The Journal of Neuroscience, July 2, 2003, 23(13):5520-5530
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Expression of Laminin Receptors in Schwann Cell Differentiation: Evidence for Distinct Roles
Stefano C. Previtali,1
Alessandro Nodari,1
Carla Taveggia,1
Celia Pardini,1
Giorgia Dina,1
Antonello Villa,2
Lawrence Wrabetz,1
Angelo Quattrini,1 and
M. Laura Feltri1
1Neuropathology Unit, Department of Neuroscience
and DIBIT, San Raffaele Scientific Institute, 20132 Milan, Italy, and
2Consorzio MIA-DNTR University MI-Bicocca, 20126
Milan, Italy
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Abstract
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Schwann cells require laminin-2 throughout nerve development, because
mutations in the 
2 chain in dystrophic mice interfere with
sorting of axons before birth and formation of myelin internodes after birth.
Mature Schwann cells express several laminin receptors, but their expression
and roles in development are poorly understood. Therefore, we correlated the
onset of myelination in nerve and synchronized myelinating cultures to the
appearance of integrins and dystroglycan in Schwann cells. Only
6
1 integrin is expressed before birth, whereas dystroglycan and
64 integrin appear perinatally, just before myelination. Although
dystroglycan is immediately polarized to the outer surface of Schwann
cells,64 appears polarized only after myelination. We showed
previously that Schwann cells lacking 1 integrin do not relate properly
to axons before birth. Here we show that the absence of 1 before birth
is not compensated by other laminin receptors, whereas coexpression of both
dystroglycan and 4 integrin is likely required for 1-null Schwann
cells to myelinate after birth. Finally, both 1-null and
dystrophic nerves contain bundles of unsorted axons, but they are
predominant in different regions: in spinal roots in dystrophic mice
and in nerves in 1-null mice. We show that differential compensation by
laminin-1, but not laminin receptors may partially explain this. These data
suggest that the action of laminin is mediated by 1 integrins during
axonal sorting and by dystroglycan, 61, and 64
integrins during myelination.
Key words: laminin receptors; Schwann cells; dystrophic mice; integrins; dystroglycan; myelination; adhesion
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Introduction
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Schwann cells (SCs) myelinate axons in the peripheral nervous system (PNS).
SC precursors originate from neural crest at embryonic day (E) 12–E13 in
mouse, migrate along neurites, and become embryonic SCs between E15 and E16
(for review, see Jessen and Mirsky,
1999
). Later, SCs segregate large axons from axon bundles and
establish a one-to-one relationship with them (promyelinating SCs). After
birth, SCs wrap axons to form myelin
(Webster et al., 1973),
whereas non-myelin-forming SCs differentiate after postnatal day (P) 15
(Jessen and Mirsky, 1999).
These events involve changes in cell shape and cytoskeletal organization that
require adhesion to the basal lamina (BL)
(Bunge, 1993) or laminin
deposition (Podratz et al.,
2001).
In mature PNS, laminin-2 (2/1/
1) and minor amounts of
laminin-8 (4/1/1) are present in the endoneurium, whereas
laminin-1 (1/1/1), -9 (4/2/1), and -11
(5/2/1) are in the perineurium
(Sanes et al., 1990;
Patton et al., 1997).
Mutations of the laminin 2 chain cause congenital muscular dystrophy
and a dysmyelinating neuropathy (Xu et
al., 1994; Shorer et al.,
1995; Sunada et al.,
1995). In dystrophic mice, the neuropathy consists of
impaired axonal sorting, mainly in the proximal PNS
(Bradley and Jenkison, 1973;
Stirling, 1975), and
myelination and paranodal abnormalities mainly in distal nerves
(Bradley et al., 1977;
Jaros and Bradley, 1979). The
alterations in axonal sorting originate prenatally and in paranodes and
internodes postnatally. The different timing and location of these
abnormalities may result from differential expression of laminin receptors in
developing nerves. Alternatively, other laminins, or their receptors, could
differentially compensate for loss of laminin function.
In keeping with the first possibility, several laminin receptors are
present in mature SCs, mainly integrins 61 and 64
and dystroglycan (DG). Integrins include and transmembrane
subunits, which form receptors for different matrix components (for review,
see Previtali et al., 2001).
DG includes an subunit and a trans-membrane subunit
(Ervasti and Campbell, 1993).
Neural crest cells and SCs synthesize 61
(Hsiao et al., 1991;
Bronner-Fraser et al., 1992;
Einheber et al., 1993).
Experiments with blocking antibody suggest a role for 1 in myelination
(Fernandez-Valle et al.,
1994), whereas selective inactivation of 1 in SCs impairs
axonal sorting (Feltri et al.,
2002). 4 integrin and DG are expressed at the outer surface
of adult SCs (Yamada et al.,
1994; Quattrini et al.,
1996), and their expression is regulated axonally
(Einheber et al., 1993;
Feltri et al., 1994;
Masaki et al., 2000).
-DG binds to laminin-2 in SC BL
(Yamada et al., 1994) and
forms a complex with periaxin and dystrophic-related protein (DRP)-2
(Sherman et al., 2001).
Genetic alterations of periaxin cause a demyelinating neuropathy
(Gillespie et al., 2000;
Boerkoel et al., 2001;
Guilbot et al., 2001). Hence,
a role for 64 integrin and DG in myelination has been proposed.
The onset of 64 and DG expression is not known.
We describe the sequential expression of laminin receptors from embryonic
nerves to the mature PNS. Precursors and immature SCs expressed only
61. DG expression immediately preceded myelination, whereas
64 appeared polarized after myelination. In the absence of
1 integrin, no compensatory expression of 64 and DG occurs
prenatally, explaining the severe prenatal defect. Dystrophic nerves,
but not roots, show upregulation of laminin-1 that may compensate for
laminin-2 defects. Thus, the complicated temporal and topographic phenotype of
laminin and receptor mutants reflects both differences in receptor expression
and differential compensation by laminins.
