Acetylcholine negatively regulates development of the neuromuscular junction through distinct cellular mechanisms.
ABSTRACT Emerging evidence suggests that the neurotransmitter acetylcholine (ACh) negatively regulates the development of the neuromuscular junction, but it is not clear if ACh exerts its effects exclusively through muscle ACh receptors (AChRs). Here, we used genetic methods to remove AChRs selectively from muscle. Similar to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs increased motor axon branching and expanded innervation territory, suggesting that ACh negatively regulates synaptic growth through postsynaptic AChRs. However, in contrast to the effects of blocking ACh biosynthesis, eliminating postsynaptic AChRs in agrin-deficient mice failed to restore deficits in pre- and postsynaptic differentiation, suggesting that ACh negatively regulates synaptic differentiation through nonpostsynaptic receptors. Consistent with this idea, the ACh agonist carbachol inhibited presynaptic specialization of motorneurons in vitro. Together, these data suggest that ACh negatively regulates axon growth and presynaptic specialization at the neuromuscular junction through distinct cellular mechanisms.
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ABSTRACT: The signaling pathways activated by Wnt ligands are related to a wide range of critical cell functions, such as cell division, migration, and synaptogenesis. Here, we summarize compelling evidence on the role of Wnt signaling on several features of skeletal muscle physiology. We briefly review the role of Wnt pathways on the formation of muscle fibers during prenatal and postnatal myogenesis, highlighting its role on the activation of stem cells of the adult muscles. We also discuss how Wnt signaling regulates the precise formation of neuromuscular synapses, by modulating the differentiation of presynaptic and postsynaptic components, particularly regarding the clustering of acetylcholine receptors on the muscle membrane. In addition, based on previous evidence showing that Wnt pathways are linked to several diseases, such as Alzheimer's and cancer, we address recent studies indicating that Wnt signaling plays a key role in skeletal muscle fibrosis, a disease characterized by an increase in the extracellular matrix components leading to failure in muscle regeneration, tissue disorganization and loss of muscle activity. In this context, we also discuss the possible cross-talk between the Wnt/β-catenin pathway with two other critical profibrotic pathways, transforming growth factor β and connective tissue growth factor, which are potent stimulators of the accumulation of connective tissue, an effect characteristic of the fibrotic condition. As it has emerged in other pathological conditions, we suggests that muscle fibrosis may be a consequence of alterations of Wnt signaling activity.Molecular Neurobiology 09/2013; · 5.29 Impact Factor
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ABSTRACT: Cumulative evidence indicates that Wnt pathways play crucial and diverse roles to assemble the neuromuscular junction (NMJ), a peripheral synapse characterized by the clustering of acetylcholine receptors (AChR) on postsynaptic densities. The molecular determinants of Wnt effects at the NMJ are still to be fully elucidated. We report here that the Wnt receptor Frizzled-9 (Fzd9) is expressed in developing skeletal muscles during NMJ synaptogenesis. In cultured myotubes, gain-and loss-of-function experiments revealed that Fzd9-mediated signaling impairs the AChR-clustering activity of agrin, an organizer of postsynaptic differentiation. Overexpression of Fzd9 induced the cytosolic accumulation of β-catenin, a key regulator of Wnt signaling. Consistently, Fzd9 and β-catenin localize in the postsynaptic domain of embryonic NMJs in vivo. Our findings represent the first evidence pointing to a crucial role of a Fzd-mediated, β-catenin-dependent signaling on the assembly of the vertebrate NMJ.Frontiers in Cellular Neuroscience 04/2014; · 4.18 Impact Factor
