of myelination
between tst-1/scip/oct-6 +/ and tst-1/scip/oct-6 +/+ mice in several litters (S. S. Scherer and E. J. Arroyo, unpublished observations).
Our analysis also requires that the expression of lacZ
mirrors that of tst-1/scip/oct-6. This is likely to be the
case. First, the design of the targeting vector did not disrupt the
tst-1/scip/oct-6 promoter, because only the open reading
frame was replaced by the expression cassette (which contained
lacZ and neo), leaving the
tst-1/scip/oct-6 promoter intact (Bermingham et al., 1996). Furthermore, the theoretical possibility that promoter elements within
tst-1/scip/oct-6 may be disrupted by the homologous
recombination of the targeting vector does not apply, because it is an
intronless gene (Kuhn et al., 1991). Second, although the half-life of
-gal and Tst-1/SCIP/Oct-6 may be different, this should not affect our interpretation. The reported half-life of E. coli
-gal in mammalian cells varies from 13 to 43 hr (Margolis et al.,
1993; Jacobsen and Willumsen, 1995); this is probably longer than the half-life of Tst-1/SCIP/Oct-6, because the expression of
Tst-1/SCIP/Oct-6 protein decreases markedly in neonatal rat sciatic
nerve by 1 d after axotomy (S. S. Scherer, unpublished
observations). Even if -gal were very stable, this should not
significantly alter the onset of -gal expression and, hence, our
conclusion that Tst-1/SCIP/Oct-6 expression peaks in promyelinating
Schwann cells. Stable -gal would, however, prolong the fall of
-gal expression, so that we may have overestimated the duration of
Tst-1/SCIP/Oct-6 expression by myelinating Schwann cells. Third, the
pattern of -gal expression in developing and injured peripheral
nerves parallels the expression of Tst-1/SCIP/Oct-6 mRNA described in
rats. In developing tst-1/scip/oct-6 +/ sciatic nerve, our
luminometric assays show an apparent peak of -gal at P5. Although
Tst-1/SCIP/Oct-6 protein levels have not been measured in mice or rats,
the peak of Tst-1/SCIP/Oct-6 mRNA in rats occurs somewhat earlier
(approximately P1) (Monuki et al., 1989, 1990; Scherer et al., 1994).
The slight delay in -gal expression compared with that of
Tst-1/SCIP/Oct-6 mRNA may reflect the time required for protein
synthesis and accumulation or a species differences.
Tst-1/SCIP/Oct-6 is mainly expressed by promyelinating
Schwann cells
The above discussion justifies the use of the -gal
histochemistry as a means of identifying which Schwann cells express
Tst-1/SCIP/Oct-6. Although one can detect Tst-1/SCIP/Oct-6 by
immunohistochemistry, the phenotype of these cells is difficult to
assess by light microscopy (Scherer et al., 1994; Blanchard et al.,
1996; Zorick et al., 1996). The differentiated state of a Schwann cell
can be established by electron microscopy, but our attempts to perform
immunoelectron microscopy with the Tst-1/SCIP/Oct-6 antibody were
unsuccessful (D. L. Sherman and S. S. Scherer, unpublished
observations). -Gal histochemistry, however, allowed us to determine
that Tst-1/SCIP/Oct-6 is mainly expressed by promyelinating Schwann
cells, both in developing and in regenerating nerves. Bundling Schwann
cells express less -gal, and myelinating Schwann cells appear to
express -gal only transiently. The peak of -gal expression
coincides with the promyelinating morphology, which is also the point
at which Schwann cell development is transiently arrested in
tst-1/scip/oct-6-null mice (Bermingham et al., 1996; Jaegle
et al., 1996). The finding that not all promyelinating and myelinating
Schwann cell have X-gal crystals, however, should not be interpreted as
evidence that these cells do not express -gal. Rather, this is
probably the result of a sampling problem inherent in the use of the
thin sections that are needed for the electron microscopic
classification of the Schwann cell phenotype. It also follows that the
absence of X-gal crystals from a particular phenotype does not exclude
the possibility of a low level of -gal expression.
These results confirm and extend previous work, in which
immunohistochemistry was used to evaluate the expression of
Tst-1/SCIP/Oct-6. Using a relatively insensitive antiserum, Scherer et
al. (1994) found Tst-1/SCIP/Oct-6 expression in developing and
regenerating rat sciatic nerves but not in adult or in permanently
axotomized nerves. With the development of more sensitive antisera, low
levels of Tst-1/SCIP/Oct-6 have also been found in Schwann cell
precursors, as well as in immature/premyelinating, myelinating,
nonmyelinating, and denervated Schwann cells (Blanchard et al., 1996;
Zorick et al., 1996). Schwann cells in developing and regenerating
nerves have the highest levels of Tst-1/SCIP/Oct-6-immunoreactivity, but the identity of these cells could not be established unequivocally by light microscopy. Our results indicate that these highly
Tst-1/SCIP/Oct-6-immunoreactive cells are probably promyelinating
Schwann cells. The low level of Tst-1/SCIP/Oct-6 immunoreactivity in
Schwann cell precursors, as well as in immature, myelinating,
nonmyelinating, and denervated Schwann cells, is consistent with the
lower frequency of X-gal and Bluo-gal crystals in bundling,
myelinating, nonmyelinating, and denervated Schwann cells. Whether
Tst-1/SCIP/Oct-6 plays an important role in these other kinds of
Schwann cells is uncertain, because the only phenotype that has been
described in tst-1/scip/oct-6-null mice is the arrested
development at the promyelinating stage (Bermingham et al., 1996;
Jaegle et al., 1996).
