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The Journal of Neuroscience, February 15, 1998, 18(4):1465-1477
Organization and Reorganization of Neuromuscular Junctions in
Mice Lacking Neural Cell Adhesion Molecule, Tenascin-C, or Fibroblast
Growth Factor-5
Lisa M.
Moscoso1,
Harold
Cremer2, and
Joshua
R.
Sanes1
1 Department of Anatomy and Neurobiology, Washington
University School of Medicine, St. Louis, Missouri 63110, and
2 Developmental Biology Institute of Marseille, Campus de
Luminy Case 907, 13288 Marseille Cedex 9, France
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ABSTRACT |
Many proteins have been hypothesized to mediate intercellular
interactions that regulate the formation, maturation, and maintenance of the skeletal neuromuscular junction. Three of the best characterized of these are a membrane-associated adhesion molecule, neural cell adhesion molecule (N-CAM), an extracellular matrix component, tenascin-C, and a soluble growth factor, fibroblast growth factor-5 (FGF-5). To assess the roles of these molecules in synaptogenesis in vivo, we examined neuromuscular junctions in
homozygous mutant mice lacking N-CAM, tenascin-C, FGF-5, or both N-CAM
and tenascin-C. End plates were 14% smaller in N-CAM-deficient mice
than in controls, and formation of junctional folds was delayed in this
mutant. In all other respects tested, however, the structure and
molecular architecture of neuromuscular junctions were normal in all
three single mutants and in the double mutant. We also tested the
abilities of damaged motor axons to reinnervate mutant muscle after
axotomy and of intact motor axons to sprout after partial denervation. Again, no significant differences among genotypes were observed. Together, these results demonstrate that N-CAM, tenascin-C, and FGF-5
are dispensable for major aspects of synaptic development and
regeneration.
Key words:
FGF; neuromuscular junction; N-CAM; reinnervation; sprouting; synapse formation; tenascin
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INTRODUCTION |
The formation of the neuromuscular
junction (NMJ) requires numerous interactions between presynaptic and
postsynaptic cells (for review, see Hall and Sanes, 1993 ). Genetic
studies in mice have recently provided support for the candidacy of two
proteins, agrin and neuregulin/ARIA, as nerve-derived mediators of
postsynaptic differentiation (Gautam et al., 1996; Sandrock et al.,
1997). Although other signals are undoubtedly involved, agrin and
neuregulin along with synaptic transmission (i.e., electrical activity)
per se, may account for much of the synaptogenetic effect of nerve on
muscle (for review, see Sanes, 1997).
In contrast, less is known about the retrograde signals that muscles
use to regulate the behavior of motor axons. Several candidates have
been proposed, primarily on the basis of their patterns of expression
in vivo and their bioactivities in vitro. These
include membrane-bound cell adhesion molecules such as the neural cell
adhesion molecule (N-CAM), basal lamina components such as
laminin- 2, interstitial matrix molecules such as tenascin-C, and
soluble growth factors such as fibroblast growth factor-5 (FGF-5).
Evidence that laminin-2 is a crucial organizer of presynaptic differentiation in vivo comes from the observation that NMJs
in mutant mice lacking this protein exhibit aberrant nerve terminals, impaired synaptic transmission, severe weakness, and an impaired ability to regenerate after injury (Noakes et al., 1995) (B. L. Patton and J. R. Sanes, unpublished observations). The purpose of the
studies reported here was to test the candidacies of N-CAM, tenascin-C,
and FGF-5 by analyzing NMJs in mice bearing null mutations in the
cognate genes.
N-CAM is an abundant cell adhesion molecule that is present on axons of
numerous types of developing and adult neurons, including motoneurons
(Goridis and Brunet, 1992). N-CAM is also expressed by muscle cells,
and this expression is regulated in remarkable parallel with muscle
susceptibility to innervation. N-CAM is found throughout the myotube
surface in the developing embryo, when synaptic contacts form.
Postnatally, N-CAM levels decrease extrasynaptically as synapses mature
and these portions of the muscle fiber surface become refractory to
innervation; in adult muscle, N-CAM is primarily confined to the
postsynaptic membrane. After denervation or paralysis, when muscle
fibers become susceptible to reinnervation, N-CAM reappears on the
surface of the myofiber. With reinnervation, as muscle fibers again
become refractory to hyperinnervation, levels of N-CAM decrease
extrasynaptically, and only synaptic expression persists (Covault and
Sanes, 1985, 1986; Moore and Walsh, 1985; Rieger et al., 1985; Sanes
and Covault, 1985; Covault et al., 1986). Because N-CAM is
predominantly a homophilic adhesion molecule, this pattern of
expression suggested the hypothesis that N-CAM on the motor axon could
interact with N-CAM on muscle surfaces to regulate synaptogenesis. In
support of this idea, anti-N-CAM inhibited interactions of neuronal
somata and myotubes in nerve-muscle cocultures (Rutishauser et al.,
1983) and reduced the outgrowth of neurites on myotubes, especially
when applied in combination with antibodies to other adhesion molecules
(Bixby and Reichardt, 1987; Bixby et al., 1987). Treatment of
denervated frog (Rieger et al., 1988) or mouse muscles
(Langenfeld-Oster et al., 1994) with anti-N-CAM in vivo
resulted in a delay in reinnervation. In rats, application of
antibodies to N-CAM decreased the extent of nerve sprouting after
paralysis of the muscle with botulinum toxin (Booth et al., 1990).
Together, these results supported the idea that N-CAM regulates
nerve-muscle interactions.
Comparable studies of expression and bioactivity in vivo and
in vitro also implicated tenascin-C in neuromuscular
development. Tenascin-C is a glycoprotein component of the
extracellular matrix that was originally isolated as an antigen
associated with myotendinous junctions (Chiquet and Fambrough, 1984)
but was later shown to be a more widely expressed molecule with both
adhesive and antiadhesive properties (Götz et al., 1996). During
early development, tenascin-C is expressed in the limb bud in patterns
that suggested a role in guiding nerves to their targets (Martini and
Schachner, 1991; Wehrle-Haller et al., 1991). After denervation of
adult muscle, tenascin levels increase in perisynaptic fibroblasts and
Schwann cells (Sanes et al., 1986; Daniloff et al., 1989; Gatchalian et al., 1989; Weis et al., 1991). Substrate-bound tenascin-C is
antiadhesive to some cells but promotes neurite outgrowth by sensory
and motoneurons in vitro (Wehrle and Chiquet, 1990;
Wehrle-Haller and Chiquet, 1993). Finally, antibodies to tenascin-C
delay reinnervation in both frog and mouse muscle in vivo
(Mège et al., 1992; Langenfeld-Oster et al., 1994). These studies
support the hypothesis that tenascin-C and N-CAM might be involved in
related neuromuscular interactions.
