Patterning of muscle acetylcholine receptor gene expression in the absence of motor innervation.

The patterning of skeletal muscle is thought to depend upon signals provided by motor neurons. We show that AChR gene expression and AChR clusters are concentrated in the central region of embryonic skeletal muscle in the absence of innervation. Neurally derived Agrin is dispensable for this early phase of AChR expression, but MuSK, a receptor tyrosine kinase activated by Agrin, is required to establish this AChR prepattern. The zone of AChR expression in muscle lacking motor axons is wider than normal, indicating that neural signals refine this muscle-autonomous prepattern. Neuronal Neuregulin-1, however, is not involved in this refinement process, nor indeed in synapse-specific AChR gene expression. Our results demonstrate that AChR expression is patterned in the absence of innervation, raising the possibility that similarly prepatterned muscle-derived cues restrict axon growth and initiate synapse formation.

Neuregulin-1-stimulated phosphorylation of GABP in skeletal muscle cells.

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated, in part, through selective transcription of AChR genes in myofiber synaptic nuclei. Neuregulin-1 (NRG-1) is a good candidate for the extracellular signal that induces synapse-specific gene expression, since NRG-1 is concentrated at synaptic sites and activates AChR synthesis in cultured muscle cells. NRG-1-induced transcription requires activation of Erk and Jnk MAP kinases, but the downstream substrates that mediate this transcriptional response are not known. Previous studies have demonstrated that a consensus binding site for Ets proteins is required both for NRG-1-induced transcription and for synapse-specific transcription in transgenic mice. This regulatory element binds GABPalpha, an Ets protein, and GABPbeta, a protein that dimerizes with GABPalpha, raising the possibility that phosphorylation of GABP by MAP kinases induces transcription of AChR genes. To determine whether MAP kinases might directly regulate the activity of GABP, we studied MAP kinase-catalyzed and NRG-1-induced phosphorylation of GABPalpha and GABPbeta. We show that GABPalpha and GABPbeta are phosphorylated in vitro by Erk and by Jnk. Using recombinant proteins containing mutated serine and threonine resides, we show that GABPalpha is phosphorylated predominantly at threonine 280, while serine 170 and threonine 180 are the major phosphorylation sites in GABPbeta. We generated antibodies specific to the major phosphorylation site in GABPalpha and show that NRG-1 stimulates phosphorylation of GABPalpha at threonine 280 in vivo. These results suggest that GABPalpha is a target of MAP kinases in NRG-1-stimulated muscle cells and are consistent with the idea that phosphorylation of GABPalpha contributes to transcriptional activation of AChR genes by NRG-1.

Src, Fyn, and Yes are not required for neuromuscular synapse formation but are necessary for stabilization of agrin-induced clusters of acetylcholine receptors.

Mice deficient in src and fyn or src and yes move and breathe poorly and die perinatally, consistent with defects in neuromuscular function. Src and Fyn are associated with acetylcholine receptors (AChRs) in muscle cells, and Src and Yes can act downstream of ErbB2, suggesting roles for Src family kinases in signaling pathways regulating neuromuscular synapse formation. We studied neuromuscular synapses in src(-/-); fyn(-/-) and src(-/-); yes(-/-) mutant mice and found that muscle development, motor axon pathfinding, clustering of postsynaptic proteins, and synapse-specific transcription are normal in these double mutants, showing that these pairs of kinases are not required for early steps in synapse formation. We generated muscle cell lines lacking src and fyn and found that neural agrin and laminin-1 induced normal clustering of AChRs and that agrin induced normal tyrosine phosphorylation of the AChR beta subunit in the absence of Src and Fyn. Another Src family member, most likely Yes, was associated with AChRs and phosphorylated by agrin in myotubes lacking Src and Fyn, indicating that Yes may compensate for the loss of Src and Fyn. Nevertheless, PP1 and PP2, inhibitors of Src-class kinases, did not inhibit agrin signaling, suggesting that Src class kinase activity is dispensable for agrin-induced clustering and tyrosine phosphorylation of AChRs. AChR clusters, however, were less stable in myotubes lacking Src and Fyn but not in PP1- or PP2-treated wild-type cells. These data show that the stabilization of agrin-induced AChR clusters requires Src and Fyn and suggest that the adaptor activities, rather than the kinase activities, of these kinases are essential for this stabilization.