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ABSTRACT: Triphosphonate cavitand, 3iPO, has been designed for the recognition of ammonium in solution. This Letter presents a study of the selective guest–host recognition between Acetylcholine and Choline ions, ACh+ and Ch+, and the host. We are able to observe that the ACh+ ion replaces the Ch+ ion in the 3iPO-Ch+ complexes with a concomitant exchange/substitution constant of 7 × 104 M−1. Conversely a replacement of the ACh+ ions by the Ch+ ions in the 3iPO-ACh+ complexes has been also observed with a concomitant exchange constant of 5.8 × 103 M−1. The structure of the 3iPO-ACh+ complex is determined by single crystal X-ray crystallography.Chemical Physics Letters 04/2012; 533:82–86. · 1.99 Impact Factor
Acetylcholine negatively regulates development of
the neuromuscular junction through distinct
Mahru C. Ana, Weichun Linb, Jiefei Yangc, Bertha Domingueza, Daniel Padgettb, Yoshie Sugiurac, Prafulla Aryala,
Thomas W. Goulda,d, Ronald W. Oppenheimd, Mark E. Hestere,f, Brian K. Kaspare,f, Chien-Ping Koc, and Kuo-Fen Leea,1
aThe Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, La Jolla, CA 92037;bDepartment of Neuroscience,
University of Texas Southwestern Medical Center, Dallas, TX 75390;cNeurobiology Section, Department of Biological Sciences, University of Southern
California, Los Angeles, CA 90089;dDepartment of Neurobiology and Anatomy, The Neuroscience Program, Wake Forest University School of Medicine,
Winston-Salem, NC 27157;eThe Research Institute, Nationwide Children’s Hospital, Columbus, OH 43205; andfIntegrated Biomedical Science and
Neuroscience Graduate Programs, Ohio State University, Columbus, OH 43210
Communicated by Stephen F. Heinemann, The Salk Institute for Biological Studies, La Jolla, CA, April 14, 2010 (received for review February 1, 2010)
Emerging evidence suggests that the neurotransmitter acetylcho-
line (ACh) negatively regulates the development of the neuromus-
cular junction, but it is not clear if ACh exerts its effects exclusively
through muscle ACh receptors (AChRs). Here, we used genetic
methods to remove AChRs selectively from muscle. Similar to the
effects of blocking ACh biosynthesis, eliminating postsynaptic
AChRs increased motor axon branching and expanded innerva-
tion territory, suggesting that ACh negatively regulates synaptic
growth through postsynaptic AChRs. However, in contrast to
the effects of blocking ACh biosynthesis, eliminating postsynaptic
AChRs in agrin-deficient mice failed to restore deficits in pre- and
postsynaptic differentiation, suggesting that ACh negatively regu-
lates synaptic differentiation through nonpostsynaptic receptors.
Consistent with this idea, the ACh agonist carbachol inhibited
presynaptic specializationofmotorneurons in vitro. Together,these
data suggest that ACh negatively regulates axon growth and pre-
synaptic specialization at the neuromuscular junction through dis-
tinct cellular mechanisms.
negative regulation|postsynaptic|presynaptic|retrograde signal|
ological function but also play a developmental role in the pat-
terning and formation of the synapses that they subserve (1–3).
For example, the neurotransmitter acetylcholine (ACh) is released
from embryonic motor neurons (MNs) at the neuromuscular
junction (NMJ), and it negatively regulates survival, axon branch-
ing, and synapse formation (4–7). ACh is also released from de-
veloping neurons even before they arrive at their target, and it acts
in an autocrine and/or paracrine fashion to regulate growth (8),
process of synapse formation, the cellular and molecular mecha-
part because the expression of ACh receptor (AChR) subtypes
varies over space and time during the development of this synapse.
For example, each of the constituent parts of the NMJ (including
the MN-derived presynaptic nerve terminal, the muscle-derived
postsynaptic apparatus, and the perisynaptic Schwann cell) expres-
ses AChR (12, 13). Therefore, in this study, we used genetic tech-
niques to study the cellular mechanism by which ACh regulates
formation of the vertebrate NMJ.