Finally, muscles are believed to secrete soluble "trophic" factors
that regulate the behavior of motor axons, and recent studies suggest
that FGFs may be such factors. FGFs are a family of >10 structurally
related, secreted polypeptides that regulate proliferation, growth, and
differentiation of many tissues, including nerve and muscle (Hannon et
al., 1996; Ozawa et al., 1996). Several FGFs (FGF-1, -2, and -4-8) are
expressed in developing skeletal muscle (Olwin et al., 1994; Hannon et
al., 1996), and motoneurons express FGF receptors (Wanaka et al.,
1990). In vitro, FGFs support the survival of chick and rat
spinal motoneurons (Hughes et al., 1993a,b), stimulate neurite
outgrowth from ciliary motoneurons (Gurney and Yamamoto, 1991), and
promote differentiation of functional motor nerve terminals (Dai and
Peng, 1995). Administration of FGFs in vivo reverses
neurochemical effects of axotomy on spinal motoneurons (Li et al.,
1994; Piehl et al., 1995), rescues motoneurons in mice with a
motoneuron disease (Ikeda et al., 1995), and promotes formation of
sprouts from motor nerve terminals on skeletal muscle fibers (Gurney et
al., 1992). Because most of the FGFs bind to all of the four known FGF
receptors (Ornitz et al., 1996), one would expect that any of several
muscle-derived FGFs could mediate retrograde effects on motoneurons.
However, studies by Hughes et al. (1993a,b) and Lindholm et al. (1994)
indicated a specific role for FGF-5 in these processes. Hughes et al.
(1993a) showed that ~65% of the total motoneuron survival activity
extractable from embryonic rat skeletal muscle was immunoprecipitated
by an antiserum to FGF-5. The antiserum was highly specific for FGF-5 and was demonstrably nonreactive with at least FGF-1, -2, and -4. Subsequently, Hughes et al. (1993b) showed that FGF-5 was more potent
than FGF-2 in promoting survival of rodent motoneurons, and Lindholm et
al. (1994) showed that FGF-5 increased choline acetyltransferase
activity of cultured cholinergic neurons from brain. Thus, although
muscles may provide numerous soluble growth factors to motor axons
(Oppenheim, 1996), these results suggested that FGF-5 might play a
prominent role.
In light of these studies, it came as a surprise to many
neurobiologists that null mutants of N-CAM (Cremer et al., 1994), tenascin-C (Saga et al., 1992; Steindler et al., 1995; Forsberg et al.,
1996; Settles et al., 1997), and FGF-5 (Hébert et al., 1994) were
all viable and fertile and, therefore, clearly had functional NMJs.
Indeed, no neural defects at all have been found in tenascin-C and
FGF-5 mutants, and only subtle defects have been found in the brain of
N-CAM mutants (Cremer et al., 1994, 1997; Ono et al., 1994). However,
the neuromuscular system was not examined in the initial studies of
these mice. It therefore remained possible that (1) the mutants
harbored neuromuscular defects that compromised synaptic structure
without preventing synaptic transmission; (2) synaptogenesis might be
delayed in the mutants; or (3) the ability of the mutant neuromuscular
system to respond to stresses such as denervation might be compromised. To explore these possibilities, we looked in detail at the size, shape,
and molecular architecture of NMJs in N-CAM, tenascin-C, and FGF-5
mutants. We also tested the ability of motor axons to elaborate sprouts
in response to partial denervation (in all three mutants and in an
N-CAM/tenascin-C double mutant), to reinnervate denervated muscle (in
N-CAM and tenascin-C mutants and in the double mutant), and to mature
on schedule (in N-CAM mutants). In no case did we find severe
abnormalities, suggesting that N-CAM, tenascin-C, and FGF-5 are all
dispensable for qualitatively normal development and regeneration of
NMJs.
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MATERIALS AND METHODS |
Mice. N-CAM-deficient mice were generated by Cremer
et al. (1994). Tenascin-C-deficient mice were generated by Saga et al. (1992) and were generously provided by Dr. S. Aizawa (Kumamoto University School of Medicine, Kumamoto, Japan). Pathogen-free colonies
of both mutants were derived via a single outcross of homozygous mutant
mice with C57BL/6 females (The Jackson Laboratory, Bar Harbor, ME).
Offspring were delivered under aseptic conditions via cesarean section
and transferred to foster mothers in a barrier facility. Subsequent
progeny were genotyped by PCR using primers that distinguished
wild-type from mutant alleles. FGF-5-deficient mice (originally called
angora; Hébert et al., 1994) were obtained from The
Jackson Laboratory and maintained as homozygotes or mated to C57BL/6
mice.
Surgery. Mice were anesthetized with ketamine (8.7 mg/100
gm) and xylazine (13 mg/100 gm) and placed supine under a dissecting microscope. The neck was shaved, and a vertical incision was made from
the chin to the superior aspect of the sternum. Skin and salivary
glands were retracted to allow access to the nerve of the
sternomastoid. To denervate the sternomastoid completely, its nerve was
crushed with a forceps just lateral to its point of entry into the
muscle. Denervation was assessed visually by noting contractions of the
muscle on nerve crush and subsequent transparency of the crushed
portion of the nerve. To prolong denervation, the nerve to the
sternomastoid was crushed at days 0 and 3 and allowed to recover until
day 7. Alternatively, a 1-2 mm portion of the sciatic nerve was
removed via an incision made in the upper thigh, and lower leg muscles
were taken on day 2 or 7. For partial denervation, animals were
prepared as above, and a tungsten needle was inserted into the nerve to
the sternomastoid to split it in half. One-half of the nerve was then
severed using a microknife. This procedure ensured that regeneration of
damaged axons was delayed, so that sprouting by intact axons could be
observed uncomplicated by reinnervation. In all cases, incisions were
closed with sutures or staples.