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Materials and Methods
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Mice and genotyping
FVB/N and C57BL6 mice were from Charles River (Calco, Italy).
Dystrophic dy2J mice (B6.WK-Lama2dy-2J) were
from The Jackson Laboratory (Bar Harbor, ME) and maintained by backcrosses to
C57BL6. P0Cre mice have been described (Feltri et al.,
1999a,
2002) and were maintained by
backcrosses to either FVB/N or C57BL6 mice. Mice lacking 1 integrin in
SCs were produced by crossing P0Cre mice
with 1 heterozygous null mice and 1-floxed homozygous mice as
described (Graus-Porta et al.,
2001; Feltri et al.,
2002). Most progeny in this study resulted from parents that were
N2–N5 generations congenic for C57BL6. Mouse genotyping was performed by
Southern blot and PCR analysis of tail genomic DNA as described previously
(Kuang et al., 1998;
Feltri et al., 1999a;
Graus-Porta et al., 2001). All
experiments involving animals were performed according to the Institutional
Animal Care and Use Committee.
In situ hybridization
cDNAs for mouse dystroglycan, 1, 4, and 6 integrins were
reverse transcribed from RNA extracted from mouse sciatic nerves as described
(Feltri et al., 1999b) using
the following primers: DAG sense: 5'-GCT CTA GAA CCC TTG AGG ACC AGG CCA
C-3'; antisense: 5'-CAG AAG CTT AAC AGT GCT TCA GAG CCA
TC-3'; 6 sense: 5'-GCT CTA GAA CTA CTT GGA CAT TCT CGT
G-3'; antisense: 5'-CAG TTC GAA GGT GTC GTC AGT CTG AAA
TC-3'; 1 sense: 5'-GCT CTA GAA TCG TGC ATG TTG TGG AGA
C-3'; antisense: 5'-CAG TTC GAA GCT TGA TTC CAA TGG TCC
AG-3'; 4 sense: 5'-GCT CTA GAC CTT GGC TAC CTG GTG ACC
T-3'; antisense: 5'-CAG TTC GAA AGC CAG TCA GAA AGA CCT
TG-3'. cDNAs were amplified by PCR, ligated into pBluescript SKII
(Stratagene, La Jolla, CA), and sequenced. Generation of RNA probes and in
situ hybridization was performed as described
(Previtali et al., 1999).
Antibodies and immunohistochemistry
The antibodies used are listed in Table
1. Secondary antibodies included the following: fluorescein
isothiocyanate (FITC)- or tetramethylrhodamine isothiocyanate
(TRITC)-conjugated goat anti-mouse or rat IgG (1:50) and FITC- or
TRITC-conjugated goat anti-rabbit IgG (1:100) (Southern Biotechnology,
Birmingham, AL). Peroxidase-conjugated secondary antibodies for Western blots
were from Sigma (Milan, Italy). A mouse-to-mouse Dako ARK-kit (Dako, Glostrup,
Denmark) was used to stain mouse tissues with mouse antibodies.
Immunohistochemistry was performed as described in Feltri et al.
(2002) and for 4
laminin chain as described in Miner et al.
(1997). For
immunocytochemistry, cells were fixed 10 min in paraformaldehyde and 15 min in
cold methanol. Slides were examined with confocal (Bio-Rad MRC 1024) or
fluorescence microscopy (Olympus AX and BX).
Northern blot analysis
Total RNA was extracted from sciatic nerves and analyzed by Northern blot
as described (Feltri et al.,
1994), using the following cDNAs as probes: (1) the 630 bp mouse
dystroglycan cDNA fragment described for in situ hybridization, (2) a
full-length cDNA of rat P0 (Lemke and
Axel, 1985), and (3) a full-length cDNA of rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
(Fort et al., 1985).
Semiquantitative RT-PCR analysis
Sciatic nerves were dissected from 2- to 3-month-old
dy2J homozygous or dy2J heterozygous
littermates and used for RT-PCR as described
(Feltri et al., 1999b), using
the following primers: laminin 1:
5'-CACCCTGGACTTACGGCAGG-3' and
5'-TTCGTTGTCTGCTCTGTAAG-3'
(Nguyet et al., 2001); GAPDH:
5'-GTATGACTCTACCCACGG-3' and
5'-GTTCAGCTCTGGGATGAC-3'). Placentas from mouse embryos were used
as positive controls.
Western blot analysis
Sciatic nerves were dissected from 2- to 3-month-old
dy2J homozygous or dy2J heterozygous
littermates and used for Western blot analysis as described
(Wrabetz et al., 2000) with
the following modifications: 60 µg of nerve homogenates was loaded on 5%
SDS-polyacrylamide gels, blotted, and stained using affinity-purified rabbit
anti-laminin 1 (E3 fragment) antibodies
(Durbeej et al., 1996).
Homogenates from mouse kidneys were used as control, and equal loading of
nerve homogenates was verified using mouse anti-neurofilament-M antibodies
(Chemicon International, Temecula, CA).