The in vitro and in vivo phosphotyrosine map of activated MuSK.

The muscle-specific receptor tyrosine kinase MuSK plays a crucial role in neuromuscular synapse formation. Activation of MuSK is induced by agrin leading to clustering of several proteins, including acetylcholine receptors, at synaptic sites. In a first step to elucidate the signal transduction cascade following MuSK activation and leading to clustering of synaptic proteins, we sought to identify the tyrosine residues that are phosphorylated in activated MuSK. We mapped the tyrosine residues that are phosphorylated in vitro and in vivo using methods that provide high sensitivity and do not require radioactive tracers. We expressed MuSK in insect cells by using a baculovirus expression vector and mapped the tyrosines that are phosphorylated in MuSK in an in vitro kinase assay using matrix-assisted laser desorption ionization MS to sequence tryptic peptides fractionated by HPLC. In addition, we isolated MuSK from Torpedo electric organ and used nanoelectrospray tandem mass spectrometry and parent ion scanning to identify the tyrosine residues that are phosphorylated in activated, endogenous MuSK in vivo. We found that six of the nineteen intracellular tyrosine residues in MuSK are phosphorylated in activated MuSK: the juxtamembrane tyrosine (Y553), the tyrosines within the activation loop (Y750, Y754, and Y755), a tyrosine near the beginning of the kinase domain (Y576), and a tyrosine (Y812) within the C-terminal lobe of the kinase domain. Our biochemical data are consistent with results from functional experiments and establish a good correlation between tyrosine residues that are phosphorylated in activated MuSK and tyrosines that are required for MuSK signaling.

The juxtamembrane region of MuSK has a critical role in agrin-mediated signaling.

MuSK is a receptor tyrosine kinase expressed selectively in skeletal muscle and localized to neuromuscular synapses. Agrin activates MuSK and stimulates phosphorylation and clustering of acetylcholine receptors (AChRs) at synaptic sites. We expressed wild-type or mutant MuSK in MuSK(-/-) myotubes and identified tyrosine residues in the MuSK cytoplasmic domain that are necessary for agrin-stimulated phosphorylation and clustering of AChRs. The activation loop tyrosines and the single juxtamembrane tyrosine were found to be essential for agrin-stimulated phosphorylation and clustering of AChRs. Further, we show that the juxtamembrane tyrosine, contained within an NPXY motif, is phosphorylated in vivo by agrin stimulation. We constructed chimeras containing extracellular and transmembrane domains from MuSK and cytoplasmic sequences from TrkA and found that inclusion of 13 amino acids from the MuSK juxtamembrane region, including the NPXY motif, is sufficient to convert a phosphorylated but inactive MuSK-TrkA chimera into a phosphorylated active chimera. These data suggest that phosphorylation of the MuSK NPXY site leads to recruitment of a phosphotyrosine-binding domain-containing protein that functions to stimulate phosphorylation and clustering of AChRs.

DNA topoisomerase IIbeta and neural development.

DNA topoisomerase IIbeta is shown to have an unsuspected and critical role in neural development. Neurogenesis was normal in IIbeta mutant mice, but motor axons failed to contact skeletal muscles, and sensory axons failed to enter the spinal cord. Despite an absence of innervation, clusters of acetylcholine receptors were concentrated in the central region of skeletal muscles, thereby revealing patterning mechanisms that are autonomous to skeletal muscle. The defects in motor axon growth in IIbeta mutant mice resulted in a breathing impairment and death of the pups shortly after birth.