Light microscopy. For immunohistochemical studies of adult
NMJs, lower leg muscles from innervated and denervated animals were
dissected, frozen without fixation in liquid nitrogen-cooled isopentane, and sectioned at 4-8 µm in a cryostat. Sections were mounted on gelatin-coated slides. Sections were then incubated with
primary antibody at 4°C overnight or at room temperature for 3 hr,
washed for 30 min, labeled with second antibody for 1 hr at room
temperature, washed again for 30 min, mounted with paraphenyldiamine
and glycerol, and viewed with epifluorescence optics.
Antibodies to the following antigens were used: mouse N-CAM,
recombinant heregulin (a gift of M. Slikowski, Genentech, San Francisco, CA), laminin-2 (JS1), neu/erbB2 (sc-284; Santa Cruz Biotechnology, Santa Cruz, CA), erbB3 (sc-285; Santa Cruz
Biotechnology), laminin-1 and S-100 (Dako, Carpinteria, CA),
neurofilaments (Sternberger Monoclonals, Baltimore, MD), synaptophysin
(a gift of A. Czernik and P. Greengard, Rockefeller University), agrin
(a gift of Z. Hall, National Institutes of Health), SV2 (a gift of K. Buckley, Harvard University), dystrophin, utrophin, and rapsyn (Moscoso et al., 1995; Noakes et al., 1995; Gautam et al., 1996).
Fluorosceinated and rhodaminated second antibodies and lectins were
obtained from Boehringer Mannheim (Indianapolis, IN) and Sigma (St.
Louis, MO).
To assay reinnervation and sprouting, animals were killed using an
overdose of sodium pentobarbitol and then perfused through the heart
with Ringer's solution followed by 2% paraformaldehyde in PBS. The
vertical incision in the neck was reopened, and the soft tissue of the
neck was bathed in 2% paraformaldehyde for 15 min. The sternomastoid
muscle was removed, rinsed in PBS, sunk in 30% sucrose, mounted in
embedding medium, and frozen in liquid nitrogen-cooled isopentane.
Longitudinal sections were cut 40 µm thick with a cryostat and
mounted on gelatin-coated slides. Sections were post-fixed in ice-cold
methanol for 10 min and stained with anti-neurofilament,
anti-synaptophysin, and rhodaminated  -bungarotoxin (Molecular
Probes, Eugene, OR). To view Schwann cell processes, sections were
stained with anti-S-100 and rhodamine--bungarotoxin. To measure end
plate size, images of rhodamine--bungarotoxin-labeled end plates
were captured using a cooled CCD camera (Photometrics, Tucson, AZ). The
major diameter of each end plate was measured using IPLab software
(Signal Analytics, Vienna, VA). Similar methods were used to measure
the length of Schwann cell processes.
Electron microscopy. Animals were killed as above and
perfused with Ringer's solution followed by 4% paraformaldehyde and 4% glutaraldehyde in 150 mM cacodylate buffer, pH 7.2. Sternomastoid muscles were dissected, fixed overnight at 4°C, washed,
and stained lightly for acetylcholinesterase using the method of
Karnovsky and Roots (1964) to facilitate location of end plates. The
end plate-rich region of the muscle was refixed in 1% OsO4
in cacodylate buffer, dehydrated, and embedded in Araldite. Thin
sections were stained with uranyl acetate and lead citrate and viewed
in a JEOL 1200 electron microscope.
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RESULTS |
N-CAM mutants
Structure of the adult NMJ
To begin this study, we compared the size and shape of NMJs in
longitudinal sections from sternomastoid muscles of adult
N-CAM / and control mice. Nerve terminals were
marked by a mixture of antibodies to neurofilaments and synaptophysin.
Neurofilments are abundant in motor axons, and synaptophysin is a major
component of the synaptic vesicles that are concentrated in the nerve
terminal. Thus, the combination of these two antibodies revealed both
the preterminal axons and their terminal arbors (Gautam et al., 1996). To stain the postsynaptic membrane, sections were incubated with rhodaminated -bungarotoxin, which binds specifically and tightly to
ACh receptors (AChRs).
A typical end plate from an adult wild-type mouse is shown in Figure
1a,a'. A single axon contacts
each muscle fiber, branches, and terminates on the muscle fiber surface
in a circumscribed spray of varicose branches (Fig. 1a).
AChRs are concentrated in the postsynaptic membrane in a pretzel-like
pattern with distinct boundaries (Fig. 1a'). Presynaptic and
postsynaptic structures overlap completely. NMJs in
N-CAM/ mice were similar in shape and complexity
to those in the wild-type mouse, and, as in controls, mutant
presynaptic and postsynaptic structures were completely apposed (Fig.
1b,b'). Morphometric analysis revealed, however, that mutant
end plates were smaller than controls. A difference was seen in each of
four age-matched pairs of animals (Fig. 1c). When data from
all animals were combined, the difference was 14%, which was
statistically significant (p < 0.001, Student's t test). However, it has been noted that N-CAM mutant mice weigh ~10% less than their wild-type littermates (Cremer et al., 1994) and might therefore be expected to have 2-3%
smaller-diameter myofibers. Because end plate size is proportional to
myofiber diameter (Balice-Gordon and Lichtman, 1993) the difference
that we observe may reflect partially the overall size of the mutant animals.
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Figure 1.
Size and shape of NMJs in adult wild-type and
N-CAM/ mice. Longitudinal cryostat sections of
sternomastoid muscles from control (a, a') and
N-CAM/ (b, b') mice were stained
with a mixture of fluorescein-tagged anti-neurofilament and
anti-synaptophysin to label preterminal and terminal portions of the
motor axon, plus rhodamine--bungarotoxin to label AChRs
concentrated in the postsynaptic membrane. The sections were viewed
through filters selective for fluorescein (a, b) or
rhodamine (a', b'). At both wild-type and mutant end plates, the terminal branches of the axon directly overlie postsynaptic specializations. c, Average length of NMJs. Each
bar represents measurements of the major diameter
(parallel to the long axis of the muscle fiber) from 50 end plates
(mean ± SEM) in the sternomastoid of a single animal. The
first four pairs of bars show data from age-matched N-CAM/ (open bars)
and control (closed bars) muscles. All data are combined in the last pair of bars. Mutant end
plates are smaller than controls (p < 0.001, Student's t test). Scale bar, 20 µm.
