* Department of Anatomy and Neurobiology, Department of Internal Medicine, Washington University School of Medicine, St.
Louis, Missouri 63110; and § Division of Neuroscience, Beckman Research Institute of the City of Hope, Duarte, California 91010
Laminins, heterotrimers of ,
, and
chains, are prominent constituents of basal laminae
(BLs) throughout the body. Previous studies have
shown that laminins affect both myogenesis and synaptogenesis in skeletal muscle. Here we have studied the
distribution of the 10 known laminin chains in muscle
and peripheral nerve, and assayed the ability of several
heterotrimers to affect the outgrowth of motor axons.
We show that cultured muscle cells express four different
chains (
1,
2,
4, and
5), and that developing
muscles incorporate all four into BLs. The portion of
the muscle's BL that occupies the synaptic cleft contains at least three
chains and two
chains, but each
is regulated differently. Initially, the
2,
4,
5, and
1
chains are present both extrasynaptically and synaptically, whereas
2 is restricted to synaptic BL from its
first appearance. As development proceeds,
2 remains
broadly distributed, whereas
4 and
5 are lost from
extrasynaptic BL and
1 from synaptic BL. In adults,
4 is restricted to primary synaptic clefts whereas
5 is
present in both primary and secondary clefts. Thus,
adult extrasynaptic BL is rich in laminin 2 (
2
1
1),
and synaptic BL contains laminins 4 (
2
2
1), 9 (
4
2
1), and 11 (
5
2
1). Likewise, in cultured muscle cells,
2 and
1 are broadly distributed but
5 and
2 are concentrated at acetylcholine receptor-rich "hot spots," even in the absence of nerves. The endoneurial
and perineurial BLs of peripheral nerve also contain
distinct laminin chains:
2,
1,
1, and
4,
5,
2,
1,
respectively. Mutation of the laminin
2 or
2 genes in
mice not only leads to loss of the respective chains in
both nerve and muscle, but also to coordinate loss and compensatory upregulation of other chains. Notably,
loss of
2 from synaptic BL in
2
/
"knockout" mice is
accompanied by loss of
5, and decreased levels of
2
in dystrophic
2dy/dy mice are accompanied by compensatory retention of
4. Finally, we show that motor axons respond in distinct ways to different laminin heterotrimers: they grow freely between laminin 1 (
1
1
1) and laminin 2, fail to cross from laminin 4 to
laminin 1, and stop upon contacting laminin 11. The
ability of laminin 11 to serve as a stop signal for growing axons explains, in part, axonal behaviors observed
at developing and regenerating synapses in vivo.
IN skeletal muscles, a continuous sheath of basal lamina
(BL)1 surrounds each muscle fiber and passes through
the synaptic cleft at the neuromuscular junction. Thus,
most of the BL separates the muscle fiber membrane from
interstitial connective tissue, whereas a small fraction
(~0.1%) separates the muscle from the nerve. These extrasynaptic and synaptic portions of the BL play several
important roles in the development and function of the muscle and the neuromuscular junction, respectively. Components of extrasynaptic BL regulate myogenesis in embryos, contribute to tensile strength in adults, and serve as
a scaffold to orient regenerating myotubes after muscle
damage. Components of synaptic BL organize differentiation of the pre- and postsynaptic membranes in embryos,
inactivate neurotransmitter in adults, and guide reinnervation after nerve damage (for reviews see Sanes, 1994 Key molecules in these processes are the laminins, glycoproteins that are major components of BLs in all tissues.
Laminin was initially isolated from tumor-derived matrix
as a trimer of Despite the strong evidence that laminins are crucial for
neuromuscular development, it has been difficult to elucidate their precise roles for several reasons. First, additional laminin chains and a total of 11 Table I.
Laminin Isoforms in Vertebrates
, 1995
).
1,
1, and
1 chains (formerly A, B1, and B2;
Chung et al., 1979
; Timpl et al., 1979
; Burgeson et al., 1994
).
A homologue of the
1 chain, originally called merosin
and now renamed
2, was subsequently isolated from
muscle and shown to be a major component of extrasynaptic BL (Lievo and Engvall, 1988; Ehrig et al., 1990
). Similarly,
2 (originally s-laminin) was identified as a component of synaptic BL (Chiu and Sanes, 1984
; Hunter et al.,
1989a
). Laminins containing the
2 chain are adhesive for
myoblasts (Schuler and Sorokin, 1995
), and recombinant
laminin
2 fragments regulate outgrowth of motor axons
(Hunter et al., 1989b
; Porter et al., 1995
). Thus, based on
their distribution in vivo and their effects in vitro, the
2
and
2 chains were hypothesized to be involved in myogenesis and synaptogenesis, respectively. Recent genetic
analyses in mice have provided strong support for these
hypotheses: targeted mutation of the laminin
2 gene
leads to aberrant structural and functional maturation of
neuromuscular junctions (Noakes et al., 1995a
), and a naturally occurring hypomorphic allele of laminin
2 (
2dy/dy)
gives rise to severe muscular dystrophy (Sunada et al.,
1994
; Xu et al., 1994b
). Some cases of human familial muscular dystrophy have also been shown to result from mutation of the laminin
2 gene (Hayashi et al., 1993
; Helbling-Leclerc et al., 1995
; Sunada et al., 1995
; Nissinen et al., 1996
).
heterotrimers
(laminins 1-11; Table I) have now been identified in vertebrates. The distribution of the new chains in muscle has
not yet been reported, so the identity of the laminin trimers in synaptic and extrasynaptic BL remains unclear. Second, mutation of a single laminin or collagen IV chain gene leads to coordinate loss of some chains and compensatory retention of others in kidney BLs (Kashtan and Kim,
1992
; Gubler et al., 1995
; Noakes et al., 1995b
; Cosgrove et
al., 1996
; Miner and Sanes, 1996
). Similar interactions
might occur in muscle, complicating analyses of the
2dy/dy
and laminin
2
/
phenotypes. Third, laminins
2 and
2
are present in the BLs of peripheral nerve as well as muscle
(Sanes et al., 1990
), so mutant phenotypes might reflect
both neurogenic and myogenic defects. Finally, functional
studies of laminin
2 have been limited to recombinant
fragments (Hunter et al., 1989b
; Porter and Sanes, 1995
;
Porter et al., 1995
) and a single heterotrimeric form (laminin 4; Brandenberger et al., 1996
). It may be inappropriate
to extrapolate from the activities of these preparations to
those of synaptic laminins.