Preparation of purified cell cultures
Neuronal cultures. Purified dorsal root ganglion (DRG) neurons
were dissociated from E15.5 Sprague Dawley rats (Charles River) as reported
previously (Kleitman et al.,
1998). After excision, DRG neurons were trypsinized (0.25%;
Invitrogen, San Giuliano Milanese, Italy) and mechanically dissociated. Cell
suspension (approximately three DRG neurons) was plated as a drop onto 12 mm
glass coverslips (Greiner) coated with rat collagen (0.2 mg/ml; Biomedical
Technologies) in CB10 media, consisting of Eagle's Minimal
Essential Medium (EMEM; Invitrogen) supplemented with 10% fetal calf serum
(FCS) (Biological Industries Kibbutz), 5 mg/ml glucose (Sigma), and 50
µg/ml crude nerve growth factor (NGF) (Harlan or Calbiochem). To remove
non-neuronal cells, CB10 media was alternated every 2 d with
E2F media for the first 12 d. E2F media contained EMEM
supplemented with 4 mg/ml glucose, 5 µg/ml insulin (Sigma), 10 µg/ml rat
transferrin (Jackson ImmunoResearch, West Grove, PA), 100 µM
putrescine (Sigma), 20 nM progesterone (Sigma), 30 nM
sodium selenite (Sigma), 10 µM 5-fluorodeoxyuridine (Sigma), 10
µM uridine (Sigma), and 50 ng/ml NGF.
SC cultures. SCs were prepared from sciatic nerves of P3 Sprague
Dawley rats by the method of Brockes et al.
(1979) and expanded as
described (Feltri et al.,
1992).
Neuron–SC cocultures
After neuronal seeding (2.5 weeks), SCs were added to coverslips (75,000
each). Cocultures were maintained in medium consisting of DMEM/F12 (1:1 vol),
5 mg/ml glucose, 5 µg/ml insulin, 10 µg/ml rat transferrin, 100
µM putrescine, 20 nM progesterone, 30 nM
sodium selenite, and 50 ng/ml NGF for 1 week. To initiate myelination,
cocultures were treated with EMEM, 15% FCS, 5 mg/ml glucose, and 50 µg/ml
ascorbic acid (Sigma).
Electron microscopy
Transgenic mice and age-matched controls were killed at P28 and 6 months
old for semithin and ultrathin analysis, as described
(Quattrini et al., 1996;
Wrabetz et al., 2000).
Image analysis
Micrographs were digitalized using an AGFA Arcus 2 scanner, and figures
were prepared using Adobe Photoshop Version 5.0.
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Results
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61 is the major laminin receptor synthesized by SCs
during embryogenesis
To study laminin receptors in the SC lineage, we examined the expression of
6, 1, 4 integrin subunits and DG by in situ
hybridization and immunohistochemistry in mouse embryos beginning at E12.5.
Anti-neurofilament antibodies were used to identify nerves. At E12.5, we only
detected expression of 6 and 1 integrin, both at the level of
message (Fig. 1, A and
E, respectively), and protein (data not shown) in DRG,
sensory and motor roots, and peripheral nerves. Identical results were
observed at E15.5 for 6 and 1
(Fig.
1B–D,F–H) (and data not shown). Double
staining with neurofilament (NF) and a marker for SCs or their precursors
(p75NTR receptor) demonstrated that 6 and 1 were
expressed in both axons and glial cells
(Fig.
1B–D,F–H) (and data not
shown). 4 integrin and DG were not detected at the level of message or
protein at E12.5 or E15.5 in the PNS (data not shown).
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Figure 1. Expression of 1 and 6 integrin subunits in early PNS
development. Cross sections of wild-type E12.5 mice (A, E) showing
spinal cord, DRG (asterisks), spinal roots (arrows), and peripheral nerves
(arrowhead), and E15.5 mice (B–H) showing motor roots. DRG
neurons, spinal roots, and spinal nerves showed mRNA expression of 1
(A) and 6 (E). Double staining of spinal roots for
1 or 6 with either NF (which identified axons) or
p75NTR (which identified Schwann cell precursors) showed that
1 and 6 were present in Schwann cells and axons. As an example,
B–D illustrate that 1 is present both in Schwann
cells and in axons; colocalization of NF (B) and 1 (C)
is merged in D. In F–H, the colocalization of
p75NTR (F) and 6 (G) is shown merged in
H. Scale bar: (in H) A, E, 50 µm;
B–H, 300 µm.
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The expression of DG briefly precedes and parallels myelination in
the PNS
At E18.5, along with 6 and 1 message and protein (data not
shown), we observed the onset of 4 integrin and DG mRNA expression in
spinal roots, DRG, and nerve trunks (Fig.
2A,D,E); however, we could not yet detect 4 and DG
protein (Fig.
2B,C,F,G). As internal positive controls, skeletal
myofibers (Fig. 2C)
stained for DG and blood vessels stained for 4
(Fig. 2G). Myelination
occurs earlier in motor than sensory roots
(Niebroj-Dobosz et al., 1980),
and expression of myelin-specific mRNAs is detectable at P1 in motor but not
sensory roots (Baron et al.,
1994; Forghani et al., 2001). Thus, to correlate the onset of DG
with the beginning of myelination, we stained motor and sensory roots at P1.
As expected, motor roots were positive and sensory roots were mostly negative
for MBP (Fig.
3B,D,G,I). Interestingly, DG staining was also stronger
in motor than sensory roots (Fig.
3A,C,F,H). Analysis of the merged staining in the roots,
and of single fibers by cross section in the nerve, showed that some fibers
stained for both DG and MBP. The staining for DG was external to that of MBP
(Fig. 3E,M) and
appeared as a ring in cross sections (Fig.
3M), indicating that DG was immediately polarized at the
ab-axonal surface of SCs. Other fibers were DG positive, with the same ring
appearance, but MBP negative (Fig.
3M, arrowhead). This indicates that DG becomes polarized
to the external SC surface (facing the basal lamina), just before the
appearance of MBP. Because MBP protein is detectable when sheaths contain at
least five to eight myelin lamellas (Hahn
et al., 1987), these data suggest that DG appears and is polarized
just before wrapping, in promyelinating SCs. At P5 and P15, expression of MBP
and DG progressively increased in sensory roots and peripheral nerves to
levels comparable with motor roots (data not shown).