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To assess synaptic ultrastructure, muscles were prepared for electron
microscopy, and NMJs were observed in thin sections. Three cells
(motoneuron, muscle fiber, and terminal Schwann cells) contribute to
the NMJ. Each of these cells is specialized at the synapse. The nerve
terminal is densely packed with synaptic vesicles, which are clustered
at active zones, whereas the preterminal axon contains few vesicles and
no active zones. The terminal Schwann cell extends thin processes to
cap the nerve terminal, whereas preterminal Schwann cells form myelin.
The postsynaptic membrane of the muscle fiber is thrown into
~1-µm-deep folds at the synapse, which are absent extrasynaptically
(Fig. 2a). In all of these respects, mutant NMJs were indistinguishable from their wild-type counterparts (Fig. 2b).
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Figure 2.
Electron micrographs of NMJs from adult control
(a) and N-CAM/
(b) mice. In both, a vesicle-laden nerve terminal
is capped by processes of a Schwann cell (S) and
overlies the enfolded postsynaptic membrane of the muscle fiber. Scale
bar, 0.5 µm.
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Molecular architecture
Although the structure of the NMJ appeared normal in
N-CAM/ mutants, its molecular composition might
have been perturbed. To test this possibility, we stained wild-type and
mutant muscle with a panel of antibodies and histological stains
specific for 13 antigens concentrated at the NMJ. Two of the antibodies
recognized synaptophysin and SV2, major components of synaptic vesicles
(Calakos and Scheller, 1996). Three antibodies were directed against
components of the synaptic basal lamina: laminin 2, agrin, and
neuregulin (Sanes, 1995). Two other components of the basal lamina were
revealed histochemically: acetylcholinesterase by an enzymatic stain
(Karnovsky and Roots, 1964) and
N-acetylgalactosaminyl-terminated carbohydrate by a lectin,
Vicia villosa agglutinin B4 (VVA-B4) (Scott et al., 1988).
Three antibodies were specific for components of the postsynaptic membrane: erbB2, erbB3, and integrin 7A (Moscoso et al., 1995; Martin et al., 1996). As noted above, -bungarotoxin labeled AChRs in
the postsynaptic membrane. Two antibodies stained cytoskeletal proteins
concentrated beneath the postsynaptic membrane: rapsyn and utrophin
(Hall and Sanes, 1993). In all cases, the distribution and approximate
levels of antigen in mutant were indistinguishable from wild type (Fig.
3 and data not shown). Thus, we detected no significant effect of N-CAM on the molecular architecture of the NMJ
in adult mice.
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Figure 3.
Distribution of synaptic antigens at NMJs in
control (a-d) and N-CAM/
(e-h) mice. Cross-sections of hindlimb muscles were
doubly stained with antibodies to N-CAM (a, e),
synaptophysin (b, f), erbB3 (c, g), or agrin (d, h) plus
rhodamine--bungarotoxin (a'-h'). Mutant end plates
were devoid of N-CAM, as expected, but did not otherwise differ from
controls. Scale bar, 10 µm.
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Development
Although the microscopic and molecular structure of adult NMJs was
not detectably affected by the absence of N-CAM, it seemed possible
that development would be delayed or perturbed in the mutant. To assess
this possibility, we examined synapses during the first 2 postnatal
weeks. NMJs are functional at birth, but they mature in at least four
respects during this early postnatal period. First, synaptic geometry
becomes more complex as the nerve terminal elaborates fine branches
within the end plate region, and the AChR-rich postsynaptic domain is
transformed from a plaque with poorly defined boundaries to a highly
branched "pretzel" with well defined boundaries (Steinbach, 1981;
Slater, 1982). Second, end plates are multiply innervated at birth and
become singly innervated as a result of synapse elimination (Colman and Lichtman, 1993). Third, molecular distinctions between synaptic and
extrasynaptic regions of the muscle fiber surface become greater; some
synaptic antigens do not become detectable until postnatally, and some
that are initially expressed throughout the myofiber become
concentrated at synapses postnatally (see below). Finally, the
junctional folds that invaginate the postsynaptic membrane form after
birth (Matthews-Bellinger and Salpeter, 1983).
To determine whether any of these developmental events were
N-CAM-dependent, we first studied the geometrical changes that occur in
presynaptic and postsynaptic structures. In wild-type mice, the nerve
terminal is poorly branched and overlies ovoid plaques of AChRs with
poorly defined borders (Fig.
4a,a'). Gradually, AChR-poor
"holes" appear within the plaques, and the nerve terminals elaborate finer branches (Fig. 4b,b'). Finally the branching
pattern of the nerve terminal becomes more distinct and overlaps a
correspondingly branched AChR-rich region with distinct boundaries
(Fig. 4c,c'). A similar progression was seen in mutant mice
(Fig. 4d-f).
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Figure 4.
Postnatal maturation of the NMJ in control
(a-c) and N-CAM/
(d-f) mice. Longitudinal sections of
sternomastoid muscles from P0 (a, d), P7 (b,
e), and P14 (c, f) mice were stained as
in Figure 1, with antibodies to neural antigens
(a-f) plus rhodamine--bungarotoxin (a'-f'). In both mutants and controls, initial
plaque-like endings are remodeled to form branched end plates during
the first 2 postnatal weeks. Scale bar, 20 µm.
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Next, we evaluated synapse elimination by counting the number of motor
axons that entered each AChR-rich end plate. At postnatal day 0 (P0),
50 and 59% of muscle fibers were singly innervated in wild-type and
mutant sternomastoid, respectively. By P14, all end plates in a
wild-type muscle but only 84% of end plates in mutant muscle were
singly innervated. All end plates were singly innervated in adult
mutant muscle. Our control values are similar to those reported
previously for wild-type sternomastoid by Balice-Gordon and Lichtman
(1993). Thus, synapse elimination appears to be slightly delayed in the
mutant, but it occurs within several days of the normal schedule.