Transcriptional pathways for synapse-specific, neuregulin-induced and electrical activity-dependent transcription.

Innervation-dependent expression of acetylcholine receptor (AChR) genes in skeletal muscle is mediated by multiple transcriptional pathways. One pathway leads to activation of AChR genes selectively in synaptic nuclei and requires an Ets binding site that binds GABP. A second pathway leads to repression of AChR transcription in nuclei throughout the myofiber and requires inactivation of E-box-binding proteins, including myogenic bHLH proteins. Taken together, these studies indicate that separate pathways regulate innervation-dependent transcription.

Neuregulins and erbB receptors at neuromuscular junctions and at agrin-induced postsynaptic-like apparatus in skeletal muscle.

We demonstrate by immunohistochemistry that at least two isoforms of neuregulin (NRG) are concentrated at neuromuscular junctions in adult rat muscles. One is NRGbeta3, a secreted protein which is bound to basal lamina that occupies the synaptic cleft. The other(s), NRG-a, is in the muscle fibers' plasma membrane. We show further that muscle NRG, including NRG-a, is concentrated at postsynaptic-like apparatus induced to form in the extrajunctional region of the soleus muscle by exposure to neural agrin. The agrin-induced postsynaptic-like apparatus also includes aggregates of the NRG receptors erbB2 and erbB3 as does postsynaptic apparatus at neuromuscular junctions. These findings together with those of others suggest a mechanism by which neural agrin induces the expression of epsilon-AChR subunits in postsynaptic-like apparatus, and they support the hypothesis that agrin has a similar function at neuromuscular junctions.

Synapse-specific and neuregulin-induced transcription require an ets site that binds GABPalpha/GABPbeta.

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated by multiple pathways. Agrin, which is the signal for one pathway, stimulates a redistribution of previously unlocalized AChRs to synaptic sites. The signal for a second pathway is not known, but this signal stimulates selective transcription of AChR genes in myofiber nuclei located near the synaptic site. Neuregulin (NRG) is a good candidate for the extracellular signal that induces synapse-specific gene expression, since NRG is concentrated at synaptic sites and activates AChR gene expression in cultured muscle cells. Previous studies have demonstrated that 181 bp of 5' flanking DNA from the AChR delta-subunit gene are sufficient to confer synapse-specific transcription in transgenic mice and NRG responsiveness in cultured muscle cells, but the critical sequences within this cis-acting regulatory region have not been identified. We transfected AChR delta-subunit-hGH gene fusions into a muscle cell line, and we show that a potential binding site for Ets proteins is required for NRG-induced gene expression. Furthermore, we produced transgenic mice carrying AChR delta-subunit-hGH gene fusions with a mutation in this NRG-response element (NRE), and we show that this NRE is necessary for synapse-specific transcription in mice. The NRE binds proteins in myotube nuclear extracts, and nucleotides that are important for NRG responsiveness are likewise critical for formation of the protein-DNA complex. This complex contains GABPalpha, an Ets protein, and GABPbeta, a protein that lacks an Ets domain but dimerizes with GABPalpha, because formation of the protein-DNA complex is inhibited by antibodies to either GABPalpha or GABPbeta. These results demonstrate that synapse-specific and NRG-induced gene expression require an Ets-binding site and suggest that GABPalpha/GABPbeta mediates the transcriptional response of the AChR delta-subunit gene to synaptic signals, including NRG.

The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo.

Formation of neuromuscular synapses requires a series of inductive interactions between growing motor axons and differentiating muscle cells, culminating in the precise juxtaposition of a highly specialized nerve terminal with a complex molecular structure on the postsynaptic muscle surface. The receptors and signaling pathways mediating these inductive interactions are not known. We have generated mice with a targeted disruption of the gene encoding MuSK, a receptor tyrosine kinase selectively localized to the postsynaptic muscle surface. Neuromuscular synapses do not form in these mice, suggesting a failure in the induction of synapse formation. Together with the results of an accompanying manuscript, our findings indicate that MuSK responds to a critical nerve-derived signal (agrin), and in turn activates signaling cascades responsible for all aspects of synapse formation, including organization of the postsynaptic membrane, synapse-specific transcription, and presynaptic differentiation.