Development of the NMJ can be divided into the following
five stages: (i) nerve-independent establishment of AChR accu-
mulations within broad central regions of muscle, (ii) nerve-
dependent refinementofthese clusters and restriction ofincoming
motor innervationto narrow central endplate bands of muscle,(iii)
motor axon branching onto specific regions of individual muscle
ecent genetic evidence in flies, fish, nematodes, and mammals
suggests that neurotransmitters not only mediate adult physi-
fibers, (iv) presynaptic specialization of motor nerve terminals, and
(v) postsynaptic stabilization of innervated AChR clusters. ACh
negatively regulates the second and third (synaptic growth) steps,
because mice lacking the ACh synthetic enzyme choline acetyl-
transferase (ChAT) exhibit increased motor endplate bandwidth
and motor axon branching (14, 15). ACh also negatively regulates
the fourth and fifth (synaptic differentiation) steps, because mice
lacking ChAT and agrin reverse the deficit in these steps that is
exhibited by mice lacking agrin alone (6, 7).
Because ACh is removed from all cell types and tissues in
ChAT mutants, it remains to be shown if ACh exerts its effects on
the NMJ through AChRs expressed by nerve, muscle, or Schwann
cells. Furthermore, because it is widely assumed that the regula-
tion of presynaptic specialization is largely dependent on stabili-
zation of the postsynaptic apparatus (16, 17), ACh may exert its
For example, nerve-derived agrin is believed to antagonize the
declustering effects of nerve-derived ACh at the postsynaptic
apparatus, and in so doing, it causes the stabilized apparatus to
release a retrograde signal that induces specialization of proximal
presynaptic terminals (6, 7). Therefore, this bottom-up model
implies that ACh negatively regulates presynaptic differentiation
indirectly and downstream of its effects on postsynaptic differ-
entiation. Alternatively, ACh could regulate presynaptic special-
ization directly by activating AChRs expressed by presynaptic
terminals or indirectly through Schwann cell-derived AChRs. To
address this issue, we studied the effect of removing exclusively
muscle-derived postsynaptic AChRs on synaptic growth and dif-
ferentiation. Available data show that embryonic muscle expres-
ses two types of nicotinic AChR complexes that use AChRα1
or AChRα7 as the ligand-binding subunits (18, 19), but no other
nicotinic AChR complexes or muscarinic AChR subtypes are used
(13, 20–22). Therefore, we analyzed mice deficient in AChRα1 and/
or AChRα7 subunits.
AChRα1 Mutants Lack a Functional Muscle AChR Complex and AChR
nor protein is detected in these mice, mRNA for other subunits,
such as the AChRδ subunit, is expressed; however, the protein is
not aggregated on the muscle cell membrane (Fig. S2 A and B).
Authorcontributions:K.-F.L.designed research; M.C.A., W.L., J.Y.,B.D., D.P.,Y.S.,P.A., T.W.G.,
B.K.K., C.-P.K., and K.-F.L. analyzed data; and M.C.A., T.W.G., and K.-F.L. wrote the paper.
The authors declare no conflict of interest.
1To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| June 8, 2010
| vol. 107
| no. 23www.pnas.org/cgi/doi/10.1073/pnas.1004956107
Electrophysiological analysis showed that spontaneous minia-
ture and nerve-evoked endplate potentials are not detected in
AChRα1 mutant muscle (Fig. 1 A and B). In contrast, mice de-
ficient in the AChRα7 subunit exhibit normal AChR clusters in
muscle (23). Consistent with the absence of synaptic transmis-
sion, when stained with Texas Red-conjugated α-bungarotoxin
(TR-αBTX), whole-mount preparations of E17.5 mouse dia-
phragm from AChRα1 mutants also fail to exhibit AChR clus-
tering (Fig. 2). These results contrast with those obtained from
mice deficient in the AChRγ subunit (23, 24), the AChRε subunit
(25, 26), and phosphorylation of the AChRβ subunit (3, 27), in
which a varying degree of AChR clustering is detected; thus, this
supports the idea that the α1 subunit is necessary for other sub-
units to be located to the membrane (28). Together, these data
provide anatomical and physiological evidence that AChRs are
absent in muscle of AChRα1 null mutant mice. Remarkably,
subunit display a similar phenotype (29, 30).