Transcription factors and the regulation of the Schwann
cell phenotype
As shown schematically in Figure 9,
several transcription factors are expressed at particular stages of
Schwann cell development (Zorick and Lemke, 1996; Scherer, 1997b).
Premyelinating, nonmyelinating, and denervated Schwann cells express
three unrelated transcription factors, Krox-24, Pax-3, and c-jun
(Kioussi et al., 1995; Nikam et al., 1995; Stewart, 1995; Blanchard et
al., 1996; Shy et al., 1996). This was shown by in situ
hybridization for Pax-3 (Kioussi et al., 1995), immunolabeling for
c-jun (Shy et al., 1996), and immunolabeling as well as histochemical
staining for -gal in heterozygous knock-out animals for Krox-24
(Nikam et al., 1995; Topilko et al., 1997).
View larger version (23K):
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Figure 9.
Schematic view of transcription factor expression
in developing rodent Schwann cells. This figure summarizes the timing
of expression of various transcription factors in developing rodent
(mouse and rat) peripheral nerve and relates the expression of these
transcription factors to the anatomical relationships of axons and
Schwann cells. The thick black lines (e.g., for Krox-20
and Tst-1/SCIP/Oct-6) indicate relatively higher levels of
expression than do the thin black lines.
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|
As shown in Figure 9, as Tst-1/SCIP/Oct-6 expression wanes in
myelinating Schwann cells, the expression of Krox-20, a zinc finger
transcription factor highly homologous to Krox-24, increases dramatically and remains highly expressed unless Schwann cells become
denervated (Topilko et al., 1994, 1997; Murphy et al., 1996; Zorick et
al., 1996). Like Tst-1/SCIP/Oct-6, Krox-20 is essential for the normal
development of myelinating Schwann cells, because Schwann cells appear
to be permanently arrested at the promyelinating stage in
krox-20-null mice (Topilko et al., 1994).
Although the phenotype of tst-1/scip/oct-6-null mice
demonstrates its importance in the development of myelinating Schwann cells, its precise function in this process remains to be determined. In their initial description of Tst-1/SCIP/Oct-6, Monuki et al. (1989) showed that its expression in developing nerves and in forskolin-stimulated Schwann cells precedes that of myelin-related genes. They postulated that Tst-1/SCIP/Oct-6 plays a central role in
the differentiation to a myelinating phenotype, but in subsequent transient cotransfection experiments, Tst-1/SCIP/Oct-6 repressed the
expression of mbp and P0 promoter
constructs in cultured Schwann cells (Monuki et al., 1990, 1993; He et
al., 1991). This repression was not observed for other POU family
members and depended on both the N terminal and the DNA binding domain
of Tst-1/SCIP/Oct-6. However, mutating the Tst-1/SCIP/Oct-6 binding
sites in the P0 promoter did not abolish this
repression, leading Monuki et al. (1993) to postulate that
Tst-1/SCIP/Oct-6 represses by "squelching," by protein-protein
interactions that occur off of DNA. In keeping with the idea that the
normal function of Tst-1/SCIP/Oct-6 is to repress the expression of
myelin-related genes, transgenic mice expressing an
N-terminal-truncated Tst-1/SCIP/Oct-6 driven by a
P0 promoter ( Tst-1/SCIP/Oct-6) have premature
myelination (Weinstein et al., 1995).
The analysis of tst-1/scip/oct-6-null mice, however, does
not support the view that the normal function of Tst-1/SCIP/Oct-6 is to
repress the expression of myelin-related genes, because the levels of
myelin-related mRNAs in neonatal tst-1/scip/oct-6-null mice
are normal or reduced (Bermingham et al., 1996; Jaegle et al., 1996).
Most myelinating Schwann cells in tst-1/scip/oct-6-null mice are arrested for more than a week at the promyelinating stage; these cells, nevertheless, express and properly localize MAG and periaxin, two myelin-related proteins that are normally expressed by
promyelinating Schwann cells. Why the myelin sheaths fails to form at
the normal time and, indeed, how the myelinating Schwann cells
eventually form them remain unanswered. The identification of the
target genes of Tst-1/SCIP/Oct-6 and Krox-20 will prove invaluable in
this regard.
| |
FOOTNOTES |
Received June 16, 1998; accepted July 10, 1998.
This work was supported by National Institutes of Health Grants NS34528
and NS37199 to S.S.S., by National Institute of General Medical
Sciences Fellowship GM15020-04 to E.J.A., and by a grant from the
National Multiple Sclerosis Society. We thank Susan Shumas and Yi-Tian
Xu for technical assistance, Victor Ming for advice on statistical
analysis, and Dr. Diane Sherman for immunoelectron microscopic
analysis.
Correspondence should be addressed to Dr. Steven S. Scherer, 460 Stemmler Hall, 36th Street and Hamilton Walk, The University of
Pennsylvania Medical Center, Philadelphia, PA 19104-6077.
| |
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Top
Abstract
Introduction