To address these issues, we have analyzed the expression of all 10 known laminin chains in developing and
adult muscles and nerves of wild type, 2dy/dy , and
2
/
mice. We have also documented expression of putative
synaptic laminins by cultured muscle, and assayed the effects of several laminin trimers on the outgrowth of motor
axons. We show that the laminin isoforms of synaptic,
extrasynaptic, and nerve BLs change as development proceeds. In adults, the
4,
5, and
2 chains are all concentrated in synaptic BL, but they are distributed and regulated in different ways, and could form three distinct trimers
(laminins 4, 9, and 11). Both the
5 and the
2 chains are
lost from synaptic sites in
2 mutants, which display severe synaptic defects, but both are retained in
2 mutants, in
which synaptic defects are mild. Moreover, laminin 11 (
5
2
1) serves as a potent stop signal for motor axons in
vitro whereas laminin 4 (
2
2
1) does not. Together, these
results focus attention on laminin 11 as a critical organizer
of synaptic development.
Animals
Mice deficient in laminin 2 and littermate controls were generated and
genotyped as described by Noakes et al. (1995a)
. When maintained on
high-fat rodent chow after weaning, the
2
/
mutants do not gain weight
but do live until P28-P35. Mice homozygous for a mutation in the laminin
2 gene (Lama2dy/dy) and littermate controls were purchased from Jackson Laboratories (C57BL/6J-Lama2dy/dy; Bar Harbor, ME). Embryos
were taken from timed pregnant ICR or C57BL6 mice, bred in our colony.
Antibodies
Monoclonal antibodies to rat laminin 1 (C21 and C22),
2 (D5, D7, D19,
and D27), and
1 (D18) chains, rabbit antisera to recombinant laminin
4
and
5 chains, and a guinea pig antiserum to laminin
2 were produced in
this laboratory and have been described previously (Sanes and Chiu, 1983
;
Hunter et al., 1989a
; Sanes et al., 1990
; Green et al., 1992
; Miner et al.,
1997
). Rat monoclonal antibodies to mouse laminin
1 (clones 198 and
200; Sorokin et al., 1992
) were gifts from L. Sorokin (Institute for Experimental Medicine, Erlangen, Germany). Rabbit antiserum to human laminin
2, which cross-reacted with the mouse protein, was provided by P. Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ; see
Miner et al., 1997
). Rabbit antiserum to mouse laminin
3 (Aberdam et
al., 1994
) was a gift from D. Aberdam (Institut National de la Sante et de
la Recherche Medicale [INSERM] U385, Nice, France). Rabbit antiserum
to laminin-5 (
3
3
2) was a gift of R. Burgeson (Massachusetts General
Hospital, Charlestown, MA). A rat monoclonal antibody to laminin
1
(5A2; Abrahamson et al., 1989
; Martin et al., 1995
) was a gift from D. Abrahamson (University of Alabama, Birmingham, AL). Rat anti-mouse
laminin
1 was purchased from Chemicon International, Inc. (Temecula,
CA). FITC- and HRP-conjugated, goat anti-rabbit antibodies were from
Boehringer Mannheim Corp. (Indianapolis, IN); FITC-conjugated, goat
anti-rat antibodies were from Cappel/Organon Teknika (Durham, NC);
Cy3-goat anti-rabbit antibodies were from Jackson Immunoresearch Laboratories (West Grove, PA); biotinylated, goat anti-guinea pig antibodies
were from Sigma Chemical Co. (St. Louis, MO); and HRP-avidin was
from Zymed Labs Inc. (South San Francisco, CA).
All results on laminin 1 were confirmed with two monoclonal antibodies, 198 and 200, which react with distinct epitopes (Sorokin et al.,
1992
). All results on laminin 5 (
3
3
2) were obtained using an antibody
that recognizes all three chains (Marinkovich et al., 1992
), and were confirmed using an antibody specific for the
3 chain, which binds an epitope
present in both
3A and
3B isoforms (Aberdam et al., 1994
; Miner et al.,
1997
). To date,
3 and
2 have been found only in association with
3
(Table I), so absence of
3 provides indirect support for absence of
3
and
2. These antibodies stained a subset of lung BLs intensely in our
hands (data not shown). All results on laminins
4 and
5 were confirmed
with two separately generated rabbit antisera to each chain; the specificity
of these sera has been documented previously (Miner et al., 1997
).
Laminin Heterotrimers
Laminin 1 (1
1
1) from the mouse EHS tumor matrix, and laminin 2 (
2
1
1) from human placenta, were purchased from GIBCO BRL/Life
Technologies (Gaithersburg, MD). Purified laminin 4 (
2
2
1) and a second sample of laminin 2, both purified from human placenta, were generous gifts of Y.-S. Cheng and P. Yurchenco (Robert Wood Johnson Medical School; Cheng et al., 1997). Laminin 11 (
5
2
1) was purified from the
conditioned medium of rat Schwannoma D6P2T cells by a modification of
the method described by Chiu et al. (1992)
. By immunoblotting using antibodies described above, the "laminin 11" fraction was found to be rich in
5,
2, and
1, but to contain little or no
1,
2,
3,
4, or
1.
Neuronal Cultures
To generate patterned substrates, plastic culture dishes were first coated
with a thin layer of nitrocellulose (type BA85; Schleicher and Schuell, Keene,
NH) (Lagenaur and Lemmon, 1987). A pattern was then formed by applying ~1-µl drops of test proteins in PBS supplemented with 2 mM EDTA
and 1 mg/ml sulforhodamine-101 (Sigma Chemical Co., St. Louis, MO).
After incubation in a humidified chamber for 1-5 h at room temperature,
dishes were flushed repeatedly to remove unbound material, and then
coated for 2 h at room temperature with laminin 1 (20 µg/ml in PBS;
GIBCO BRL) to support cell attachment and initiate neurite outgrowth.
Dishes were rinsed with PBS or PBS/BSA and used immediately for neurite outgrowth assays.
Chick ciliary ganglia were dissected from E8-9 embryos, digested in
0.05% trypsin in Ca2+/Mg2+-free HBSS for 15 min at 37°C, and dissociated
by trituration. Cells were washed in MEM (No. 11095; GIBCO BRL) containing 10% FCS (Hyclone, Logan, UT), and resuspended in MEM supplemented with 25 mM Hepes, 7% heat inactivated horse serum (Hyclone),
3% FCS, 1 mM glutamine, 1 mM glucose, penicillin, and streptomycin. Aliquots of 30 µl containing 0.2 ganglion equivalent (~800 neurons) were
suspended from culture dish lids in a 37°C, 7% CO2 incubator. Cell clusters formed in 3-6 h within the hanging drops. The clusters were then
transferred to culture dishes and positioned near patches of test substrates. Neurons were grown for 30-36 h in culture medium containing 1%
chick eye extract (Nishi and Berg, 1981), and then fixed with formaldehyde.