Loss of Muscle Receptors Results in Increased Nerve Branching and
Motor Neuron Number. Mice lacking ChAT display increased
endplate bandwidth, motor axon branching, and MN number (14,
15). To determine if these effects are mediated by postsynaptic
AChRs, we immunostained diaphragm muscles with neurofila-
ment (NF) antibodies (to assess branching) and quantified the
number of vesicular acetylcholine (VAChT) mRNA-positive
cells in the lumbar lateral motor column (to assess MN number)
of AChRα1 mutant mice. In contrast to the centrally restricted
pattern of innervation observed in the control diaphragm at
E17.5, AChRα1 mutant mice exhibit a greater number of nerve
branches that supply a wider region of muscle (Fig. 1 C–F).
Additionally, MN number is increased by about 60% in both
ChAT and AChRα1 mutants compared with wild type (WT) (Fig.
1G). These results are similar in magnitude to those reported in
ChAT mutants and support the idea that postsynaptic AChRs
limit the growth, branching, and survival of developing MNs;
additionally, they are consistent with those results observed in
chicken embryos treated with a toxin that selectively blocks fetal
muscleAChRs(31), butthey contrast withthe resultsobtained on
studies of primary MNs in zebrafish nic-1 mutants (29).
Muscle Receptors Are Not Required for Presynaptic Specialization. In
mice lacking muscle-specific kinase (MuSK) or agrin, AChR is not
or elaborate specialized synaptic terminals, suggesting that AChR
clustering may be required for presynaptic differentiation (16, 17).
To test this idea, we costained E17.5 diaphragm with TR-αBTX
and antibodies against the synaptic vesicle protein synaptophysin
(Syn) (Fig. 2). In control diaphragms, Syn is highly concentrated in
nerve terminals closely opposed to AChR clusters. Surprisingly,
Syn immunoreactivity is accumulated in presynaptic nerve termi-
the higher level ofpunctate staining observed in the nerve terminal
is clearly distinct from the lower level of diffuse reactivity charac-
teristic of axonal staining (32, 33). Furthermore, when antibodies
against the synaptic vesicle protein SV2 were used, similar results
were obtained (Fig. S3B). Finally, accumulations of synaptic vesi-
cles within presynaptic nerve terminals are observed by electron
microscopy (EM) in AChRα1 mutant mice (Fig. S4B; SI Results
and Table S1 show additional EM analysis). Similar to the pattern
of NF-immunostaining, Syn-rich nerve terminals occupy a broader
region of the diaphragm. These results indicate that although
agrin–MuSK signaling is necessary for both expression of AChR in
postsynaptic clusters and specialization of presynaptic nerve ter-
minals, expression of AChR in clusters per se is not an essential
intermediary in this process. Interestingly, synaptic vesicles are
which are deficient in the AChRδ subunit (34, 35). Although
syntrophin isoforms, and rapsyn (36–38), we found that AChE and
MuSK are, whereas rapsyn is not, clustered at the postsynaptic
membrane (Fig. S3), consistent with previous findings (35, 39–41).
perinnervation, and increased motor neuron number in
AChRα1 mutant mice. (A) Spontaneous miniature end-
plate potentials (MEPPs) were observed in control (+/+)
diaphragm but not in AChRα1 mutant (AChRα1−/−) di-
aphragm. One MEPP is expanded below. (B) Nerve-evoked
endplate potentials (EPPs) were observed in control but
not in mutant muscle. (C–F) E17.5 whole-mount di-
aphragm muscles from controls and AChRα1 mutants
were immunostained with antineurofilament (anti-NF)
antibodies (green). Both (C and D) low- and (E and F)
high-power magnifications showed that the phrenic
nerve is highly branched in mutant muscle (D and F).
(Scale bars: 200 μm for C and D; 100 μm for E and F.) (G) A
similar increase in the number of lumbar motor neurons
was observed in ChAT (ChAT−/−) and AChRα1 mutants
compared with controls. **, P < 0.01.