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Figure 2. Expression of 4 integrin and -DG in the PNS of E18.5 wild-type
mice. Cross sections of whole-mount fetuses (A, D, E) including
spinal cord, DRG (asterisk), spinal roots (arrows), and peripheral nerves
(arrowheads). Magnification of motor roots (B, F) and intramuscular
peripheral nerves (C, G) is shown. In situ hybridization
showed -DG (A) and 4 (D, E) mRNA expression in
DRG, spinal roots, and spinal nerves. Double immunostaining for -DG
(red) and NF (green) or 4 (red) and NF (green) showed the absence of
both DG and 4 proteins in motor roots (B, F) and peripheral
nerves (C, G). As internal control, muscle stained positively for
-DG (C) and vessels stained positively for 4 (G,
double arrowheads). Scale bar: (in G) A, D, E, 50 µm;
B, C, F, G, 350 µm.
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Figure 3. Expression of -DG protein in the PNS of P1 normal mice. Cryosections
of motor roots (A–E), sensory roots (F–J), and
peripheral nerve (K–M), double stained for -DG and MBP.
Onset of -DG paralleled that of myelin proteins, beginning first in
motor roots (compare A, B with F, G). -DG was
expressed with a polarized, ab-axonal pattern on the surface of myelinating
SCs (MBP positive) (E, M, arrows). -DG staining was also
detectable with a similar ab-axonal (or ring) distribution in SCs still
negative for MBP (E, M, arrowheads). Scale bar: (in J)
A, B, F, G, 100 µm; C–E,
H–J, 35 µm; K–M, 15 µm.
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To further test the hypothesis that DG onset is in promyelinating SCs, we
stained peripheral nerves at P1 for both NF and DG. As shown in
Figure 4B, only SCs
that surrounded a single axon (arrows) expressed DG. By the same logic, we
stained mutant animals in which the absence of either laminin 2 or
integrin 1 causes a subset of SCs to arrest at the stage of axonal
sorting. We asked whether the SCs "frozen" at this developmental
stage expressed DG. Indeed, SCs around bundles of unsorted axons
(Fig. 8E) did not show
any DG staining (Fig.
4C–E, arrowhead), whereas SCs containing
only one axon expressed DG (Fig.
4C–E). To directly visualize ensheathing,
DG-negative SCs, we triple stained P1 sciatic nerves for DG, NF, and
p75NTR receptor. As shown in
Figure
4F–I, axon bundles are surrounded by
DG-negative, p75NTR-positive SCs (arrowhead), whereas some single
axon are surrounded by a DG-positive SC (arrow), which has already
downregulated p75NTR.
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Figure 4. Expression of DG in postnatal development and relative to SC–axonal
relationships. A, Northern analysis of DG, P0 glycoprotein, and GAPDH
expression in developing rat sciatic nerve. The ages of the rats are indicated
above the lanes. The blot was successively hybridized with
32P-labeled cDNA probes for DG, P0, and GAPDH and exposed to film
for 48, 3, and 24 hr, respectively. DG mRNA levels were low at P2 and P5 and
rose at P15, similarly to a myelin gene, P0. GAPDH signal demonstrates equal
loading of the RNA (10 µg total RNA per lane). Densitometric values of
DG/GAPDH ratios are indicated at the bottom of each lane, relative to the P2
value arbitrarily indicated as 1. B–E, Cryosections of
P1 normal mouse nerve (B), adult dy2J motor root
(C), sciatic nerve (D), and adult 1-null sciatic nerve
(E) double stained for NF (green) and DG (red). During development,
only SCs around single axons express DG (B, arrow). Similarly in
laminin 2-deficient or integrin 1-deficient mature PNS, only
single axons in the mutant mice are surrounded by DG staining, whereas
unsorted axons (C, D, E, arrowheads) are not surrounded by DG
staining. F–G, cryosections of normal P1 mouse nerve,
triple stained for NF (red), DG (blue), and p75NTR (green). SCs
identified by staining with anti-p75NTR antibodies surround bundles
of axons (stained by anti-NF antibodies; arrowhead) and do not express DG; in
contrast only SCs in a 1:1 relationship with axons lose p75NTR and
begin to express DG (arrow). Other Schwann cells associated with single axons
are in a transition state, with p75NTR still detectable and DG
synthesis just beginning. Scale bar: B, C, F–I, 10
µm; D, E, 12µm.
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Figure 8. Consequences of deletion of 1 integrin in Schwann cells on the
expression of laminins and their receptors. A–L,
Ultrathin (E) and cryosections (A–D,
F–L) of sciatic nerves from E17.5 (A–D) and
P28 (E–I) and motor roots from P28 (J–L)
conditional 1-null mice. In E, an electron micrograph of a
bundle of unsorted axons of mixed caliber surrounded by a Schwann cell is
shown. In A–D, expression of DG (B) and
4 (D) was not prematurely induced in embryonic 1-null
nerves identified by neurofilament staining (A, C). In F, an
area from a cross section of a P28 sciatic nerve showing MBP-positive and
-negative fibers is shown. MBP and 1 integrin are double stained on one
section; DG and 4 integrin are double stained on a serial section.
MBP(+) fibers (marked 1–6) showed DG and 4 positivity. Incontrast,
MBP(–)fibers (arrow) of ten expressed DG but usually not 4
(arrow). Laminin chains 2 and 4 were expressed in the
endoneurium of sciatic nerves (G–I) and in motor roots
(J–L). Laminin chains were present around, but not within,
bundles of unsorted axons (arrowhead). Laminin 1 was also detected
around axonal bundles in sciatic nerve (see Results). Asterisk identifies
spinal cord. Scale bar: (inL) A–D, 40 µm;
E, 2.5µm; F, 20µm; G–L,
80µm.