For analysis, cultures were viewed with phase optics to visualize neurites and rhodamine optics to visualize the substrate border. Two categories of neurites were counted. Group A were those that extended on laminin 1 and terminated on or near the substrate border, in a swath extending
from 50 µm onto the laminin 1 to 10 µm over onto the test substrate.
Group B were neurites that began on laminin 1 and extended greater than
10 µm beyond the border. Neurites initiated on the test substrate, and
neurites that did not approach a border were not scored. Inhibition of crossing was calculated as (A [A + B]).
Because this assay was designed to test the behavior of neurites in response to various laminins, it was crucial to rule out the possibility that
test substrates were acting indirectly by reducing the concentration of
laminin 1 available to the neurons. To this end, we stained patterned substrates with antisera specific for laminin 1, and found that laminin 1 immunoreactivity was not detectably decreased in regions that had previously
been spotted with BSA solutions of 1 mg/ml or laminins 2, 4, or 11 at 40 µg/ml. Biological effects reported below for laminins 4 and 11 were observed at <40 µg/ml.
Muscle Cell Culture
The RMo rat muscle cell line (Merrill, 1989) was cultured in F10 medium
containing 15% FCS and 3% chick embryo extract. Nearly confluent cultures were induced to differentiate and form myotubes by medium replacement with DME containing 4% horse serum. After 6 d a soluble 95-kD
fragment of rat agrin (Ferns et al., 1993
) was added to cultures for 24 h to
promote clustering of acetylcholine receptors (AChRs) and associated
proteins. C2 mouse muscle cells were cultured as described in Martin et al.
(1995)
.
Immunohistochemistry
Freshly frozen tissues were sectioned in a cryostat at 4-8 µm and fixed
with 2% paraformaldehyde in PBS for 10 min. Fixed sections were
blocked with 0.1 M glycine in PBS (10 min), and incubated overnight at
4°C with antibodies diluted in a solution of 2% BSA and 0.1% (wt/vol) saponin (Sigma Chemical Co.) in PBS. After washing, bound antibodies were
detected with species-specific, fluorochrome-conjugated secondary antibodies, and then washed, mounted in glycerol containing p-phenylenediamine, and observed with epifluorescent illumination. Where appropriate,
rhodamine--bungarotoxin (50 nM; Molecular Probes, Eugene, OR) was
included with the second antibodies, to label AChRs. Rabbit antilaminin
4 and guinea pig anti-
2 recognized only denatured antigen (Miner et al.,
1997
), so sections to be labeled with these antibodies were pretreated with
0.05% SDS in PBS at 50°C for 20 min. AChR were labeled in denatured
sections by incubating sections in rhodamine-
-bungarotoxin both before
fixation and after denaturation.
Immunoblotting and Immunoprecipitation
Samples were heated to 95°C for 5 min in SDS-PAGE sample buffer, with or without the reducing agent DTT, then subjected to SDS-PAGE on 7% (reduced) or 3.5% (nonreduced) gels. Proteins were transferred to nitrocellulose membranes (Schleicher and Schuell) in 25 mM Tris, pH 9.5, 130 mM glycine, 0.1% SDS, and 10% (vol/vol) methanol at 320 mA for 24 h at 4°C. Positions of major bands were visualized with Ponceau S and marked. After blocking with a solution of 5% nonfat dry milk (Schnucks, St. Louis, MO) and 0.3% Tween-20 in PBS, filters were cut into strips and incubated with antibodies overnight. Bound mouse and rabbit antibodies were detected with HRP second antibodies and chemiluminescent substrates (Renaissance; DuPont-NEN, Boston, MA). Guinea pig antibodies were detected with biotinylated second antibody and HRP-avidin.
For immunodepletion, aliquots of a protein A-Sepharose CL-4B conjugate (Pharmacia Biotechnology Inc., Piscataway, NJ) were loaded with
either antilaminin 5 or preimmune serum (1.5 µl/µl resin), blocked with
2 mg/ml BSA (immunoglobulin-free; Sigma Chemical Co.), and then
washed extensively with PBS plus 2 mM EDTA. Samples of laminin 11 were then added (0.25 µg/µl resin) and incubated 6 h or overnight at room
temperature. The supernatant was withdrawn and tested for effects on
neurite outgrowth as described above.
Diversity of Laminin Chains in Adult Muscle and Nerve
We first asked which of the 10 known laminin chains (1-5,
1-3,
1, and
2) were present in the BL that ensheathed
adult mouse muscle fibers. Antibodies to the
2,
1, and
1 chains intensely stained this BL (Fig. 1, a, b, and e). In
contrast,
1,
3,
3, and
2 were undetectable in muscle
(Fig. 1, d and f). The
4,
5, and
2 chains were also undetectable in extrasynaptic BL (Fig. 1, c, g, and h), although
they were present at synaptic sites, as detailed below.
Thus, consistent with previous reports (Engvall et al., 1990
;
Sanes et al., 1990
; Vachon et al., 1996
) and subject to caveats discussed below (see Discussion), the predominant
laminin in adult muscle fiber BL appears to be the
2
1
1
heterotrimer, laminin 2 (Table I).
We also examined intramuscular nerves, which are almost invariably present in sections of skeletal muscle. Such
nerves contain two distinct types of BL. One is the multilammelar perineurial BL that coats the fibroblast-derived
perineurium, which in turn surrounds fascicles of Schwann
cell axon units (Bunge et al., 1989). The second is the endoneurial BL that surrounds individual Schwann cells,
which in turn ensheathe one myelinated or several unmyelinated axons. Perineurial BL was rich in laminins
4,
5,
2, and
1, and was devoid of detectable
1-3,
1,
3, or
2. Endoneurial BL, in contrast, was rich in
2,
1, and
1
but contained little or no
1,
3-5,
2,
3, or
2 (Fig. 2).
Thus, the predominant laminin of endoneurial Schwann
cells, like that of muscle fibers, is likely to be laminin 2, whereas perineural BL contains laminins 9 and 11.
Finally, three distinct vascular BLs were readily identifiable in muscle: those of capillaries, arterioles, and venules.
Capillary BL contained laminins 4,
5,
1, and
1 but
not
1-3,
3, or
2;
2 was detected with some but not all
antibodies, as previously described in rat muscles (Sanes
et al., 1990
). Arteriolar BL contained
5,
2, and
1, but
little
4 and no detectable
1-3,
1,
3, or
2. Venous BL
contained
1 instead of
2 but was otherwise similar to arteriolar BL (Fig. 1; and data not shown). Thus, capillary BL
is likely to be rich in laminins 8 and 10, arteriolar BL in laminin 11 and venous BL in laminin 10; capillary BL may
also contain laminins 9 and 11.