Third, we stained muscles to reveal four components that become
confined to the postsynaptic membrane postnatally: agrin, neuregulin,
N-acetylgalactosaminyl-terminated carbohydrates (recognized by the lectin VVA-B4), and integrin 7A. Agrin, neuregulin, and N-acetylgalactosaminyl-terminated carbohydrates are present
throughout the myofiber membrane at birth, but expression becomes
restricted to the postsynaptic surface by P14 (Hoch et al., 1993;
Moscoso et al., 1995) (P. T. Martin and J. R. Sanes,
unpublished observation). Integrin 7A is undetectable at birth and
appears specifically at the synapse during the first postnatal week
(Martin et al., 1996). The distribution of each of these components was
indistinguishable in wild-type and mutant muscles examined at P0, P7,
and P14 (data not shown). Thus the synaptic localization of these
molecules occurs via an N-CAM-independent mechanism.
Finally, we used electron microscopy to assess the ultrastructure
of the developing NMJ. No qualitative differences were seen between the
mutant and wild-type nerve terminals at P7 or P15; synaptic vesicles
filled the nerve terminal, Schwann cell processes capped each terminal,
and junctional folds invaginated the postsynaptic membrane. However, we
were struck by a relative paucity of folds in mutant end plates (Fig.
5a,b). We had shown previously
that N-CAM is localized at the depths of the folds in the adult, and we
hypothesized that N-CAM plays a role in the formation or maintenance of
folds (Covault and Sanes, 1986). We therefore counted the number of
folds in 45 synaptic profiles from two mutant mice and 72 synaptic profiles from three control mice at P7. There were approximately threefold more folds per profile in control mice (2.26 folds per profile) than in the mutant (0.82 folds per profile). This difference was statistically significant (p < 0.001) and
did not merely reflect delayed overall growth of mutant end plates,
because the difference persisted when values were normalized to the
size of the junction (Fig. 5c). The difference was
transient, however; it was statistically insignificant
(p > 0.2) by P15 and absent in the adult (Fig.
5c). Thus, formation of junctional folds is delayed, but not
prevented, in N-CAM/ mice.
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Figure 5.
Formation of junctional folds
is delayed in the absence of N-CAM. a, b, Electron
micrographs of NMJs from wild-type (a) and N-CAM/ (b) mice at P7.
c, Number of postsynaptic folds per micrometer of
nerve-muscle apposition, counted from electron micrographs such as
those in a and b. Each
point represents the mean ± SEM measurements from
20-72 end plates in two or three mice. Indentations of the
postsynaptic membrane >50 nm deep were counted as folds. Scale bar,
0.5 µm.
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Reinnervation
As noted above, N-CAM is regulated in parallel with muscle
susceptibility to innervation (references in introductory remarks). Based on this correlation, it was suggested that N-CAM on denervated muscle fibers promotes reinnervation, perhaps by interacting with N-CAM
on the motor axon. Consistent with this possibility, antibodies to
N-CAM delay reinnervation in frogs and mice (Rieger et al., 1988;
Langenfeld-Oster et al., 1994). To test this hypothesis further, we
assayed reinnervation of sternomastoid muscles in N-CAM/ mice. Muscles were sectioned and doubly
stained for nerve terminals and AChRs 3-7 d after nerve crush. It is
known that AChR aggregates persist for months after denervation, and
that regenerating axons exclusively reinnervate original synaptic sites
under these conditions (Rich and Lichtman, 1989). Therefore, the extent
to which AChR-rich membranes are reoccupied by axons provides a
reliable measure of reinnervation. Using this measure, we found that
end plates in both control and mutant muscles were nearly completely
reinnervated by 1 week after nerve crush (Fig.
6a,b). Moreover, the precise apposition of presynaptic and postsynaptic sites indicated that original synaptic sites were as selectively reinnervated in mutant as
in wild-type mice. Quantitation of the fraction of synaptic sites
reoccupied between 3 and 7 d after nerve crush indicated that the
rate of reinnervation was similar in mutants and controls (Fig.
6c).
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Figure 6.
Reinnervation of denervated end plates occurs at a
normal rate in N-CAM mutant mice. a, b, Longitudinal
sections of 7 d reinnervated sternomastoid muscles from control
(a) and N-CAM/
(b) mice were stained as in Figure 1. The nerve
terminal (a, b) reoccupies AChR-rich original synaptic
sites (a', b') during reinnervation. c,
Fraction of end plates reinnervated at various times after nerve crush
in mutant (filled diamonds) and control (open circles) animals. Each point
represents an average of 116 end plates from a single animal.
d, Fraction of AChR-rich postsynaptic membrane covered
by nerve terminal for three pairs of mutant (shaded) and
control (open) mice at 5-7 d after nerve crush. In
c, end plates were counted as reinnervated if any
portion of the AChR-rich postsynaptic membrane was apposed by a nerve
terminal. In d, only reinnervated end plates were
scored. Scale bar, 20 µm.
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Much of the time between axotomy and the initiation of
reinnervation involves regeneration of axons from the point of injury to the myofiber surface. If this time was unaffected by N-CAM, it is
possible that our measurements would have been insensitive to a delay
in synapse formation per se. To test this idea, we asked whether the
fraction of postsynaptic surface covered by regenerating axons differed
between mutant and wild-type mice at a time when many end plates had
been reached just recently. The reinnervation of single end plates was
quantified by estimating the amount of end plate region covered by
nerve terminal. This distribution was similar in three pairs of control
and mutant animals (Fig. 6d). These results showed that the
rate at which axons reoccupied end plates was not detectably
N-CAM-dependent.
Schwann cells
Recent studies have shown that terminal Schwann cells elaborate
processes after denervation of rat muscle (Reynolds and Woolf, 1992),
and that reinnervating axons follow these processes (Son and Thompson,
1995). Because Schwann cells as well as motoneurons and muscle fibers
express N-CAM, it seemed possible that interactions of Schwann cells
with nerve, muscle, or both would be affected by N-CAM. For example,
Schwann cells might have been unable to sense the loss of the nerve on
denervation or to interact properly with denervated muscle. To test
this possibility, we stained denervated muscles with antibodies to the
Schwann cell-specific marker S-100. Schwann cells formed processes
after denervation of mouse muscle (Fig.