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To determine whether the levels of DG expression were regulated during
postnatal development, we performed Northern blot analysis of developing rat
sciatic nerve, from P2 to adulthood. Figure
4A shows that the steady-state levels of DG mRNA
increased between P10 and P15, in parallel with the increase of myelin mRNAs,
such as P0 (Fig. 4). Thus, the
perinatal appearance and rise of DG protein is likely transcriptionally
regulated during myelination, similar to 4 integrin
(Feltri et al., 1994).
To correlate the appearance of DG with myelination in a more synchronized
system, we stained rat SC–neuron cocultures (for review, see
Bunge, 1993) in which the onset
of myelination is triggered by the addition of ascorbic acid
(Bunge, 1993). No DG staining
was found when SCs were either cultured alone or cocultured with DRG neurons
in non-myelinating medium (Fig.
5A–C). Only 6 and 1 integrins
were detectable on SCs (data not shown). Staining for DG appeared in SCs 3 d
after addition of ascorbic acid, when a minority of fibers also stained for
MBP. Again, DG staining preceded and paralleled MBP expression and appeared
outside the MBP-stained myelin sheaths (shown at 7 d)
(Fig. 5D–F,
F'–F''', arrow). As we observed
in vivo, DG was polarized even in MBP-negative fibers
(F'', arrowhead). DG staining extended longitudinally in the
paranodal region (F''–F'''). The in
vivo and in vitro data indicate that DG expression immediately
precedes myelination in single fibers.
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Figure 5. Expression of -DG and 4 integrin in Schwann cell–neuron
cocultures. Primary rat Schwann cells were seeded onto cultures of dissociated
sensory neurons. Ascorbic acid was added at day 0 to promote myelination. At
day 0 (A–C, J–L), day 7
(D–F), and day 14 (G–I,
M–O), cultures were fixed and double immunostained for MBP
(A, D, G, J, M, F', O') and -DG (B, E,
H, F'') or 4 (K, N, O''). Merges are shown in
C, F, I, L, O, F''', and O'''). At day 0, MBP, DG,
and 4 were not expressed (A–C,
J–L). At day 7, MBP and DG were expressed in few fibers
(D–F). DG was expressed at the ab-axonal surface of few Schwann
cells, both in the absence of MBP staining or externally to MBP and in
paranodal regions (F'–F'''; arrow indicates
a DG + MBP + fiber; arrowhead indicates a DG + MBP – fiber). At day 14,
the number of fibers MBP + DG + (G–I) increased. 4
staining was diffuse and weak in MBP-negative fibers but more intense and
polarized in MBP-positive fibers (O'–O''',
arrow). Scale bar: (in O) A–O, 100 µm.
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Polarized expression of integrin 64 occurs after onset
of myelination
Next, we determined the onset of 4 integrin synthesis. At P1, we
observed a faint and diffuse expression of 4 protein in spinal roots and
peripheral nerves (Fig. 6). The
diffuse staining of 4 preceded myelination but did not correlate with
its onset, because there were no differences in 4 expression between
sensory and motor roots (Fig.
6A,D). 4 staining became stronger only after MBP
appeared in a single fiber and polarized outside the compact myelin sheath,
resembling its mature ab-axonal distribution
[Fig.
6G–I, compare left (') with right
('') magnified insets].
In vitro, SCs alone or cultured with DRG neurons under
non-myelinating conditions expressed negligible amounts of 4
(Fig.
5J–L). After adding myelin-promoting
medium, 4 integrin staining was low in MBP-negative fibers but stronger
and polarized in MBP-positive internodes
(Fig. 5M–O,
O'–O''') (and data not shown), in
agreement with the findings of Einheber and co-workers
(1993). Thus, these data
confirm the in vivo observation that both DG and 4 integrin are
expressed just before myelination, but 4, in contrast to DG, is not
polarized until myelin synthesis begins.
Expression of 2, 3, and 7 integrin
So far we focused on pure laminin receptors, but other laminin receptors
expressed by SCs include 11 integrin, a dual
collagen–laminin receptor expressed by non-myelin-forming SCs in mature
nerves (Stewart et al., 1997),
21 integrin (Hsiao et al.,
1991; Milner et al.,
1997), which binds collagen, laminin, or tenascin depending on the
cell type (Santoro, 1986;
Elices and Hemler, 1989;
Languino et al., 1989), and
71 integrin. We therefore looked at the expression of 2,
3, and 7 integrin during nerve development. We found that SCs
expressed very low levels of 2 and 3 integrin subunits, and
their expression did not change during development
(Fig. 7). 7 integrin
instead is synthesized by SCs in mature nerves
(Fig. 7I) but not in
fetal nerves (Fig.
7J–L) and appears 1 or 2 d after
myelination begins in postnatal nerves
(Previtali et al., 2003).
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Figure 7. Expression of 2, 3, and 7 integrin subunits in PNS
development. Cryosections of peripheral nerves of adult (A, E, I) and
E17.5 (B–L) mice were double labeled with
neurofilament (B, F, J) and anti-2 (A, C), 3
(E, G), or 7 (I, K) integrin antibodies. Low levels
of 2 and 3 staining are present in fetal and adult nerves,
whereas 7 integrin is absent in fetal SCs but robustly synthesized by
adult SCs. Scale bar: (in L) A–L, 50
µm.
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Compensation by laminins or their receptors
In adult 1-null sciatic nerves, most axons are grouped in large
unsorted bundles, resembling the bundles normally found in fetal nerves
(Fig. 8E)
(Martin and Webster, 1973;
Feltri et al., 2002) and in
roots of dystrophic mice (Bradley
and Jenkison, 1973). Interestingly, 1-null roots are less
affected than nerves (Feltri et al.,
2002), whereas dystrophic roots are more affected than
nerves. The differential expression of laminin-2 receptors could explain why
laminin-2 alterations produce variable effects across development. However,
diverse receptor function cannot account for the varying topography of the
phenotypes within laminin-2 and 1 integrin mutants (roots 
nerves)
and between them (roots worse in dystrophic; nerves worse in
1-null). To investigate this question, we searched for compensatory
expression of laminins or their receptors in dystrophic or
1-null roots and nerves.