Differential Distribution of Laminin Chains in Synaptic BL
Three BLs with distinct cellular origins are joined at the
edge of the neuromuscular junction (Fig. 3 a): extrasynaptic BL (produced by muscle cells and fibroblasts), Schwann
cell BL (produced by Schwann cells and fibroblasts), and
the BL of the synaptic cleft (produced by nerve and muscle) (Sanes, 1994, 1995
). Moreover, ultrastructural studies
of membrane and cytoskeletal proteins have defined two
distinct domains within the synaptic cleft; AChRs, rapsyn,
and utrophin are concentrated in what are called primary clefts, at the crests of junctional folds, whereas neural cell adhesion molecule (N-CAM), sodium channels, and ankyrin
are concentrated along the sides of the folds in secondary
clefts (Fertuck and Salpeter, 1976
; Covault and Sanes,
1986
; Flucher and Daniels, 1989
; Bewick et al., 1992
). It
therefore seemed possible, but had not been shown, that
BLs of the primary and secondary clefts were molecularly
distinct as well.
We assessed the distribution of laminins in synaptic regions by using confocal microscopy to examine sections
double labeled with chain-specific antibodies to laminins plus
rhodamine--bungarotoxin, which binds to AChRs at the
crests of junctional folds. This single counterstain allowed
us to resolve all four domains in synaptic and perisynaptic
BL, as is evident in micrographs of sections stained for
1,
which is present throughout the BL (Fig. 3, c and j
; see
C24 antigen in Sanes and Chiu, 1983
). The BL of the primary synaptic cleft is seen as a fine line adjacent and external to the AChR-rich domain. BL extending into the depths
of the folds appears as fine struts that run ~1 µm from the
crests toward the interior of the muscle fiber (Fig. 3 j
).
Stretches of BL lateral to the AChR-rich region are extrasynaptic. Finally, Schwann cell BL is a few microns external to the AChR-rich crest.
Three laminin chains (
2,
4, and
5) were present at
synaptic sites, but each had a distinct distribution. The
2
chain was codistributed with
1, being present in the extrasynaptic, primary cleft, junctional fold, and Schwann cell
BLs (Fig. 3, c and g). The
4 chain was present in Schwann
cell and primary cleft BLs, but was absent from extrasynaptic BL and from the BL of junctional folds (Fig. 3 i). In
contrast,
5 was present in both primary cleft and junctional fold BLs, but was absent from extrasynaptic and
Schwann cell BLs (Fig. 3 k).
1 was present in extrasynaptic and Schwann cell but not synaptic BLs (Fig. 3 d; see
Sanes et al., 1990
), whereas
2, like
5, was present in crest and fold BLs (Fig. 3 e). Laminins
1,
3,
3, and
2
were undetectable (Fig. 3, f and h; and data not shown).
To confirm these localizations, we used species-specific
second antibodies to compare the distributions of pairs of
laminin chains. For example, Fig. 3 j shows a section double
labeled with antibodies to 4 and
1. Codistribution of the
two chains in primary cleft and Schwann cell BLs is evident,
as is the extension of
1 into the
4-free junctional folds.
Double labeling for
2 and
5 confirmed that these two
chains are codistributed throughout synaptic BL (Fig. 3 l).
Together, these observations reveal that each of the four
BLs that abut synaptic sites bears a distinct complement of
laminin chains: 2,
1, and
1 extrasynaptically (laminin
2);
2,
4,
5,
2, and
1 in the primary cleft (laminins 4, 9, and 11);
2,
5,
2, and
1 in the BL of junctional folds
(laminins 4 and 11); and
2,
4,
1, and
1 in the BL that
covers Schwann cells (laminins 2 and 8; Fig. 3 b).
Laminin Isoform Transitions in Developing Muscle
In some tissues, it has been shown that the complement of
laminin chains in individual BLs changes during development (Jaakkola et al., 1993; Miner and Sanes, 1994
; Virtanen et al., 1995
, 1996
; Miner et al., 1997
). We therefore
asked whether the laminin chains present when muscles
and neuromuscular junctions are forming differed from
those present in adult synaptic and extrasynaptic BL. We used intercostal muscles for this study because myogenesis, synaptogenesis, and BL formation have all been intensively studied in these muscles (see Kelly and Zacks, 1969a
,
b
; Chiu and Sanes, 1984
; Rosen et al., 1992
).
Intercostal myoblasts begin fusing to form an initial cohort of myotubes, called primary myotubes, on embryonic
days (E) 10 and 11, and the myotubes soon begin to assemble a BL. By E 11.5, numerous patches of BL are present
on myotube surfaces, but a continuous lamina is not yet
present (see Rosen et al., 1992 for references). These
patches of BL contained
2,
5,
1, and
1 chains (Fig. 4,
a, c, and f; and data not shown). The
3,
4,
2,
3, and
2
chains were undetectable (Fig. 4, d and e; and data not
shown). Thus, the BL of newly formed myotubes contains laminin 10, as well as the adult trimer, laminin 2.
Interestingly, 1 was also present in muscle at E 11.5, but
was largely restricted to the ends of myotubes, in regions
abutting the ribs (Fig. 4 b). Double labeling with anti-
1
plus anti-N-CAM (a marker of myogenic cells; Sanes et al.,
1986
; Rosen et al., 1992
) confirmed that the
1 was associated with muscle (rather than with tendons or cartilage; data
not shown), and double labeling with anti-
1 plus anti-
2
confirmed that
1 was associated with
2-containing BLs
(Fig. 4, b and c). Because myogenesis is believed to proceed in large part at the ends of fibers (Zhang and McLennan, 1995
), we speculate that laminin
1 may be expressed
by myoblasts just as they fuse with myotubes.
BL deposition around primary myotubes continues between E 11 and 13. In addition, a second cohort of myotubes, called secondary myotubes, begins to form on E 14. By E 15, most myotubes bear a continuous BL sheath. As
on E 11.5, this BL was rich in laminins 2,
5,
1, and
1,
and
1 remained confined to the ends of myotubes (Fig. 4,
g-i, and l; and data not shown). In contrast, levels of
4
increased dramatically after E 11.5, and this chain was present throughout all myotube BLs by E 15 (Fig. 4 k).
Thus, laminin 8 appears to join laminins 2 and 10 as primary myotubes mature and secondary myotubes form.
Synaptogenesis begins in intercostal areas at E 13, and
rudimentary neuromuscular junctions are readily detectable by E 14 (Kelly and Zacks, 1969b; Noakes et al., 1993
).
To assess early stages in the formation of synaptic BL, we
double-labeled sections of E 15 intercostal with antilaminins plus rhodamine-
-bungarotoxin. The
2,
4,
5 and
1 subunits were all present in synaptic as well as extrasynaptic areas. Interestingly,
2,
4, and
1 appeared to be
enriched in synaptic BL, but
5 did not (Fig. 4, m-r). As reported previously for rat intercostals (Chiu and Sanes,
1984
), the
2 chain appeared soon after AChR clusters
formed, and was restricted to synaptic sites at all stages,
whereas
1 was present both synaptically and extrasynaptically at early stages of synaptogenesis (data not shown;
note that the C1/C4 and C21/C22 antigens studied by Chiu
and Sanes, are now known to be laminins
2 and
1, respectively [Sanes et al., 1990
]).