7a,b) as reported previously
for rat. Similar numbers of terminal Schwann cells formed processes in
mutant and control muscles [sprouts at 94 of 101 end plates (93%) in
two mutant animals and at 55 of 70 (79%) in two controls]. Moreover,
the length of processes did not differ significantly between wild-type
and mutant mice (Fig. 7c). Thus, N-CAM is not required for
Schwann cell sprouting after denervation.
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Figure 7.
Terminal Schwann cells extend
processes in denervated N-CAM/ muscle. a,
b, Longitudinal sections of 7 d denervated muscle from control (a) and N-CAM/
(b) mice were stained with anti-S-100 (a,
b) and rhodamine--bungarotoxin (a', b'). In
both controls and mutants, terminal Schwann cells extend processes
(arrowheads) after double nerve crush. c,
Extension of Schwann cell processes beyond the AChR-rich postsynaptic
membrane, measured from micrographs such as those in a
and b. Schwann cell sprouts [130 in 3 control mice
(open bars) and 138 in 3 mutant mice (hatched
bars)] were measured. The first six bars
represent single animals (mean ± SEM). All data are combined in
the last pair of bars. Scale bar, 20 µm.
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In studying muscles treated with anti-N-CAM, Rieger et al. (1988) found
that Schwann cells often failed to cap nerve terminals after
reinnervation. To see whether N-CAM plays a role in the adhesion of the
terminal Schwann cell to the nerve terminal, we assessed the
ultrastructure of reinnervated end plates with electron microscopy in
two mutant and two control sternomastoid muscles. Nerve terminals were
capped by Schwann cell processes at all reinnervated end plates in both
mutants and controls (data not shown). Thus, N-CAM is not essential for
Schwann cell processes to cap reinnervating nerve terminals.
Sprouting
After nerve crush, most axons regenerate through Schwann cell
tubes in intramuscular nerves and are thereby guided to original synaptic sites (for review, see Sanes and Covault, 1985). Consequently, some ingrowing axons may not interact with extrasynaptic portions of
the muscle membrane. In contrast, nerve terminals sprout beyond the
boundaries of the end plate in partially denervated or paralyzed muscle, in which case they do interact with extrasynaptic regions (Hoffman, 1950; Brown et al., 1981). Because denervation and inactivity lead to appearance of N-CAM in extrasynaptic membrane, it was suggested
that N-CAM promotes such sprouting (Covault and Sanes, 1985). In
support of this idea, anti-N-CAM partially inhibits growth of motor
axons on myotubes in vitro (Bixby et al., 1987) and
partially inhibits sprouting after paralysis in vivo (Booth et al., 1990). Accordingly, we asked whether motor axons were capable
of terminal sprouting in mutant mice. To this end, we partially
denervated the sternomastoid muscle (see Materials and Methods), waited
7 d for sprouts to form, and then doubly stained sections to
reveal axons and AChRs. Seven days after partial denervation, innervated end plates had elaborated sprouts in both control and N-CAM/ muscles. Consistent with previous
descriptions, sprouts in controls were thinner than nerve terminals and
bore occasional swellings along their length (Fig.
8a). In
N-CAM/ mice, sprouts were also present and had
similar morphology (Fig. 8b). Moreover, nerve terminal
sprouting occurred in the mutant to the same extent as in the control;
terminal sprouts were present at 36% of innervated end plates in
mutants (18 of 51 in three muscles) and at 37.5% in controls (38 of 91 in four muscles). These results show that N-CAM is dispensable for
terminal sprouting.
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Figure 8.
Intact motor nerve terminals sprout after partial
denervation of N-CAM-deficient muscles. Sternomastoid muscles were
sectioned longitudinally 7 d after partial denervation, and
sections were stained as in Figure 1, with antibodies to neural
antigens (a, b) and rhodamine--bungarotoxin
(a', b'). Intact end plates in both control
(a) and N-CAM/
(b) muscle extended terminal sprouts.
Arrowheads in b point to terminal sprout.
Scale bar, 20 µm.
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Tenascin-C mutants
Like the N-CAM/ mutants, tenascin-C-mutant
mice are externally normal (Saga et al., 1992; Forsberg et al., 1996),
but previous studies of their neuromuscular system have not been
reported. We therefore examined synapses in adult mutant mice but found no significant difference between tenascin-C/
mice and controls (Fig. 9; data not
shown). Because the protocols were identical to those described above,
the results can be summarized briefly: (1) staining with
rhodamine--bungarotoxin plus antibodies to neural antigens showed
that end plates were normal in size and shape in
tenascin-C/ mutants (Fig. 9a); (2)
electron microscopy revealed normal ultrastructure of nerve terminals,
Schwann cells, and muscle fibers at mutant NMJs (data not shown); (3)
all synaptic antigens tested (synaptophysin, SV2, neuregulin, N-CAM,
laminin 2, erbB2, erbB3, agrin, rapsyn, and integrin 7A) were
localized identically in mutants and controls (data not shown); (4)
intact mutant nerve terminals formed sprouts within 7 d of partial
denervation; the sprouts in the mutant were similar in morphology to
those of controls (Fig. 9b), and the extent of sprouting was
similar in mutant and control muscles (37.5% of 91 end plates in four
control muscles and 50.5% of 128 end plates in four mutant muscles);
and (5) >80% of end plates in the sternomastoid were reinnervated
7 d after nerve crush (Fig. 9c). Moreover, in mutant as
in control animals, axons reoccupied original synaptic sites precisely,
although, as in controls, some original sites were only partially
reoccupied (data not shown). Together, these data show that NMJs can
form and regenerate in the absence of tenascin-C.
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Figure 9.
NMJs of mice lacking tenascin-C. a,
b, NMJs from adult tenascin-C/
sternomastoid muscles, stained for nerve (a, b) and
AChRs (a', b') as in Figure 1. a, The
mutant NMJ is indistinguishable from controls (compare with Fig.
1a). b, Terminal sprouting from an intact
end plate in a tenascin-C/ muscle 7 d after
partial denervation. The sprouts resemble those seen in controls
(compare with Fig. 8a). c, Fraction of
end plates reinnervated 7 d after nerve crush in
tenascin-C/ (shaded) and control
(open) muscle. Each bar represents
48-185 end plates from a single animal. The first three
pairs of bars represent age-matched pairs of
animals. All data are combined in the last pair of
bars. Scale bar, 20 µm.