No compensatory mechanisms protect prenatal SCs from the absence of
1 integrins
1-null nerves manifest more axonal sorting defects in nerves than
spinal roots; however, the expression of neither DG nor 4 integrin was
upregulated in either 1-null roots (data not shown) or nerves
(Fig.
8A–D) before birth, when axonal sorting
normally occurs. Not surprisingly, expression of 2, 3, and
7 integrin, which normally pair only with 1, is similarly not
upregulated before birth in 1-null nerves (data not shown). Thus,
compensation by these laminin receptors does not explain the difference
between 1-null nerves and roots.
Laminin receptors may cooperate during myelination
After birth in 1-null nerves, a few SCs achieve a one-to-one
relationship with axons and proceed to form myelin, albeit with delay. The few
SCs that myelinated axons were clearly 1-negative
(Fig. 8F)
(Feltri et al., 2002). To
explore the expression of DG and 4 integrin in promyelinating versus
myelinating 1-null Schwann cells, we first stained cross sections of
1-null nerves with neurofilament and then asked whether single axons
were surrounded by DG, 4 integrin, or MBP-positive SCs. Almost 70% of
single axons were surrounded by a DG-positive SCs (378 fibers double stained
with DG and NF out of 540 NF-positive fibers; data not shown). Using serial
sections in which DG-positive fibers were recognizable, we determined that
among the DG-positive fibers, 30% were MBP positive and 70% were MBP negative.
Interestingly, the MBP-positive fibers were always 4 positive
(Fig. 8F, 1–6),
whereas MBP-negative fibers were almost always 4 negative
(Fig. 8F, arrow). Only
1–2% of fibers DG positive and MBP negative were faintly 4
positive (data not shown). These data suggest that all three receptors
cooperate in myelination, because in the absence of 1, integrin
myelination is possible but delayed, and the presence of both 64
and DG correlates with myelination in 1-null SCs.
Laminins in 1-null nerves
Because 1 integrin, in association with DG, has been proposed to play
a role in BL organization in other tissues
(Henry et al., 2001), we
investigated the effects of 1 deletion on laminins. Normally, the
endoneurium contains laminin chains 2, low levels of 4, 1,
and 1, and the perineurium contains 1, 4, 5,
1, 2, and 1. Laminin chains 2, low levels of
4, and 1 were present in the endoneurium of 1-null nerves,
around single fibers and around bundles of nonsorted axons
(Fig. 8H,I) (and data
not shown). Instead, 1 and 2 laminin chains were present in the
perineurium but also in the endoneurium, primarily around bundles of unsorted
axons (Fig. 8G) (and
data not shown) where perineurial-like cells are abnormally present
(Feltri et al., 2002). No
laminins were detectable within the bundles of nonsorted axons
(Fig.
8G–I) (and data not shown). Thus, no
alteration in the organization of normal endoneurial laminins was detectable
at this resolution, other than the presence of perineurial laminins. In spinal
roots, laminin chains were present similarly to controls. Laminin chains
2, 4, and 1 were detected around SCs, whereas 1,
4, and 2 chains were detected in meninges
(Fig.
8J–L, compare
Fig.
9A–C) (and data not shown).
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|
Figure 9. Expression of laminins and laminin receptors in the PNS of
dy2J mice. Cryosections of motor root (A–K)
and the sciatic nerve (L–U) from wild-type
(A–C, L–N) or dy2J
mice (D–K, O–U) were immunostained for
laminin chain 1 (A, D, L, O), 2 (B, E, M, P),
4 (C, F, N, Q), and integrin subunit 6 (J, R),
1 (I, T), 4 (K, U), and -DG (H,
S). In both roots and sciatic nerve, the staining for 2 laminin
chain was reduced in dy2J as compared with controls
(compare B with E and M with P). The
staining for 4 laminin chain was similar in dy2J
and control mice. The 1 laminin chain was undetectable in spinal roots
in both dy2J and controls, whereas it was upregulated in
the sciatic nerve of dy2J mice (compare A with
D and L with O). Laminin receptors were normally
expressed in the endoneurium of dy2J mice
(H–K, R–U). V, RT-PCR analysis
of laminin 1 expression. Total RNA was extracted from either homozygous
(dy2J/dy2J) or heterozygous
(dy2J/+) dystrophic sciatic nerves and from
mouseplacenta (Pl) as positive control. cDNA was synthesized and amplified
with GAPDH-specific primers using 32P-dCTP. On the basis of
quantitative autoradiography for GAPDH product, equal amounts of cDNA template
were reamplified with primer pairs specific to laminin 1. Laminin
primers amplify the expected 201 nucleotide band in placenta and in
dystrophic nerves but not in heterozygous control nerves. Cycles of
amplification are indicated by 22, 24, 26, 28 and 30. W, Western blot
analysis of laminin 1 expression. Homogenates from mouse kidney
(K) as a source of laminin 1 and from either homozygous
(dy2J/dy2J) or heterozygous
(dy2J/+) dystrophic sciatic nerves were detected
by anti-laminin 1 antibodies, and anti-NF-M antibodies were used to
show equal loading of the nerve samples. Low levels of laminin 1 are
present in dystrophic nerves but not in heterozygous control nerves.