Further changes in the composition of muscle BL occurred perinatally as myotubes matured into myofibers.
Extrasynaptic levels of 4 and
5 were markedly lower at
birth than at E 15, and neither subunit was detectable by
the end of the first postnatal week. At the synapse, the intensity of
5 staining rose postnatally, while levels of
4
remained modest, and the
1 subunit was gradually lost
from these regions. The
2 subunit remained confined to
synaptic sites, and levels of
2 and
1 remained high both
synaptically and extrasynaptically throughout development
(data not shown). We also noted one developmental change
in the BL of intramuscular nerves during this period: in
embryos and during the first 2 postnatal wk, endoneurial
BL contained
4 as well as
2, whereas adult endoneurium contained only
2 (data not shown; compare Figs. 2 g
and 8 k).
In summary, the laminin chain composition of both extrasynaptic and synaptic BL changes during development,
but in different ways. The 2 and
1 chains are present in
both domains at all stages of development;
4 and
5 are
initially present throughout the BL, and then lost from extrasynaptic BL; the
1 chain is initially ubiquitous, and then
lost from synaptic BL; and
2 is confined to synaptic sites
from its first appearance. From the perspective of deduced
trimeric structure, laminin 2 predominates extrasynaptically at all stages, but is joined transiently by laminin 1 near
the ends of fibers and by laminins 8 and 10 throughout the
fiber length. Synaptically, the embryonic presence of
1 suggests that the
1-containing trimers (laminins 2, 8, and 10)
are present initially but then lost, whereas the
2-containing trimers (laminins 4, 9, and 11) appear slightly later and
are retained.
Production of Laminins 4 and
5 by Muscle Cells
To understand how laminins act, it is important to know
which cells make them. This issue is of particular importance for synaptic BL, which is known to contain contributions from both muscle and nerve (Sanes, 1995), and might
also contain products of Schwann cells. Myogenic cells are
known to synthesize the laminin
1,
2,
1,
2, and
1
chains (Green et al., 1992
; Kroll et al., 1994
; Schuler and
Sorokin, 1995
; Vachon et al., 1996
), but synthesis of
4 or
5 by muscle has not yet been reported. To address this issue, we used the rat muscle cell line, RMo (Merrill, 1989
). We have previously shown that RMo cells express
1,
2,
and
1 chains, and hitherto unidentified
-like chains (Green
et al., 1992
). Here, we asked whether the
-like chains corresponded to
4 or
5.
Proteins of RMo myotubes were separated by SDS-PAGE, and then immunoblotted. Antisera to laminin 4
recognized a protein of ~200 kD, and antisera to
5 recognized a protein of ~400 kD (Fig. 5 a, lanes 3 and 4).
These apparent molecular weights were similar to those
obtained previously from rat lung and kidney tissue extracts immunoblotted with these same antisera (Miner et al.,
1997
). Antiserum to laminin 1 recognized bands of ~400,
~220, and ~200 kD (Fig. 5 a, lane 2), presumably representing the
1,
1, and
1 chains, respectively. As expected, anti-
2 bound to a protein of ~190 kD (Fig. 5 a,
lane 5). Non-immune serum showed no reaction with any
of the laminin chains (Fig. 5 a, lane 1). Thus, muscle cells synthesize not only the laminin
1,
2 (see below),
1,
2,
and
1 chains, but also
4 and
5.
We and others have previously shown that myotubes
can differentially localize laminin chains in the absence
of nerves:
1 is broadly distributed throughout the myotube BL, whereas
2 is largely restricted to small patches
of BL that cover AChR-rich domains ("hot spots") of the
plasma membrane (Silberstein et al., 1982
; Sanes and
Lawrence, 1983
; Martin et al., 1995
). Here, we used RMo
cells to ask whether the ability to differentially distribute
chains is also cell autonomous, or whether it requires nerves and/or Schwann cells. Unfortunately, available anti-
4 sera
require antigen denaturation, which turned out to be incompatible with determination of surface localization. However, extracellular deposits of laminins
5 and
2 were
both clearly concentrated in AChR-rich regions (Fig. 5, b
and c). Double labeling with anti-
5 and anti-
2 showed
that the two chains were generally codistributed (Fig. 5 d).
In contrast, the
2,
1, and
1 chains were broadly distributed on the myotube surface (Fig. 5, e and f; and data not
shown). Thus, myotubes can not only synthesize multiple
laminin
chains, but also differentially distribute them.
Moreover, the nerve is not necessary for the selective association of laminin 11 with regions that resemble postsynaptic membrane.
We also immunostained C2 and primary mouse myotubes with antibodies to laminin 5. In contrast to results
obtained with RMo cells,
5 was associated with both
AChR-rich and AChR-poor regions of myotubes in these
preparations (data not shown). Since
5 becomes restricted to synaptic sites in vivo as development proceeds, it may be that postsynaptic differentiation progresses to a
later stage in RMo than in C2 or primary myotubes.
Compensation and Coregulation in Laminin 2 and
2 Mutants
Mice with mutations in two laminin chain genes are available: naturally occurring 2dy/dy mice, in which levels of
2
are markedly reduced (Arahata et al., 1993
; Sunada et al.,
1994
, 1995
; Xu et al., 1994a
,b), and
2
/
mice, in which a
null mutation was introduced by homologous recombination (Noakes et al., 1995a
). The
2dy/dy mice exhibit severe
muscular dystrophy with only minor perturbation of neuromuscular junctions (Carbonetto, 1977
; Banker et al.,
1979
; Law et al., 1983
; Desaki et al., 1995
) whereas synaptic maturation is markedly aberrant but muscles are nearly
normal in
2
/
mice (Noakes et al., 1995a
). These results
implicate the laminin
2 and
2 chains in myogenesis and
synaptogenesis, respectively. However, interpretation of
the mutant phenotypes requires understanding which laminin trimers are present in the BLs of mutant muscle and
peripheral nerve. We therefore assessed the distribution of other laminin chains in
2dy/dy and
2
/
muscle.
In extrasynaptic BL of 2dy/dy muscles, laminin
2 immunoreactivity was markedly reduced in intensity and was
patchy rather than continuous in distribution (Fig. 6 j).