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FGF-5 mutants
Hébert et al. (1994) generated mutant mice with a null
allele of the FGF-5 gene. Although FGF-5 is expressed in numerous embryonic and adult tissues, the only phenotype detected in mutant homozygotes was a marked increase in hair length. Analysis of the
mutants indicated that FGF-5 is an inhibitor of hair elongation and
revealed that a previously identified and commercially available spontaneous mutant, angora (go), was
another null allele of FGF-5 (Hébert et al., 1994). In light of
suggestions that FGF-5 might be a muscle-derived regulator of motor
axons (see introductory remarks), we examined neuromuscular junctions
in adult go homozygotes. As for the N-CAM and tenascin-C
mutants described above, synaptic geometry was normal in FGF-5 mutants
(Figure 10a,a'). Further
immunochemical characterization of cross-sectioned material failed to
reveal any abnormalities of the motor nerve terminal (synaptophysin and SV2) terminal Schwann cell (S-100), synaptic basal lamina (neuregulin, laminin 2, and agrin), or postsynaptic membrane (N-CAM, erbB3, integrin 7A, and AChRs; data not shown).
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Figure 10.
NMJs in sternomastoid muscles of adult mice
lacking FGF-5 stained for nerve (a, b) and AChRs
(a', b') as in Figure 1. a, The mutant
NMJ is indistinguishable from controls (compare with Fig. 1a). b, Terminal sprouting from an intact
end plate in an FGF-5/ muscle 7 d after
partial denervation. The sprout (arrow) resembles those
seen in controls (compare with Fig. 8a). Scale bar, 20 µm.
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Because soluble factors have been repeatedly implicated as promoters of
sprouting (Brown et al., 1981; Hall and Sanes, 1993), we also partially
denervated sternomastoid muscles in two go mice and then
counted sprouts 7 d later (Fig. 10b,b'). Approximately half of the intact nerve terminals extended sprouts in the mutant muscles (40 and 58% in the two animals), a frequency similar to that
observed in the control (37.5%), N-CAM/ (36%),
and tenascin-C/ (50.5%) muscles described
above.
An N-CAM/tenascin-C double mutant
One explanation for unexpectedly subtle phenotypes of null mutants
is that the mutated gene was only one of two or more capable of
performing the function in question. When this is the case, double
mutants lacking both genes often have far more severe phenotypes than
either single mutant (see Discussion). As a first step in evaluating
this possibility, we mated tenascin-C/ and
N-CAM/ mice to produce compound heterozygotes
(N-CAM+/ and tenascin-C+/)
and then mated these to generate mice doubly homozygous for the N-CAM
and tenascin-C null mutations. This combination was chosen because
antibodies to N-CAM and tenascin-C have similar (although not
identical) effects on reinnervation (Rieger et al., 1988; Mège et
al., 1992; Langenfeld-Oster et al., 1994). Several litters of
offspring, totaling ~100 pups, were genotyped by PCR shortly after
weaning. We expected to find approximately six double mutants (1/4 × 1/4 × 100) in this cohort but found only one. This low number
might reflect a statistical anomaly, increased mortality of the double
mutant (either embryonically or postnatally), or the fact that a subset
of animals was discarded because we were unable to genotype them
definitively for technical reasons. Nonetheless, the only double mutant
was studied in some detail.
In a single operation, one sternomastoid muscle was completely
denervated, and the contralateral sternomastoid was partially denervated. As before, complete denervation was performed by crushing the nerve, whereas partial denervation was done by severing a portion
of the nerve. The crushed axons regenerated rapidly, whereas reinnervation by cut axons was delayed, permitting assessment of
sprouting by the remaining intact axons. Seven days after surgery, the
animal was killed, and the double-staining protocol was used to reveal
nerve terminals and AChRs in both sternomastoids and several leg
muscles. We detected no differences between double mutant and control
NMJs (Fig. 11a, compare with
Fig. 1a). Original synaptic sites were reinnervated in the
muscle that had been completely denervated (Fig. 11b,
compare with Fig. 6a). Intact terminals had formed sprouts
in the partially denervated muscle, and the sprouts were similar in
shape to those observed in the single mutants and in controls (Fig.
11c, compare with Fig. 8). Moreover, the extent of
reinnervation and terminal sprouting was not lower in the double mutant
than in controls or single mutants; all of the denervated end plates
(40 of 40) had been reinnervated, and 29 of 43 (67%) intact end plates
in the partially denervated muscle bore terminal sprouts. From these
data, we conclude that N-CAM and tenascin-C are not functionally
redundant in terms of molecular architecture, reinnervation, and
sprouting.
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Figure 11.
NMJs from a mouse lacking both N-CAM and
tenascin-C. Muscles from a doubly mutant animal were sectioned
longitudinally and stained for nerves (a-c) and AChRs
(a'-c'), as in Figure 1. a, The nerve
terminals and AChRs of an adult NMJ are indistinguishable from those in
controls (compare with Fig. 1). b, Original synaptic sites are completely reoccupied by regenerated axons 7 d after nerve crush. c, Seven day partially denervated muscles
elaborate terminal sprouts from innervated end plates. Scale bar, 20 µm.
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DISCUSSION |
There is abundant evidence that muscles regulate the behavior of
motor axons as NMJs form in embryos, mature postnatally, are maintained
in adulthood, and regenerate after injury (Hall and Sanes, 1993).
Numerous molecules have been suggested as retrograde mediators of these
interactions, based on three types of evidence: (1) they are expressed
at relatively high levels in embryonic and/or denervated muscles, which
are most susceptible to innervation; (2) when presented to motoneurons
in vitro, the purified proteins promote survival, growth,
adhesion, neurite extension or differentiation; and (3) administration
of exogenous molecules or antisera to them perturbs neuromuscular
interactions in vivo. As detailed in the introductory
remarks, all three lines of evidence have supported the candidacy of
N-CAM, tenascin-C, and FGF-5 as regulators of synaptogenesis at the
vertebrate NMJ.