Postsynaptic transmission deficiency, muscle hy-
An et al.PNAS
| June 8, 2010
| vol. 107
| no. 23
ACh Inhibits Pre- and Postsynaptic Differentiation Through Nonpost-
synaptic AChR. Previous studies have shown that whereas agrin
mutant mice lack both pre- and postsynaptic differentiation (42),
removing ACh from agrin mutants by deleting ChAT restores
both of these deficits (6, 7). The presence of presynaptic special-
ization in ChAT/agrin double mutants may be indirect and result
from the restoration of postsynaptic differentiation, or ACh may
be direct and inhibit presynaptic specialization through non-
postsynaptic receptors. In striking contrast to ChAT/agrin double
mutants (Fig. 3C and Table S2), AChRα1/agrin double mutants
fail to exhibit punctate Syn immunostaining and thus, presynaptic
specialization (Fig. 3A). To exclude the possibility that the
AChRα7 subunit, which is transiently expressed in embryonic
muscle at low levels and nonpostsynaptic sites, plays a compensa-
tory role to mediate inhibition by ACh in AChRα1/agrin double
mutants, we analyzed AChRα1/AChRα7/agrin triple mutant mice.
Presynaptic specialization was not observed in either AChRα7/
agrin double or AChRα1/AChRα7/agrin triple mutants (Fig. S5).
Finally, to determine if AChRα1/agrin double mutants maintain
postsynaptic differentiation even in the absence of presynaptic
differentiation, we examined the expression of AChE and MuSK
in E17.5 muscle. In contrast to results obtained from ChAT/agrin
double mutant mice (7), both AChRα1/agrin (Fig. 3B and Fig. S6)
and AChRα1/AChRα7/agrin mutants fail to exhibit clustering of
either AChE or MuSK. Therefore, because (α7)5and (α1)2βδγ
pentamers are the only AChR complexes present in embryonic
muscle (18, 19), our results indicate that ACh inhibits both pre-
and postsynaptic differentiation by a mechanism that does not
involve postsynaptic receptor clusters; instead, we suggest that the
inhibitory activity is likely mediated by AChRs on nerve terminals
or Schwann cells.
Agrin Induces Expression of Fibroblast Growth Factors. The presence
of an alternative pathway by which ACh negatively regulates
synaptic differentiation prompted us to examine if and how agrin
might antagonize this pathway. Recent studies show that muscle-
derived organizing molecules, including selective members of the
fibroblast growth factor (FGF) family, play essential roles in
regulating presynaptic specialization (43, 44). To investigate if
the retrograde signal induced by agrin was a member of this
family, RNA was isolated from C2C12 myotubes treated with
trol (+/+; A, C, and E) and AChRα1 mutant (AChRα1−/−; B, D, and F) E17.5
whole-mount diaphragm muscles were immunostained with antisynapto-
physin antibodies (A, B, and green in E and F) and costained with Texas
Red-conjugated α-bungarotoxin (C, D, and red in E and F). Synaptophysin
immunoreactivity is accumulated at the nerve terminals (arrows in A)
and colocalized with receptor clusters in control diaphragms. Mutant dia-
phragms lack receptor clusters but maintain synaptophysin accumulations
at the nerve terminals. (Scale bar: 50 μm.)
Presynaptic nerve terminals differentiate in AChRα1 mutants. Con-
AChRα1/AGD mutants were immunostained with antibodies against synaptophysin. Presynaptic specialization is not observed in either AGD single or AChRα1/
AGD double mutants. (Scale bar: 100 μm.) (B) Absence of AChE clusters in AGD and AChRα1/AGD mutants. (Scale bar: 200 μm.) (C) Summary analysis of
accumulation of synaptic vesicles in control, AGD, AChRα1−/−, AChRα1−/−, AGD, and ChAT−/−, AGD mutants.