This incomplete loss has been noted previously, and is
consistent with dy/dy being an allele that decreases
2 levels but does not affect the size of the
2 polypeptide (Sunada et al., 1994
; Xu et al., 1994a
). In contrast, levels of
1
and
1 immunoreactivity were only slightly lower in
2dy/dy
muscles than littermate control muscles (Fig. 6, a, b, g, and h), consistent with previous reports in
2dy/dy mice (Sunada et al., 1994
; Xu, et al., 1994a) and in human merosin-deficient dystrophy (Hayashi et al., 1993
; Sewry et al.,
1995
). Assuming that native laminins are all heterotrimers
(Burgeson et al., 1994
), this pattern implies a compensatory increase in the level of another
chain. Indeed, immunoreactivity for laminin
4 was undetectable in controls
but intense in
2dy/dy extrasynaptic BL (Fig. 6, e and k).
This compensation was specific, in that the laminin
1,
3,
5, and
2 chains remained undetectable extrasynaptically
(Fig. 6, c, f, i, and l; and data not shown). Thus, laminin 8 may compensate for the loss of laminin 2 in
2dy/dy muscle.
In normal adult muscle, levels of laminin 2 were significantly higher in synaptic than in extrasynaptic regions of
the muscle fiber surface (see above). Paradoxically, levels
of
2 were more strikingly reduced in synaptic than in extrasynaptic regions of
2dy/dy muscle, leaving synaptic and
Schwann cell BLs nearly devoid of
2 immunoreactive
material (Fig. 7 a). As if in compensation, levels of
4
were more markedly increased synaptically than extrasynaptically in
2dy/dy muscle (Fig. 7 b). Levels of
5 and
2
were similar in synaptic BL of wild-type and
2dy/dy mice,
and
1,
3, and
1 were absent from control and mutant synapses alike (Figs. 6, c and i; and 7, c and d; and data not shown). Thus, the apparent ratios of synaptic laminins are
altered in
2dy/dy. Levels of laminin 11 are similar at control and mutant synapses, but levels of laminin 4 are dramatically reduced and levels of laminin 9 are increased in
the mutant. Therefore, laminin 4 is not required for qualitatively normal synaptic structure and function.
A different pattern of compensation was observed in the
BL of laminin 2
/
mutants. No alterations were detectable in extrasynaptic laminins, consistent with the restriction of
2 to synaptic sites at all stages of development
(Fig. 6, m-r). However, the laminin composition of mutant
synaptic BL was altered in three ways. First, as expected, laminin
2 was absent (Fig. 6 o). Second, and unexpectedly, laminin
5 was undetectable at synapses in
2
/
mutants at all stages examined, from P10 though P35 (Fig.
7 g; and data not shown). Third, laminin
1, which is undetectable at control and
2dy/dy synapses, was clearly
present in
2
/
synaptic BL (Fig. 7 h). These changes in
synaptic BL were specific, since levels of
2 and
1 were
not greatly reduced at
2
/
synapses (Fig. 7 e; and data
not shown). Laminin
4 also remained concentrated at
synaptic sites in
2
/
muscle (Fig. 7 f); however, we were
unable to determine whether this
4 was present in synaptic BL per se as well as in the closely apposed BL of
Schwann cells, which protrude into synaptic clefts in
2
/
mutants (Noakes et al., 1995a
). Together, these alterations
suggest that the normal synaptic laminins (laminins 4, 9, and 11) are replaced by laminin 2 and possibly laminin 8, but not laminin 10 in
2
/
mutants. In that laminin 4 is apparently dispensable for synaptogenesis (as shown by the
2dy/dy mice; see above), these results focus attention on
laminins 9 and 11 as regulators of neuromuscular development.
To extend our analysis of compensation and coregulation, we assessed the distribution of laminin chains in 2dy/dy
and
2
/
intramuscular nerves. As detailed above,
2 is
normally found in endoneurial BL and
2 in perineurial
BL. In endoneurial BL of the
2dy/dy mutant, levels of
2
were markedly reduced and levels of
4 were increased
(Fig. 8, d and e). Thus,
4 appears to compensate for
2 in
nerve as it does in muscle. In perineurial BL of the
2
/
mutant,
2 and
5 were both absent and
4 was considerably reduced (Fig. 8, i-l). Thus,
5 and
2 appear to be coregulated in nerve as in muscle. The laminin compositions
of perineurial BL in the
2dy/dy mutant and of endoneurial
BL in the
2
/
mutant were qualitatively normal (compare Figs. 2 and 8).
Distinct Response of Motor Axons to Laminins 1, 2, 4, and 11
As neuromuscular junctions mature in embryos or regenerate after nerve injury in adults, motor axons encounter
synaptic BL. Analysis of the laminin 2
/
mutant mice
suggests that laminin
2 is one of the components that arrests the growth of motor axons and promotes their differentiation into nerve terminals (Patton, B.L., and J.R. Sanes,
in preparation). Likewise, we have shown that recombinant
2 fragments have outgrowth-stopping and differentiation-promoting activities in vitro (Hunter et al., 1991;
Porter et al., 1995
; Patton, B.L., and J.R. Sanes. 1995. Soc.
Neurosci. Abstr. 13:799). Here, we have presented evidence that synaptic
2 may be associated with three distinct heterotrimers: laminins 4, 9, and 11. It was therefore
important to assess the effects of native
2 in heterotrimeric form, and to ask whether the
2-containing heterotrimers have distinct bioactivities. Accordingly, we assessed
outgrowth from embryonic chick ciliary neurons on substrates coated with one of four different heterotrimers:
laminins 1, 2, 4, or 11. (Purified laminin 9 was not available
to us.) Ciliary neurons, like spinal motor neurons, innervate striated muscle in vivo. Moreover, they are easily isolated, recognize original synaptic sites on skeletal muscle
fibers (Covault et al., 1987
), and stop growing in response
to recombinant laminin
2 in vitro (Porter et al., 1995
).
Initially, we plated dissociated ciliary neurons on substrates coated with purified laminin 1, 2, 4, or 11, and then
fixed and viewed them 30 h later. Most neurons extended
neurites on laminins 1, 2, or 4, as reported previously
(Weaver et al., 1995; Brandenberger et al., 1996
) (Fig. 9, a
and b). In contrast, neurons adhered to but did not extend
neurites on laminin 11 (Fig. 9 c). Likewise, little outgrowth
was observed when neurons were plated on mixtures of
laminins 1 and 11, indicating that laminin 11 inhibited neurite outgrowth and did not merely lack outgrowth-promoting activity (data not shown). In this respect, laminin 11 behaved like recombinant
2 fragments, which we have previously shown to support adhesion of, but inhibit outgrowth from ciliary motoneurons (Porter et al., 1995
).