Scale bar: (in U) A–U, 50 µm.
|
|
Laminin receptors and laminins in dystrophic roots and
nerves
Dystrophic mice show failure of axonal sorting that is more severe
in the spinal roots than in distal PNS. This regional difference in severity
could be attributable to a differential function of laminin-2 in roots versus
nerves, e.g., via interaction with different receptors in the two locations.
To address this, we asked whether the expression of laminin receptors was
different in roots versus nerves of normal and dystrophic animals. We
found no differences in the expression of 6, 1, and 4
integrin or DG in spinal roots and peripheral nerves of both wild-type and
dy2J mice (Fig.
9G–J,Q–T) (and data not
shown). Although the activation state of the receptors, particularly in
dy2J mice, cannot be evaluated by this technique, our data
do not support the hypothesis that laminin has a different function in roots
versus nerves.
Increased laminin-1 in sciatic nerve but not roots of
dy2J mice
An alternative explanation for the regional differences observed in
dystrophic mice is that another laminin compensates for the lack of
laminin-2 in nerves but not roots of dystrophic mice. Thus, we
compared the expression of laminin chains in the roots and peripheral
nerves of normal and dy2J mice.
In the spinal roots and sciatic nerves of wild-type mice, we found
expression of laminin 2, with low levels of 4 in the endoneurium
(Fig. 9B,C,M,N).
Laminin 1 was restricted to the perineurium and meninges (Figs.
9A,L). In
dy2J mice, immunostaining of 2 chain was reduced,
as reported previously (Sunada et al.,
1995). Interestingly, 1 laminin chain was upregulated in
the endoneurium of sciatic nerves but not in roots of dy2J
mice (Fig. 9, compare A with
D, L with O). This upregulation was confirmed by
semiquantitative RT-PCR and Western blot analysis.
Figure 9, V and
W, shows that message and protein for 1 laminin
chain were detectable in dy2J homozygous but not
dy2J heterozygous sciatic nerves. Thus, compensation by
laminin receptors does not explain the difference between roots and nerves in
1-null animals, but compensation by laminins may well explain this
difference in dystrophic mice.
|
Discussion
|
|---|
Laminin-2 deficiency causes several alterations in PNS development,
probably mediated by interaction with different receptors in SCs. We show that
laminin receptors have a hierarchy of expression, positioning them as possible
downstream effectors of laminin-2 at different times. Also, we show that
laminin-1 may compensate for the deficiency of laminin-2 in distal nerves but
not roots of dystrophic mice. In contrast, redundancy and
compensation of laminin receptors play no role in prenatal development,
whereas cooperation among receptors may allow myelination postnatally. These
data are necessary to interpret the results of gene inactivation experiments
and to produce working hypotheses that such experiments can test.
Integrin 61 is the first laminin receptor expressed by
SCs
We show that 61 is the major laminin receptor expressed by
precursors and immature SCs, in addition to 21. The exclusive
expression of 1 integrins prenatally explains why SCs are incapable of
sorting axons in fetal 1-null nerves. Because this phenotype strongly
resembles the proximal PNS of dystrophic mice, binding of laminin-2
is likely required in early SC–axon interactions. Loss-of-function
experiments have not shown an obvious role for 1 and 7 integrins
in PNS and were not informative because of early lethality for integrin
2 (De Arcangelis and
Georges-Laboeusse, 2000;
Previtali et al., 2003). Thus,
61 integrin is likely the receptor involved in this process;
however, because many other partners such as 4 and 5
dimerize with 1 in SCs, we cannot exclude the possibility that receptors
for other extracellular matrix components are involved. Conditional
inactivation of 6 integrin in SCs will test this hypothesis.
DG and the onset of myelination
Myelination requires cytoskeletal rearrangements in SCs as they advance the
mesaxon, eliminate cytoplasm from spirals, and compact myelin. We observed
that DG appears in a SC just before initiation of myelination. Similarly to
myelin proteins, DG was detected first in motor and then in sensory fibers
(Fig. 3) (Baron et al., 1994; Forghani
et al., 2001). The polarized pattern of DG expression, with a single axon
within the DG ring, suggested that SCs express DG at the promyelinating stage.
Also, we show that during myelination, DG mRNA expression is upregulated.
These data extend previous reports on DG expression in mature SCs (Yamada et
al., 1994,
1996) and agree with recent
data by Masaki et al. (2002)
who reported continuous DG staining by immunoelectron microscopy beginning in
SCs in a one-to-one relationship with axons. It is possible that DG is
important for the onset of myelination. Indeed DG interacts with utrophin,
Dp116 (Saito et al., 1999),
DRP-2, and periaxin, and both periaxin mutations and conditional inactivation
of DG in SCs cause a dysmyelinating neuropathy
(Saito et al., 2003).
64 integrin distribution correlates with later events of
myelination.
64 integrin is required for the assembly of hemidesmosomes in
epithelial cells to stabilize a link between the cytoskeleton and the BL
(Nievers et al., 1999).
Several authors suggested a correlation between 64 expression and
myelination. 4 is polarized to the ab-axonal surface of SCs, parallels
myelination in vivo and in vitro, and is induced by axonal
contact in development and regeneration
(Einheber et al., 1993;
Feltri et al., 1994;
Quattrini et al., 1996).
4-negative SCs from null mice form rudimental myelin in SC–neuron
cocultures (Frei et al.,
1999), however, suggesting that 64 is not strictly
required for the onset of myelination. We show that shortly before
myelination, 4 staining was weak and diffuse, whereas after the
beginning of myelin synthesis it was stronger and polarized. Thus,
64 may stabilize the link-age of the BL to SCs by participating
in a specialized adhesion system similar to hemidesmosomes for epithelia. The
generation of mice null for 4 in SCs will address this.