Agrin acts via a MuSK receptor complex.

Formation of th neuromuscular junction depends upon reciprocal inductive interactions between the developing nerve and muscle, resulting in the precise juxtaposition of a differentiated nerve terminal with a highly specialized patch on the muscle membrane, termed the motor endplate. Agrin is a nerve-derived factor that can induced molecular reorganizations at the motor endplate, but the mechanism of action of agrin remains poorly understood. MuSK is a receptor tyrosine kinase localized to the motor endplate, seemingly well positioned to receive a key nerve-derived signal. Mice lacking either agrin or MuSK have recently been generated and exhibit similarly profound defects in their neuromuscular junctions. Here we demonstrate that agrin acts via a receptor complex that includes MuSK as well as a myotube-specific accessory component.

Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses.

Neuregulin (NRG) is concentrated at synaptic sites and stimulates expression of acetylcholine receptor (AChR) genes in muscle cells grown in cell culture. These results raise the possibility that NRG is a synaptic signal that activates AChR gene expression in synaptic nuclei. Stimulation of NRG receptors, erbB3 and erbB4 initiates oligomerization between these receptors or between these receptors and other members of the epidermal growth factor (EGF) receptor family, resulting in stimulation of their associated tyrosine kinase activities. To determine which erbBs might mediate synapse-specific gene expression, we used antibodies against each erbB to study their expression in rodent skeletal muscle by immunohistochemistry. We show that erbB2, erbB3 and erbB4 are concentrated at synaptic sites in adult skeletal muscle. ErbB3 and erbB4 remain concentrated at synaptic sites following denervation, indicating that erbB3 and erbB4 are expressed in the postsynaptic membrane. In addition, we show that expression of NRG and erbBs, like AChR gene expression, increases at synaptic sites during postnatal development. The localization of erbB3 and erbB4 at synaptic sites is consistent with the idea that a NRG-stimulated signaling pathway is important for synapse-specific gene expression.

Neuregulins and their receptors.

The recent identification of an activator for the ErbB2/Neu receptor has uncovered a new family of polypeptide growth factors that undoubtedly play a major role in the regulation of neuronal growth and differentiation. These factors, called the neuregulins, are expressed in neural and mesenchymal tissues, and activate members of the epidermal growth factor family of receptor tyrosine kinases. The identification and characterization of the neuregulins and their receptors will facilitate the dissection of the biochemical pathways regulating nervous system development.

Drosophila glutamate receptor RNA expression in embryonic and larval muscle fibers.

Glutamate is the excitatory transmitter at neuromuscular synapses in Drosophila, and electrophysiological studies indicate that the receptors for glutamate are concentrated in muscle fibers at synaptic sites. Acetylcholine is the excitatory transmitter at vertebrate neuromuscular synapses, and previous studies have shown that accumulation of acetylcholine receptors (AChRs) at synaptic sites is controlled both by transcriptional and post-translational mechanisms. The transcriptional pathway culminates in selective expression of AChR subunit genes in nuclei near the synaptic site, causing AChR mRNA to accumulate in the synaptic region of the muscle fiber. We used a cDNA encoding a subunit of the Drosophila muscle glutamate receptor (DGluR-II) to determine the temporal and spatial expression pattern of the DGluR-II gene during embryogenesis and in larval muscle. We show that DGluR-II mRNA is first expressed at stage 12 of embryogenesis and that expression is detected in developing dorsal, lateral, and ventral somatic muscles within the next 2 hr. By stage 16 DGluR-II mRNA is expressed in all somatic muscles and in pharyngeal muscles. In third instar larvae DGluR-II mRNA is expressed in all body-wall muscle fibers. DGluR-II mRNA, however, is expressed throughout the larval muscle fibers and is not concentrated within muscle fibers at neuromuscular synapses. These results indicate that although the DGluR-II gene is expressed in somatic muscle cells it is not selectively expressed in nuclei near the synaptic site.