Based on these results, we plated clusters of ciliary neurons on a uniform field of laminin 1, and then observed
neurites that encountered a patch containing a mixture of
laminin 1 plus laminins 2, 4, or 11. Neurites crossed freely
onto either laminin 2 or laminin 4 (Fig. 9, e and f). Likewise, mixtures of laminin 1 plus either BSA or additional
laminin 1 had no discernible effect on outgrowth (Fig. 9, d
and i). In contrast, neurites seldom grew from laminin 1 onto a mixture of laminins 1 and 11 (Figs. 9 h and 10 a).
The effect of laminin 11 was abolished by thermal denaturation or UV irradiation (as in Porter et al., 1995) (Fig.
9, j and k), further indicating that the native preparation
was actively inhibitory rather than merely inactive. The inhibitory activity was depleted by precipitation with antisera to
5 (Fig. 10 b), confirming that the bioactivity was
attributable to laminin 11 itself rather than to a contaminant in the preparation. From these results, we conclude that laminin 11 can serve as a stop signal for motor neurites.
In the assays scored in Fig. 10, ciliary neurons were plated onto laminin 1 and their growth onto another laminin was monitored. In some cases, however, clusters of ciliary neurons were plated onto the mixed substrate, so that neurites encountered the border from the opposite direction. For mixtures of laminins 1 and 2, neurites readily traversed the border in both directions. As noted above, outgrowth was sparse in mixtures of laminins 1 and 11, so crossing onto laminin 1 was difficult to evaluate. Interestingly, however, neurites seldom crossed from laminins 1 and 4 onto laminin 1 (Fig. 9 g). In that laminin 1 clearly promotes neurite outgrowth (i.e., is not inhibitory) on its own, this result indicates that ciliary neurons prefer laminin 4 to laminin 1 as a substrate. Thus, two different synaptic laminins have distinct effects on neurite outgrowth.
In previous studies, we have analyzed the distribution, regulation, and function of the laminin chains in muscle
(Chiu and Sanes, 1984
; Hunter et al., 1989a
,b; Sanes et al.,
1990
; Noakes et al., 1995a
; Porter et al., 1995
). Here, we
have extended these analyses to the
chains, with special
emphasis on the newly discovered
4 and
5. Our main
results are as follows. (a) Cultured muscle cells express
four different
chains (
1,
2,
4, and
5), and developing muscles incorporate all four into BLs, each in a distinct pattern. (b) Synaptic and extrasynaptic BL acquire distinct
complements of laminin chains as development proceeds:
2,
4,
5, and
1 are initially present both synaptically
and extrasynaptically, whereas
2 is restricted to synaptic
BL from its first appearance;
2 remains broadly distributed;
4 and
5 become restricted to synaptic BL; and
1
becomes restricted to extrasynaptic BL. (c) Likewise, cultured muscles cells restrict
5 and
2 to AChR-rich hot
spots but broadly distribute
2 and
1, even in the absence of nerves. (d) Laminin isoforms mark two distinct
domains within adult synaptic BL:
2,
5,
2, and
1 are
present in both the primary cleft and junctional folds,
whereas
4 is restricted to the primary cleft. (e) The endoneurial and perineurial BLs of peripheral nerve each contain distinct laminin chains (
2,
2,
1, and
4,
5,
1,
1,
respectively). (f) Mutation of the laminin
2 and
2 genes
leads to coordinate loss and compensatory upregulation of
other chains. (g) Motor axons respond in distinct ways to
different laminin heterotrimers: they grow freely between
laminin 1 and laminin 2, fail to cross from laminin 4 to
laminin 1, and stop upon contacting laminin 11. The ability
of laminin 11 to serve as a stop signal for growing axons
explains, at least in part, axonal behaviors observed at developing and regenerating synapses in vivo.
Fig. 11 summarizes the main patterns of laminin chain
expression that we have documented in muscle. The figure
also indicates the heterotrimers (as named in Table I) that
we deduce to be present at various stages and sites. This
interpretation depends, however, on two assumptions. The
first is that all possible heterotrimers are formed from
1-5,
1,
2, and
1, whenever the constituent chains are
present. We know of no evidence against this idea, but it
has not been critically tested. The second is that no other
laminin chains are present in muscle besides those for
which we have probes. In fact, there are some indications that other chains exist. For example, perineurial BL of
2
/
muscle contains
4 and
1 but little or no
1-3. If all
laminins are heterotrimers, there may be another
chain
to be discovered in perineurium. Thus, the assignments of
trimers made in Fig. 11 and throughout the text must be
regarded as provisional.
Previously, we showed that synaptic BL contains two chains:
2, which is present throughout muscle fiber BL,
and a synapse-specific
chain that reacted with the 4C7
monoclonal antibody (Sanes et al., 1990
). At the time, we
believed that 4C7 reacted solely with the
1 chain, but this
now seems unlikely (Miner et al., 1997
). Indeed, we show
here that
1 is not found in synaptic BL, but that
4 and
5 are. Thus, subject to the caveats above, we conclude that there are three synaptic laminins: 4, 9, and 11. Any or
all may play important roles in the development and maintenance of synapses, but several results focus our attention
on laminin 11. First, isolated muscle cells can localize both
the
5 and the
2 subunits even in the absence of nerves,
thus suiting this laminin to a role as a synaptic retrograde
signal. Second, both subunits are lost from synaptic sites in
2
/
mutants, which show severe synaptic defects (Noakes
et al., 1995), whereas both are retained in
2dy/dy mice,
which show mild synaptic defects (Carbonetto, 1977
; Banker et al., 1979
; Law et al., 1983
; Desaki et al., 1995
). In contrast, laminin 4 is lost from
2dy/dy synapses. Third, laminin
11 serves as a stop signal for elongating motor neurites, a
property previously ascribed to synaptic BL (Sanes et al.,
1978
).
The bioactivity of laminin 11 is consistent with the synaptic defects observed during reinnervation of 2
/
mutant muscle, in which regenerating axons often extend beyond original synaptic sites (Patton, B.L., and J.R. Sanes,
in preparation). However, the coordinate loss of the
5
and
2 chains from mutant synaptic BL raises the question
of which chain is responsible for the stopping activity. In
support of
5 is the observation that laminin 4 (
2/
2/
1)
does not inhibit neurite outgrowth. In support of
2 is the
observation that recombinant
2 fragments exert a potent
stopping activity very much like that shown here for native
laminin 11 (Porter et al., 1995
). One intriguing possibility is that the inhibitory activity resides in the
2 chain, but
that it is context dependent, being favored by combination
with
5 but opposed by combination with
2.
Recently, Brandenberger et al. (1996) also showed that
2-containing laminin 4 promotes outgrowth of neurites
from ciliary neurons and argued against the idea that synaptic laminins serve as stop signals for motor axons. Our
results are consistent with theirs, but our new data lead us
to question several of their conclusions. First, contrary to
their assertion, laminin 4 is not the sole synaptic laminin.