Absence of presynaptic differentiation in AChRα1/agrin double mutants. (A) Diaphragm muscles from control, agrin-deficient (AGD), AChRα1, and
| www.pnas.org/cgi/doi/10.1073/pnas.1004956107An et al.
agrin and subjected to real-time quantitative PCR analysis. As
shown in Fig. 4B, mRNA levels for FGF7 and FGF9 are sig-
nificantly elevated after agrin treatment. Conversely, levels of
FGF7 and FGF9 mRNA are reduced in agrin mutant diaphragm
compared with control (Fig. 4C). These results show that agrin
regulates expression of FGFs in muscle and suggest that these
molecules may oppose the negative effects of nonpostsynaptic
ACh signaling on presynaptic differentiation.
ACh Agonist Disperses FGF-Induced Synaptic Vesicle-Rich Varicosities.
To show that ACh inhibits presynaptic specialization through non-
postsynaptic AChRs, we used embryonic stem (ES) cell-derived
MNs to determine if ACh inhibits FGF-induced presynaptic spe-
cialization (45). The HBG3 (HB9-green fluorescence protein) ES
cell line was derived from transgenic mice expressing an enhanced
GFP under the control of the MN-specific homeobox protein 9
promoter (45). MNs were treated overnight with FGF9 or FGF22
to induce aggregation of synaptic vesicles, washed with medium to
remove FGFs, and then treated with the ACh agonist carbachol
(CCh). Consistent with previous studies (43, 44), FGFs induce
formation of Syn- and SV2-immunoreactive varicosities (Fig. 5A,
arrows), but treatment with CCh markedly reduces the number of
these varicosities (Fig. 5 A and B). Interestingly, FGFs are in-
capable of preventing inhibition of CCh-induced dispersion of
varicosities (Fig. 5B). These results support the idea that ACh
inhibits presynaptic specialization directly by activating presynaptic
AChRs such as those present on motor axons.
In this study, we provide genetic evidence that ACh negatively
regulates synaptic growth and differentiation by distinct cellular
mechanisms. Specifically, ACh inhibits motor endplate band-
width and motor axon branching (synaptic growth) by activating
postsynaptic AChRs, and it inhibits presynaptic nerve terminal
specialization and postsynaptic AChR clustering (synaptic dif-
ferentiation) by activating nonpostsynaptic AChRs. A schematic
model summarizing these findings is presented in Fig. S7. These
results are unexpected and have several important implications.
First, they strengthen the hypothesis that aneural AChR clusters
detected at E14.5 along a narrow central band of muscle are
a component of the muscle intrinsic mechanism for prepattern-
ing of neuromuscular synapses (46). We suggest that their func-
tion is to restrict nerve branching and nerve terminal growth
within a limited, central region of muscle fiber, thereby con-
tributing to the control of the boundary for the formation and
distribution of mature synapses (15). Second, the effects of
AChRα1 inactivation on branching and survival strengthen the
idea that MN activity regulates these aspects of development
through postsynaptic AChR and thus, a peripheral mechanism,
at least in chick and mouse (47). These findings are, therefore,
consistent with the neurotrophic tenet that retrograde distribu-
tion by muscle of branching- and survival-promoting molecules is
regulated by MN activity. Interestingly, results obtained from
studies of primary MNs in zebrafish AChRα1 mutants failed to
show an effect on MN branching and terminal arborization,
which may reflect species differences or unique characteristics
of zebrafish primary MNs (29). Indeed, recent evidence suggests
that the cues regulating primary and secondary motor axon
branching in zebrafish are different (48, 49).
In contrast to its effects on endplate bandwidth and branching,
ACh inhibits pre- and postsynaptic differentiation through non-
postsynaptic AChRs in the absence of postsynaptic AChR and
agrin. This is unexpected, because ACh directly inhibits post-
synaptic differentiation by activating and dispersing postsynaptic
AChR clusters (50). Together with the findings that agrin is re-
quired not only for postsynaptic but also presynaptic differenti-
ation (42) and that ChAT/agrin double mutants restore these
pre- and postsynaptic deficits, these data led to the proposition
that agrin at the NMJ inhibits the declustering activity of ACh (6,
7). According to this model, presynaptic specialization occurs
only in nerve terminals closely opposed to these agrin-stabilized
postsynaptic AChR clusters and thus, is downstream of post-
synaptic differentiation (16). This bottom-up model is also con-
sistent with the idea that agrin-stabilized AChR clusters release
a retrograde factor that specializes presynaptic terminals (43,
44). However, the current results suggest that this model may be
too simple and that a parallel top-down pathway also exists to
regulate NMJ development. Therefore, whereas ACh may dis-
perse agrin-unstabilized AChR clusters directly through inter-
actions with postsynaptic AChRs, ACh also eliminates these
clusters indirectly in the absence of agrin through interactions
with nonpostsynaptic AChRs, presumably through an indirect,
orthograde signal released from unspecialized nerve terminals.