In studies reported here, we attempted to define roles of N-CAM,
tenascin-C, and FGF-5 further by documenting abnormalities in the
structure, molecular architecture, development, or regeneration of NMJs
in mutant mice lacking each of these three proteins and in a single
mouse lacking both N-CAM and tenascin-C. Surprisingly, the only
abnormalities we detected were a delay in the formation of junctional
folds, a possible slowing of synapse elimination, and a slight decrease
in end plate size in N-CAM mutant mice. Three potential explanations
for the apparent discrepancies between these and previous results are
that (1) we failed to detect some neuromuscular defects; (2) other,
related molecules may have been able to compensate for loss of the
mutated gene products; and (3) some previous perturbation studies may
have given misleading results. We shall consider the first two of these
possibilities.
Clearly, our search for neuromuscular defects in these mice was not
exhaustive. Although we probably would have detected major changes in
the geometry or molecular architecture of the synapse, modest changes
in antigen levels would have gone undetected. In mutants lacking the
synaptic cytoskeletal component utrophin, for example, AChR density is
decreased significantly but the difference was not striking visually
(Deconinck et al., 1997a; Grady et al., 1997a). Likewise, although we
failed to detect differences after complete or partial denervation, the
proteins could play roles in other stressful situations in murine life
that we did not test, such as muscle damage, exercise or aging. In
addition, tenascin-C/ and
FGF-5/ mutants were studied in less detail than
N-CAM/ mice, so subtle defects were more likely
to have been overlooked. Perhaps most important, we did not study some
phenomena relevant to neuromuscular development in which the molecules
have been implicated. For example, N-CAM has been proposed to be
required for some aspects of peripheral nerve guidance (Landmesser et
al., 1988; Tang et al., 1993) and myogenesis (Fredette et al., 1993; Fazeli et al., 1996), tenascin-C may contribute to the stability of
myotendinous junctions or muscle spindles (Chiquet and Fambrough, 1984;
Pedrosa-Domellof et al., 1995), and FGF-5 may be required for survival
of some motoneurons (Hughes et al., 1993a). Further studies would be
needed to test these possibilities.
Another general explanation for unexpectedly subtle phenotypes of null
mutants is that the mutated gene is only one of several that can
perform the function in question. If two or more gene products are
normally present and play similar roles, they are said to be redundant.
Alternatively, loss of one gene may lead to enhanced expression or
accumulation of another, which then compensates for lack of the first.
Multiple examples of redundancy and compensation have been documented
in nerves and muscles of mutant mice. For example, although forced
expression of either myoD or myf5 can transform undifferentiated cells
into muscle, myogenesis proceeds almost normally in mice carrying null
mutations for either gene. In contrast, animals lacking both myoD and
myf5 never form muscle (Rudnicki and Jaenisch, 1995). In this case, myoD and myf5 have functionally redundant roles in myogenesis. Similarly, loss of dystrophin from muscle leads to compensatory enhanced retention of its homolog, utrophin, attenuating the muscular dystrophy that would otherwise occur (Deconinck et al., 1997b; Grady et
al., 1997b).
Because N-CAM, tenascin-C, and FGF-5 are all members of large multigene
families, compensation and redundancy are plausible explanations for
the lack of dramatic defects. With regard to N-CAM, numerous
immunoglobulin superfamily molecules are expressed by motoneurons,
myotubes, interstitial cells, or Schwann cells; these include thy-1,
L1/Ng-CAM, TAG-1/axonin, and neurofascin (Booth et al., 1984; Moscoso
and Sanes, 1995; Fazeli and Walsh, 1996). In the case of tenascin-C,
the closely related homologs tenascin-X and -Y are both normally
expressed in developing muscle and could play roles (Erickson, 1993;
Burch et al., 1995; Chiquet-Ehrismann, 1995; Hagio et al., 1996).
Likewise, at least five FGFs in addition to FGF-5 are expressed in
developing or adult muscle or both, and most, if not all, of these are
capable of interacting with the FGF receptors that motoneurons bear
(Hannon et al., 1996; Ornitz et al., 1996).
We made two attempts to detect redundancy and compensation. First, we
stained innervated and denervated muscles with antibodies to several
immunoglobulin superfamily molecules (thy-1, L1/Ng-CAM, TAG-1/axonin-1,
F3/F11/contactin, Nr-CAM/BRAVO, and neurofascin) but detected no
differences in their expression between wild-type and N-CAM-deficient
mice (data not shown). Second, we examined innervation and
reinnervation in an N-CAM/tenascin-C double mutant, based on previous
results indicating that both N-CAM and tenascin-C interact with highly
homologous members of the immunoglobulin superfamily (Brümmendorf
and Rathjen, 1993; Chiquet-Ehrismann, 1995) and that antibodies to
N-CAM and tenascin-C have similar effects on reinnervation (Rieger et
al., 1988; Mège et al., 1992; Langenfeld-Oster et al., 1994).
Again, no significant abnormalities were apparent in the single animal
tested.
Despite these negative results, we favor the possibility that
redundancy, compensation, or both may explain some differences between
previous results and those reported here. Thus, function-blocking antibodies might be more effective at perturbing reinnervation than
genetic ablation, because blockade occurs rapidly and may not provide
sufficient opportunity for compensation to occur. Moreover, recent
studies of fasciclin II, the Drosophila N-CAM ortholog, have
demonstrated roles for this molecule in formation and plasticity of the
fly NMJ (Schuster et al., 1996; Davis et al., 1997); perhaps the
relative simplicity of the fly genome minimizes masking by redundancy
or compensation. On the other hand, it is important to realize that
most of the hypotheses about roles of N-CAM, tenascin-C, and FGF-5 in
the vertebrate neuromuscular system were derived from analysis of
expression patterns, from studies in vitro, and from
pharmacological (overexpression) studies. A painful lesson of genetic
studies in organisms from yeast to flies to mice is that these lines of
indirect evidence are at best imperfect indicators of roles that
molecules actually play in developmental processes in
vivo.
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FOOTNOTES |
Received Oct. 14, 1997; revised Nov. 20, 1997; accepted Nov. 26, 1997.
This work was supported by grants from the National Institutes of
Health. We thank Jeanette Cunningham and Mia Nichol for expert
assistance.
Correspondence should be addressed to Dr. Joshua R. Sanes, Department
of Anatomy and Neurobiology, Washington University School of Medicine,
Box 8108, 660 South Euclid Avenue, St. Louis, MO 63110.
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Top
Abstract
Introduction