Neuregulins are concentrated at nerve-muscle synapses and activate ACh-receptor gene expression.

Two different signalling pathways mediate the localization of acetylcholine receptors (AChRs) to synaptic sites in skeletal muscle. The signal for one pathway is agrin, a protein that triggers a redistribution of previously unlocalized cell surface AChRs to synaptic sites. The signal for the other pathway is not known, but this signal stimulates transcription of AChR genes in myofibre nuclei near the synaptic site. Neuregulins, identified originally as a potential ligand for erbB2 (Neu differentiation factor, NDF), stimulate proliferation of Schwann cells (glial growth factor, GGF), increase the rate of AChR synthesis in cultured muscle cells (AChR-inducing activity) and are expressed in motor neurons. These results raise the possibility that neuregulin is the signal that activates AChR genes in synaptic nuclei. Here we show that neuregulin activates AChR gene expression in C2 muscle cells and that the neuregulin response element in the AChR delta-subunit gene is contained in the same 181 base pairs that confer synapse-specific expression in transgenic mice. We use antibodies to show that neuregulins are concentrated at synaptic sites and that, like the extracellular signal that stimulates synapse-specific expression, neuregulins remain at synaptic sites in the absence of nerve and muscle. We show that C2 muscle cells contain erbB2 and erbB3 messenger RNA but little or no erbB4 mRNA, and that neuregulin stimulates tyrosine phosphorylation of erbB2 and erbB3, indicating that neuregulin signalling in skeletal muscle may be mediated by a complex of erbB2 and erbB3.

AML1 is expressed in skeletal muscle and is regulated by innervation.

Although most skeletal muscle genes are expressed at similar levels in electrically active, innervated muscle and in electrically inactive, denervated muscle, a small number of genes, including those encoding the acetylcholine receptor, N-CAM, and myogenin, are expressed at significantly higher levels in denervated than in innervated muscle. The mechanisms that mediate electrical activity-dependent gene regulation are not understood, but these mechanisms are likely to be responsible, at least in part, for the changes in muscle structure and function that accompany a decrease in myofiber electrical activity. To understand how muscle activity regulates muscle structure and function, we used a subtractive-hybridization and cloning strategy to identify and isolate genes that are expressed preferentially in innervated or denervated muscle. One of the genes which we found to be regulated by electrical activity is the recently discovered acute myeloid leukemia 1 (AML1) gene. Disruption and translocation of the human AML1 gene are responsible for a form of acute myeloid leukemia. AML1 is a DNA-binding protein, but its normal function is not known and its expression and regulation in skeletal muscle were not previously appreciated. Because of its potential role as a transcriptional mediator of electrical activity, we characterized expression of the AML1 gene in innervated, denervated, and developing skeletal muscle. We show that AML1 is expressed at low levels in innervated skeletal muscle and at 50- to 100-fold-higher levels in denervated muscle. Four AML1 transcripts are expressed in denervated muscle, and the abundance of each transcript increases after denervation. We transfected C2 muscle cells with an expression vector encoding AML1, tagged with an epitope from hemagglutinin, and we show that AML1 is a nuclear protein in muscle. AML1 dimerizes with core-binding factor beta (CBF beta), and we show that CGF beta is expressed at high levels in both innervated and denervated skeletal muscle. PEBP2 alpha, which is structurally related to AML1 and which also dimerizes with CBF beta, is expressed at low levels in skeletal muscle and is up-regulated only weakly by denervation. These results are consistent with the idea that AML1 may have a role in regulating gene expression in skeletal muscle.