The extent to which this indirect, top-down pathway occurs ex-
(i.e., only in the absence of the bottom-up pathway) in control
animals is still unclear, and it will require the selective removal of
presynaptic and/or perisynaptic AChRs in agrin mutants.
These studies also suggest that, in addition to opposing the
direct postsynaptic ACh pathway by stabilizing nerve terminal-
FGF7 and FGF9 mRNAs are expressed in E18.5 diaphragm muscles. The results illustrate that the primer sets used for the RT-PCR experiments are specific
because no signal is detected when reverse transcriptase (RT) is omitted (No RT) in the reaction. For the simplicity of presentation, we cropped the original
scan to show only FGF7 and FGF9 expression in control samples. (B) Real-time quantitative RT-PCR showed that agrin induces expression of FGF7 and FGF9
mRNA in C2C12 myotube cultures. Expression level of control without agrin treatment is set as 100%. The results were expressed as percentage of control
(n = 4). *, P < 0.05; **, P < 0.01. (C) RNA was isolated from E18.5 controls or agrin mutant muscles for real-time quantitative RT-PCR analysis. Expression level
of control embryos is set as 100%. The results were expressed as percentage of control embryos. The results showed reduced levels of FGF7 and FGF9 mRNA in
muscle from agrin mutants, relative to controls (n = 4). *, P < 0.05; **, P < 0.01.
Induced FGF mRNA expression by agrin in C2C12 myotubes and decreased FGF mRNA expression in agrin mutant muscle. (A) RT-PCR showed that
An et al.PNAS
| June 8, 2010
| vol. 107
| no. 23
opposedAChR clusters,agrinis capableofinhibiting theindirect,
presynaptic ACh pathway; mice lacking only AChRα1 exhibit
normalexpression of MuSK,AChE, andSyn at theNMJ, whereas
those lacking both AChRα1 and agrin do not. Because nerve-
derived agrin acts on muscle receptors and because the indirect
ACh pathway originates within Schwann cells or motor axon
terminals, the ability of agrin to inhibit this latter pathway is likely
mediated by the induction of a retrograde signal from muscle to
presynaptic nerve terminal. However, compared with the retro-
grade factor hypothesized to induce presynaptic specialization
(bottom-up pathway), this signal more likely achieves presynaptic
specialization through the disinhibition of ACh effects through
nonpostsynaptic AChRs. Moreover, whereas the retrograde fac-
tor in the bottom-up pathway is proposed to be dependent on
agrin-mediated stabilization of postsynaptic AChR clusters, the
retrograde signal in the top-down pathway is released through an
agrin-induced mechanism independent of cluster stabilization;
this is because clusters are not stabilized in the AChRα1/agrin
double mutants in which this pathway is revealed (Fig. S7). In
support of the idea that agrin induces the release of a retro-
grade signal capable of disinhibiting presynaptic specialization,
we found that agrin induces FGF expression in muscle and con-
versely, that muscle from agrin-deficient mice expresses less
FGF than muscle from control mice. However, other retrograde
factors such as β-catenin are required for antagonizing ACh-
induced inhibition of presynaptic differentiation such as β-catenin–
dependent signals (51).