Previous findings suggested the existence of at least one
additional synaptic laminin (Sanes et al., 1990
), and our
new results suggest that laminins 4, 9, and 11 are all
2-containing synaptic laminins. Moreover, the phenotypes of
the
2dy/dy and
2
/
mutants discussed above suggest that
laminins 9 and/or 11 are more critical for synaptic function
than is laminin 4. Second, based on their finding that laminin 4 does not inhibit neurite outgrowth, Brandenberger et
al. (1996)
concluded that laminin
2 is unlikely to be involved in the process that leads motor axons to stop growing at synaptic sites. In contrast, our studies of laminin-coated substrates demonstrate that
2-containing laminin 11 is a potent inhibitor of neurite outgrowth. Third, they
confirmed our previous reports (Hunter et al., 1989b
; Porter et al., 1995
) that the tripeptide sequence LRE affects the
adhesiveness of a recombinant
2 fragment, and that mutation of this sequence decreases the ability of the fragment to inhibit neurite outgrowth. However, they showed
that LRE is inactive when inserted into a recombinant fragment of a chicken cartilage matrix protein, which is
predicted to form a coiled-coil structure similar to that of
the LRE-containing domain of
2. They therefore argue
that the LRE site is unlikely to be active in the native protein. Our results with laminin 11 raise the alternative possibility, that the activity of the LRE site may be context
dependent. For example, the juxtaposition of
2 with
5
might disrupt the coiled-coil conformation of the former.
Finally, Brandenberger et al. (1996)
noted that ciliary neurons extended neurites of the same mean length on substrates of laminins 2 and 4, and therefore concluded that
neurite outgrowth on laminin 2 was indistinguishable from
that on laminin 4. However, when we observed neurites
growing from either laminins 1 and 2 or 1 and 4 onto laminin 1, we found that neurites were blind to laminin 2 but
sensitive to laminin 4 borders. Thus, these two trimers do
have distinguishable effects on neurites.
We and others have previously documented complex
patterns of regulation for the laminins and collagens IV of
renal BLs (Kashtan and Kim, 1992; Miner and Sanes, 1994
,
1996
; Gubler et al., 1995
; Noakes et al., 1995b
; Cosgrove
et al., 1996
; Miner et al., 1997
). Results presented here extend these phenomena to muscle. First, as in kidney (Miner
et al., 1997
), individual adult intramuscular BLs can express one (extrasynaptic:
2), two (perineurial:
4+5), or
three (synaptic:
2+4+5)
chains, along with a single
and
chain. Second, the
and
chain complements of individual BLs can change as development proceeds. For example, extrasynaptic muscle BL may progress through at
least three compositions with regard to its
chains (
1+2
to
2+5, to
2+4+5, and then to
2). Likewise, in renal
glomerular BL, developmental transitions occur in collagen IV chains (
1+2 to
1-5, and then to
3-5), laminin
chains (
1+4 to
1+4+5 to
4+5, and then to
5), and
laminin
chains (
1 to
1+2, and then to
2) (Ekblom et
al., 1990
; Miner and Sanes, 1994
; Virtanen et al., 1995
;
Miner et al., 1997
). Third, loss of a single laminin chain
from muscle leads to an apparently compensatory appearance of others. For example, decreased expression of
2 in
muscle and endoneurial BLs in
2dy/dy mice leads to increased levels of
4, and loss of
2 from synaptic BL leads
to increased levels of
1. These changes are reminiscent of
those seen in kidney, where deletion of laminin
2 or collagen IV
3-5 from glomerular BL results in increased levels of laminin
1 or collagen IV
1+2, respectively (Kashtan
and Kim, 1992
; Noakes et al., 1995b
; Cosgrove et al., 1996
;
Miner and Sanes, 1996
). Interestingly, in all of these cases,
the compensating chain is one that was normally expressed in embryos and then lost in adults; whether the
compensation is best viewed as reexpression or retention of the embryonic phenotype will require further studies of
mechanism. Finally, loss of laminin
2 from synaptic and
vascular BLs leads to coordinate loss of
5. Likewise, in
kidney, mutations in genes encoding any of three collagen
IV chains,
3-5, leads to loss of all three chains from
glomerular BL (Gubler et al., 1995
; Cosgrove et al., 1996
;
Miner and Sanes, 1996
). Together, these results suggest
that colocalization of multiple isoforms, isoform transitions during development, and compensation and coregulation
in specific deficiency states represent general features of
BL assembly and maintenance.
These complexities in composition, development, and
regulation are important in considering the roles that laminins play in formation and maintenance of nerve, muscle, and
synapse. Potential roles of synaptic laminins are discussed
above. As regards muscle, the upregulation or retention of
4 in
2dy/dy mice may be of particular interest. The
2dy/dy
mouse exhibits severe muscular dystrophy, and has long
been used as an animal model of human dystrophies. The
recent findings that the
2 gene is mutated both in
2dy/dy
mice and in some humans with congenital dystrophies (Hayashi et al., 1993
; Sunada et al., 1994
, 1995
; Xu et al., 1994b
;
Helbling-Leclerc et al., 1995
; Nissinen et al., 1996
) demonstrates that it is a genotypically as well as phenotypically
valid model of human disease. In humans with
2 deficiency, levels of another
-like chain are increased (Mundegen et al., 1995; Sewry et al., 1995
; Connolly et al., 1996
);
the identity of this chain remains uncertain, but our results
suggest that it might be
4. If so, this pattern would resemble that seen in dystrophin-deficient dystrophies (mdx in mice, and Duchenne and Becker in humans), in which
utrophin, the autosomal homologue of dystrophin, is expressed transiently in developing normal muscles, but retained or upregulated in adult mutant muscle. Recent studies have shown that utrophin attenuates the severity of
dystrophy in mdx mice, and raised the possibility that further upregulation of utrophin could be therapeutically beneficial in humans with Duchenne dystrophy (Tinsley et al., 1996
; Deconinck et al., 1997
; Grady et al., 1997
). Likewise,
it will be important to ask whether
4 partially compensates for
2 functionally as well as structurally, and
whether it may provide an avenue for intervention in the
human disease.
Received for publication 15 August 1997 and in revised form 10 October 1997.
Address all correspondence to Joshua R. Sanes, Department of Anatomy and Neurobiology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. Fax: (314) 747-1150. E-mail: sanesj{at}thalamus.wustl.eduWe are grateful to Dr. J. Lichtman for advice on confocal imaging, and J. Cunningham, and J. Ko for technical assistance.
This work was supported by grants from the National Institutes of Health. J.H. Miner was supported by a Damon Runyon-Walter Winchell Cancer Research Fund fellowship.
AChR, acetylcholine receptors; BL, basal lamina; E, embryonic day.
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