Redundancy and compensation
Redundancy, the presence of molecules with similar function, or
compensation, the new expression of such molecules, may explain why
inactivation of genes for extracellular matrix and their receptors often
produces phenotypes less severe than expected
(Hynes, 1996). We investigated
redundancy and compensation in loss-of-function of laminin-2 and one of its
receptors. In the receptor mutant, 1-null, we previously described
defects in late fetal development. Here we show that only 61
integrin was normally present in SCs before birth, and there was no
compensation by earlier expression of the other receptors, accounting for the
severe defect. In contrast, we show that DG, 64, and
71 are coexpressed with 61 at various points after a
normal SC reaches the promyelinating stage and that 1-null SCs can
myelinate (with delay) when they express both DG and 64. Thus DG,
64, and possibly 61 and 71 probably
cooperate in the formation of a myelin sheath, even if their function in
myelination is partially redundant. Conditional inactivation of multiple
receptors will be necessary to test these hypotheses.
Second, we investigated the expression of laminins and laminin receptors in
dystrophic mice. Interestingly, we observed ectopic expression of
laminin chain 1 in the endoneurium of dystrophic nerves but
not in roots. This may explain the more severe sorting defect seen in roots
and proximal nerves of dy2J mice when compared with their
distal nerves, because new expression of laminin-1 may compensate for the
function of laminin-2 in axonal sorting. Our data on 4 laminin agrees
with those reported on roots but differs from data reported in nerves
(Patton et al., 1997;
Nakagawa et al., 2001), in
that we found a similar amount of 4 staining in the endoneurium of
normal nerves and dystrophic mice. Possibly this difference is
attributable to the different districts examined (roots and sciatic nerves in
our study vs intramuscular nerves in their studies) or the different allelic
variants of dystrophic mice (dy2J/dy2J
in our study vs dy/dy and dy3k/dy3k in
their studies).
BL assembly in SCs
Polymerization of laminin triggers assembly of the BL and induction of a
matrix–receptor– cytoskeletal network that activates signaling
(Colognato et al., 1999).
Interaction between laminin and its receptors facilitates polymerization
(Colognato and Yurchenco,
2000), and specific receptors, such as DG and 1 integrins,
facilitate BL assembly, either directly
(DiPersio et al., 1997;
Henry and Campbell, 1998;
Sasaki et al., 1998;
Henry et al., 2001) or through
the induction of laminin itself (Li et
al., 2002). Tsiper and Yurchenko
(2002) reported that DG is
reorganized with laminin during assembly of BL of SCs in vitro,
whereas 1 integrin mediates the formation of a fibrillar matrix
organization, distinct from a BL and before real BL assembly. Although the SCs
used in their study are removed from the physiological state of SCs in nerves
(high passage number, absence of axons), these results are consistent with our
observations and previous in vivo observations. In vivo, a
discontinuous BL first appears before birth around "families" of
SCs that surround bundles of axons
(Ziskind-Conhaim, 1988;
Masaki et al., 2002), whereas
a continuous basal lamina appears only at birth
(Webster et al., 1973). We
show that only 1 integrins are present in SCs before birth, suggesting
that they participate in the deposition of the immature BL at the time of
axonal sorting. Consistent with this, sorting is impaired in1-null
nerves; however, those 1-null SCs that generate myelin form an
apparently normal BL. Therefore, 1 is not required for mature basal
lamina formation. Instead, expression of DG first, and then recruitment of
64, at P1 may facilitate the formation of the mature BL during
myelination. In support of this, we show that DG and 64 onset
coincide with the appearance of a mature basal lamina, that DG is first
polarized to the outer surface of single SCs just before myelination, and that
4 integrin polarization follows the expression of DG and MBP. In
conclusion, we postulate that 1 favors the deposition of the immature BL
in families of premyelinating SCs, whereas DG organizes, and recruitment of
64 and 71 stabilizes, the mature BL in
promyelinating and myelinating SCs.
|
Footnotes
|
|---|
Received Jul. 22, 2002;
revised Apr. 9, 2003;
accepted Apr. 14, 2003.
This work was supported by Telethon Grants D93 (M.L.F.) and 1177 (L.W.),
Progetto Finalizzato Ministero della Sanitá (M.L.F., L.W.), Multiple
Sclerosis Society of Great Britain (L.W.), Fondazione Italiano Sclerosi
Multipla (L.W.), National Institutes of Health Grants NS 41319 (L.W.) and
NS45630 (M.L.F.), and Amici Centro Sclerosi Multipla (S.C.P., A.Q.). We thank
C. Ferri and M. Fasolini for excellent technical assistance, Dr. G. Zanazzi
for help with cocultures, Alembic for confocal microscopy, and Drs. A.
Sonnenberg (Netherlands Cancer Institute, Amsterdam, The Netherlands), V.
Quaranta (The Scripps Research Institute, La Jolla, CA), V. Lee (University of
Pennsylvania Medical School, Philadelphia, PA), K. Rubin (Uppsala University,
Uppsala, Sweden), S. J. Kennel (Oak Ridge National Laboratory, Oak Ridge, TN),
F. Giancotti (Memorial Sloan-Kettering Cancer Center, New York, NY), L.
Sorokin (University of Erlangen-Nuremberg, Erlangen, Germany), J. H. Miner
(Washington University School of Medicine, St. Louis, MO), U. Majer (School of
Biological Sciences, Manchester, UK), and K. Campbell (Howard Hughes Medical
Institute, University of Iowa, Iowa City, IA) for the gift of antibodies.
Correspondence should be addressed to M. Laura Feltri, San Raffaele
Scientific Institute, DIBIT, Via Olgettina 58, 20132 Milan, Italy. E-mail:
feltri.laura{at}hsr.it.
Copyright © 2003 Society for Neuroscience
0270-6474/03/235520-11$15.00/0
|
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Top
Abstract
Introduction
Gene