Although the data in this study strongly support the idea that
ACh regulates NMJ formation in nAChRα1-deficient mice
by nonpostsynaptic nicotinic or muscarinic AChRs, it remains
a formal possibility that ACh regulates postsynaptic muscarinic
AChRs, although evidence supporting the embryonic expression
of muscle-derived mAChRs has not been reported. ACh may also
mediate these effects by activating nonpostsynaptic muscarinic
AChRs in embryonic MNs or Schwann cells, because distinct
muscarinic AChR subtypes are expressed by adult MNs, Schwann
cells, and muscle (13, 21, 22, 52) and play a role in the stability of
adult NMJs (52). A more detailed analysis of both nicotinic and
muscarinic AChRs in embryonic NMJ cell types will help clarify
the molecular mechanism by which ACh regulates presynaptic
differentiation in nAChRα1-deficient mice. Similarly, whereas
the similarity of electrophysiological results between nAChRα1
and ChAT null mutant mice strongly suggests that the effects of
deleting nAChRα1 are caused by the lack of ACh-mediated
neurotransmission at the NMJ, it is possible that other ACh-in-
dependent events, such as altered muscle development, may
contribute to these effects.
In conclusion, our results show that although ACh nega-
tively regulates synaptic growth exclusively through postsynaptic
AChRs, ACh inhibits synaptic differentiation by activating non-
postsynaptic AChRs.Thesedata revealanunexpectedcomplexity
in the mechanism by which ACh regulates synaptic differentiation
and suggest that ACh directly prevents the specialization of pre-
synaptic terminals not opposing a postsynaptic apparatus in ad-
dition to directly eliminating postsynaptic AChR clusters that are
not opposed to presynaptic terminals,. Conversely, agrin directly
terminals and indirectly stimulates the presynaptic specialization
of nerve terminals opposing a postsynaptic apparatus. This re-
ciprocal control of pre- and postsynaptic elements of the deve-
loping NMJ by positive and negative nerve-derived signals may
represent a homeostatic mechanism preventing the development
of inappropriate and thus deleterious neuromuscular circuits.
Materials and Methods
Mice. AChRα1 mutant mice were generated by standard procedures. For
details, see SI Materials and Methods.
Electrophysiology. Intracellular sharp-electrode recording was performed
blind to genotype on phrenic nerve/diaphragm preparations from E17.5
embryos. For details, see SI Materials and Methods.
Motor Neuron Counts. The total number of VAChT mRNA-positive moto-
neurons in the lumbar spinal cord was quantified according to details avail-
able in SI Materials and Methods.
gates of synaptic vesicles by the ACh ago-
nist carbachol in ES-derived motor neu-
rons. (A) Compared with control cultures
of ES cell-derived motor neurons (Con-
trol), treatment with the ACh agonist
CCh did not induce aggregation of syn-
aptic vesicles in motor neurons (CCh). ES-
derived motor neurons treated with FGF9
exhibited synaptic vesicle-rich varicosities
(FGF9; arrows). CCh destabilized FGF-
induced aggregation of synaptic vesicles
(FGF9/CCh). (B) Quantitative analysis of
the effect of CCh in the maintenance of
synaptic varicosity (n = 4). **, P < 0.01.
Dispersion of FGF-induced aggre-
| www.pnas.org/cgi/doi/10.1073/pnas.1004956107An et al.
Embryonic Stem Cell-Derived Motor Neurons, FGF, and CCh Treatments. HBG3
mouse ES cells were used to generate motor neurons according to details
described in SI Materials and Methods.
for labeling fasciculin with fluorochrome to detect AChE clusters, Steven
Burden for the in situ probes and anti-MuSK antibodies, Zuo-Zhong Wang for
anti-AChRδ subunit antibodies, Stanley Froehner and Margaret Maimone for
Jessell for HBG3 cells. This work was supported by a fellowship from the
Muscular Dystrophy Association (J.Y.) and grants from the Robert Packard
Center for ALS Research (R.W.O.) and the Muscular Dystrophy Association
(K.-F.L.). This work was also supported by National Institutes of Health Grants
HD034534, NS047345, and NS044420 (to K.-F.L.) and NS055028 (to W.L.).
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An et al.PNAS
| June 8, 2010
| vol. 107
| no. 23